Drug carrier having self-assembled 3-D nucleic acid nanostructure

Abstract

The present invention relates to a molecule delivery technology and a carrier technology, which may selectively deliver a material to a desired specific cell and living tissue. The present invention may be utilized in the field of a drug carrier which effectively delivers an imaging probe and a therapeutic agent to an affected part.

Claims

1. A method of in vivo delivering a pharmaceutically active ingredient to cancer tissue, comprising: administering a self-assembled 3-D nucleic acid nanostructure as a pharmaceutically active ingredient carrier to a subject in need thereof, wherein the self-assembled 3-D nucleic acid nanostructure comprises: double strand nucleic acids; and single strands form the sides of the self-assembled 3-D nucleic acid nanostructure, wherein the pharmaceutically active ingredient carrier has an L-DNA triangular prism structure composed of 5 strands of L-DNA self-assembled from the combination of: i) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:15; ii) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:16; iii) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:17; iv) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:18; and v) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:19, and wherein the pharmaceutically active ingredient carrier does not comprise a targeting ligand for the tissue.

2. The method of claim 1, wherein the pharmaceutically active ingredient is an anticancer agent.

3. The method of claim 1, wherein the pharmaceutically active ingredient encapsulated is within the nucleic acid nanostructure or bonded to the backbone of the nucleic acid nanostructure, and thereby delivered to the tissue.

4. A method of in vivo delivering an anticancer agent comprising: administering a pharmaceutical composition comprising: a self-assembled 3-D nucleic acid nanostructure as an anticancer agent carrier to carry the anticancer agent to a subject in need thereof, wherein the self-assembled 3-D nucleic acid nanostructure comprises: double strand nucleic acids; and single strands form the sides of the self-assembled 3-D nucleic acid nanostructure, wherein the anticancer agent carrier has an L-DNA triangular prism structure composed of 5 strands of L-DNA self-assembled from the combination of: i) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:15; ii) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:16; iii) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:17; iv) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:18; and v) a single strand of an L-DNA nucleotide sequence of SEQ ID NO:19, and wherein the anticancer agent carrier does not comprise a targeting ligand for the tissue.

5. The method of claim 1, wherein each single strand is connected by linker.

6. The method of claim 2, wherein the anticancer agent is doxorubicin.

7. The method of claim 4, wherein the anticancer agent is doxorubicin.

8. The method of claim 1, the self-assembled 3-D nucleic acid nanostructure is mixed with a pharmaceutical acceptable carrier selected from the group consisting of gum acacia, methyl hydroxybenzoate, and propyl hydroxybenzoate.

9. The method of claim 4, the self-assembled 3-D nucleic acid nanostructure is mixed with a pharmaceutical acceptable carrier selected from the group consisting of gum acacia, methyl hydroxybenzoate, and propyl hydroxybenzoate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

(2) 1. FIG. 1 is a schematic view illustrating a principle in which a DNA polyhedron is assembled from 4 DNA strands by hybridization.

(3) 2. FIG. 2a is a schematic view illustrating a process in which a library of DNA nanostructures is prepared by changing the sugar backbone and size, and the cell and biotissue specificity thereof is confirmed, and FIG. 2b illustrates a result in which 4 DNA tetrahedrons (DSS, LSS, D92, and L92) are prepared and confirmed by PAGE.

(4) 3. FIGS. 3a to 3d illustrate the results in which cells transfected with 4 DNA tetrahedrons (DSS, LSS, D92, and L92) are observed through fluorescent microscopy (FIG. 3a: Hela cells, FIG. 3b: HepG2 cells, FIG. 3c: AS49 cells, and FIG. 3d: MCF7 cells).

(5) 4. FIG. 4 illustrates the results in which cells transfected with 4 DNA tetrahedrons (DSS, LSS, D92, and L92) are subjected to flow cytometry.

(6) 5. FIGS. 5a to 5g illustrates fluorescent images obtained by injecting 4 DNA polyhedrons (DSS, LSS, D92, and L92) into mice. Specifically,

(7) FIG. 5a: Fluorescent image obtained by injecting SS Td,

(8) FIG. 5b: Fluorescent image obtained by injecting D92 Td,

(9) FIG. 5c: Comparison of in vivo fluorescent images obtained by injecting LSS and L92 and leaving the mice to stand for 0 minute to 24 hours after the injection,

(10) FIG. 5d: Fluorescent image obtained by injecting LSS and leaving the mouse to stand for 0 minute to 5 hours (in vivo+ex vivo 5 hours after injection),

(11) FIG. 5e: Fluorescent image obtained by injecting L92 and leaving the mouse to stand for 0 minute to 7 hours (in vivo+ex vivo 7 hours after injection),

(12) FIG. 5f: Comparison of ex vivo fluorescent images obtained by injecting LSS and L92 (L55 and L92 are obtained 5 hours after injection and 7 hours after injection, respectively), and

(13) FIG. 5g: Comparison of ex vivo fluorescent images obtained by injecting L55 and L92 (after adjusting scale).

(14) 6. FIGS. 6a to 6d illustrate the results in which the formation of the 4 (L-Td, L-TP, L-Cb, and L-Od) structures are confirmed by PAGE (FIG. 6a: L-Td, FIG. 6b: L-TP, FIG. 6c: L-Cb, and FIG. 6d: L-Od).

(15) 7. FIGS. 7a to 7f illustrate the results in which L-DNA nanostructures fluorescently labeled are injected into a mouse tumor model, and a change in distribution of nanostructures in the organism is observed (FIG. 7a: Free dye, FIG. 7b: L-Td, FIG. 7c: L-TP, FIG. 7d: L-Cb, FIG. 7e: L-Od, and FIG. 7f: Comparison of in vivo images of 4 structures on the same scale).

(16) 8. FIGS. 8a to 8f illustrate the results in which L-DNA nanostructures fluorescently labeled are injected into a tumor model, the mouse was sacrificed, and 6 organs of brain, heart, lung, liver, kidney, and spleen and tumor were removed and observed (FIG. 8a: Free dye, FIG. 8b: L-Td, FIG. 8c: L-TP, FIG. 8d: L-Cb, FIG. 8e: L-Od, and FIG. 8f: Comparison of ex vivo images of 4 structures on the same scale).

(17) 9. FIGS. 9a and 9b illustrate the results in which an optimal binding ratio is searched using a Job plot in order to load a doxorubicin (DOX) drug into L-Td55 (FIG. 9a) and L-Td92 (FIG. 9b).

(18) 10. FIG. 10 illustrates a schematic view of selective DOX delivery and therapy of a cancer tissue using a Xenograft cancer mouse animal model.

(19) 11. FIG. 11 illustrates photographs of external parts of mice which were classified into total 6 groups (PBS, L-Td55, L-Td92, free DOX, DOX@L-Td55, and DOX@L-Td92) and treated for 18 days, and a photograph of tumors removed at the final day of therapy.

(20) 12. FIGS. 12(a-d) are graphs illustrating changes in mice which were classified into total 6 groups (PBS, L-Td55, L-Td92, free DOX, DOX@L-Td55, and DOX@L-Td92) and treated for 18 days during the therapy period (FIG. I2a: Change in volume of tumor, FIG. 12b: Change in body weight, FIG. 12c: Number of survived mice, and FIG. 12d: Final tumor weight).

(21) 13. FIG. 13. illustrates the results in which mice were classified into total 6 groups (PBS, L-Td55, L-Td92, free DOX, DOX@L-Td55, and DOX@L-Td92), treated for 18 days, and sacrificed at the final day of therapy, and lung, liver, kidney, and cancer removed were observed through optical microscopy.

DETAILED DESCRIPTION OF INVENTION

(22) Hereinafter, the present invention will be described in more detail through the Examples. These Examples are provided only for more specifically describing the present invention, and it will be obvious to a person with ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these Examples.

EXAMPLES

(23) I. In Vivo Biodistribution of D/L-DNA Nanostructures

(24) 1. Preparation of DNA Nanostructures Through Synthesis and Self-Assembly of D/L-DNA Oligonucleotides 13

(25) DNA oligonucleotides required for the Tds structure were synthesized by using a DNA synthesis standard protocol. The base sequence of the 55mer DNA standard was adopted from the Tuberfield Td, and the base sequence of the 92mer DNA standard was adopted from the Anderson (Lee et al. Nat. Nanotechnol. 2012, 7, 389-393). The oligonucleotide sequences used to constitute total 4 DNA nanostructures (D-DNA 55mer Td, L-DNA 55mer Td, D-DNA 92mer Td, and L-DNA 92mer Td) are shown in the following Table 1.

(26) TABLE-US-00001 TABLE 1 Sequence (5′ to 3′) Cy5.5- ACATTCCTAAGTCTGAAACATTACAGCTTGC SEQ ID labeled  TACACGAGAAGAGCCGCCATAGTA-Cy5.5 NO: 1 D/L  55_S1 Fluores- ACATTCCTAAGTCTGAAACATTACAGCTTGC SEQ ID cein- TACACGAGAAGAGCCGCCATAGTA- NO: 2 labeled fluorescein D/L  55_S1 D/L TATCACCAGGCAGTTGACAGTGTAGCAAGC SEQ ID 55_S2 TGTAATAGATGCGAGGGTCCAATAC-NH.sub.2 NO: 3 D/L TCAACTGCCTGGTGATAAAACGACACTACGT SEQ ID 55_S3 GGAATCTACTATGGCGGCTCTTC-NH.sub.2 NO: 4 D/L TTCAGACTTAGGAATGTGCTTCCCACGTAG SEQ ID 55_S4 TGTCGTTTGTATTGGACCCTCGCAT-NH.sub.2 NO: 5 Cy5.5- CTCAACTGCCTCAGACGGACAGGTGATACGA SEQ ID labeled GAGCCGGATGGGCATGCTCTTCCCGTAGAGA NO: 6 D/L TAGTACGGTATTGGACCGAGTCCTCGCATG- 92_S1 Cy5.5 Fluores- CTCAACTGCCTCAGACGGACAGGTGATACGA SEQ ID cein- GCCGGATGGGCATGCTCTTCCCGTAGAGATG NO: 7 labeled AAGTACGGTATTGGACCGAGTCCTCGCATG- D/L  fluorecein 92_S1 D/L CGTATCACCTGTCCGTCTGAGGCAGTTGAGAG SEQ ID 92_S2 ATCTCGAACATTCCTAAGTCTGAAGATCCATT NO: 8 TATCACCAGCTGCTGCACGCCATAGTAG-NH.sub.2 D/L GGATCTTCAGACTTAGGAATGTTCGAGATCAC SEQ ID 92_S3 ATGCGAGGACTCGCTCCAATACCGTACTAACG NO: 9 ATTACAGATCAAAGCTACTTGCTACACG-NH.sub.2 D/L CTCTACGGGAAGAGCATGCCCATCCGGCTCAC SEQ ID  92_S4 TACTATGGCGTGCAGCAGCTGGTGATAAAACG NO: 10 TGTAGCAAGTAGCTTTGATCTGTAATCG-NH.sub.2

(27) A library of DNA nanostructures was prepared by performing the Td assembly as described in the document (Kim et al. Chem. Sci., 2014, 5, 1533-1537) to change the sugar backbone and size (see FIG. 2a), and then the self-assembled D and L-Td (D-DNA 55mer Td, L-DNA 55mer Td, D-DNA 92mer Td, and L-DNA 92mer Td) were confirmed by performing 6% non-denaturing polyacrylamide gel electrophoresis (PAGE) (FIG. 2b).

(28) 2. Cell Selectivity of DNA Nanostructures

(29) (1) Transfection of DNA Nanostructures into HeLa, HepG2, A549 and MCF7 Cells

(30) In order to confirm the cell selectivity of each DNA nanostructure, cancer cells were treated with the DNA nanostructures prepared above.

(31) Specifically, HeLa, HepG2, A549 and MCF7 cells were each inoculated into a glass-bottom 35-mm petri dish including a DMEM medium (Gibco, USA) containing 10% fetal bovine serum inactivated with heat, 1% penicillin, and streptomycin, and then the dish was cultured in a wet atmosphere including 5% CO2 at 37° C. The growth medium was removed from each cell sample and washed twice with PBS (Gibco, USA), and the DNA nanostructures prepared were subjected to transfection treatment in each cell.

(32) (2) Microscopic Images of DNA Nanostructures in Cell

(33) Cells transfected with the DNA nanostructures were observed under a fluorescent microscope (DeltaVision, Applied Precision, USA) and living cells were imaged, and the results are each shown in FIG. 3 (FIGS. 3a to 3d).

(34) As a result of the experiment, it could be seen that the DNA nanostructures were entered into the cells, and it could be confirmed that the DNA nanostructures were in the cytoplasm region without being delivered to the cell nucleus.

(35) (3) Flow Cytometry

(36) The HeLa, HepG2, A549 and MCF7 cells were cultured with DNA molecules fluorescently labeled using the method which is the same as that adopted in the transfection experiment, the fluorescence intensity of the cells was evaluated using a flow cytometer (FC500, Beckman coulter, USA), and then, the result is shown in FIG. 4.

(37) Based on the result that the amount of each nanostructure delivered into the cells was quantified through a flow cytometry, the amounts delivered for each cell could be compared with each other, and as a result of the experiment, it was confirmed that D55 had HepG2 cell selectivity, L55 simultaneously had strong HeLa cell selectivity and considerable HepG2 cell selectivity, and L92 had HeLa and HepG2 cell selectivity (FIG. 4).

(38) As described above, since the kind of cell delivered in a large amount for each structure is present, it was confirmed that a structure having cell selectivity could be discovered through the construction of a library composed of various DNA nanostructures.

(39) 3. Tissue Selectivity of DNA Nanostructures

(40) (1) In Vivo Imaging

(41) The animal experiment was approved by the Institutional Animal Care and Use Committee of Korean Institute of Science and Technology, and all the mice were treated according to the regulations of the committee. For in vivo imaging and establishment of a disease model, a mouse was anesthetized by intraperitoneally injecting 0.5% pentobarbital sodium (0.01 m L/g). An animal disease model was established by using a BALB/c nude mouse (5 weeks old, male, Orient Bio Inc., Korea). A tumor was produced by subcutaneously inoculating SCC7 cells (1.0×10.sup.6 cells suspended in a culture medium) into the thigh of the mouse.

(42) The DNA structures prepared were injected into the caudal vein of the mouse, and fluorescent images obtained by using a CCD camera performed in a highly sensitive imaging system (IVIS-spectrum, Perkin-Elmer, USA) are shown in FIG. 5 (FIGS. 5a to 5e).

(43) (2) Ex Vivo Imaging and Histological Analysis After an in vivo imaging study, ex vivo near-infrared fluorescence images for excised organs and the other sites of the body were obtained by using an IVIS-spectrum imaging system including the same obtaining set as that used for in vivo imaging, and the results are shown in FIG. 5 (the second photograph in FIGS. 5a and 5b, the last photograph in FIGS. 5d and 5e, FIG. 5f, and FIG. 5g).

(44) As a result of the experiment, it was confirmed that D55 was delivered more selectively to the liver, and distributed even in the skin tissue. On the contrary, D92 was delivered more selectively to the kidney. It was shown that L55 and L92 were accumulated, exhibiting high selectivity for the cancer tissue In addition, it was observed that as time elapsed, L-Td's had accumulated been more selectively in cancer instead of being distributed throughout the tissue than D-Td (after about 6 to 7 hours), and had escaped after 24 hours.

(45) II. Evaluation of In Vivo Biodistribution of L-DNA Nanostructures

(46) 1. Formation of Structure Labeled with Fluorescent Dye

(47) (1) Formation of 4 Structures (L-Td, L-TP, L-Cb, and L-Od)

(48) The oligonucleotide sequences used to self-assemble total four L-DNA nanostructures having tetrahedron (L-Td), triangular prism (L-TP), cube (L-Cb) and octahedron (L-Od) shapes as an L-DNA nanostructure are shown in the following Tables 2 to 5.

(49) TABLE-US-00002 TABLE 2 Structure Sequence (5′ to 3′) Tetra- S1 CGATGTCTAAGCTGACCG/iSp18/GGAC SEQ ID hedron CGTGATTCCATGAC/iSp18/CTTAGAGT NO: 11 (L-Td) TGCCACCAGG S2 GTCATGGAATCACGGTCC/iSp18/GGCT SEQ ID CACATTGGCTACAG/iSp18/CTATCCGA NO: 12 TCGAGGCATG S3 CATGCCTCGATCGGATAG/iSp18/CGG SEQ ID TCAGCTTAGACATCG/iSp18/GCAAGT NO: 13 GCTGCGTCATAC S4 CCTGGTGGCAACTCTAAG/iSp18/GTA SEQ ID  TGACGCAGCACTTGC/iSp18/CTGTAG NO: 14 CCAATGTGAGCC

(50) TABLE-US-00003 TABLE 3 Structure Sequence (5′ to 3′) Trian- S1 CGATGTCTAAGCTGACCG/iSp18/ SEQ ID gular GGACCGTGATTCCATGAC/iSp18/ NO: 15 prism CTTAGAGTTGCCACCAGG/iSp18/ (L-TP) GAATCCTATGCTCGGACG S2 CGGTCAGCTTAGACATCG/iSp18/ SEQ ID GGCTCACATTGGCTACAG/iSp18/ NO: 16 CTATCCGATCGAGGCATG/iSp18/ CATACTGAGAGCGTTCCG S3 CCTGGTGGCAACTCTAAG/iSp18/ SEQ ID GCGTATCTGAACTGCGAC/iSp18/ NO: 17 CATGCCTCGATCGGATAG/iSp18/ CCACCGAATGGTGTATCG S4 CTGTAGCCAATGTGAGCC/iSp18/ SEQ ID CGTCCGAGCATAGGATTC/iSp18/ NO: 18 CGATACACCATTCGGTGG S5 GTCGCAGTTCAGATACGC/iSp18/ SEQ ID GTCATGGAATCACGGTCC/iSp18/ NO: 19 CGGAACGCTCTCAGTATG

(51) TABLE-US-00004 TABLE 4 Struc- ture Sequence (5′ to 3′) Cube S1 CGATGTCTAAGCTGACCG/iSp18/ SEQ ID (L-Cb) GGACCGTGATTCCATGAC/iSp18/ NO: 20 CTTAGAGTTGCCACCAGG/iSp18/ GAATCCTATGCTCGGACG S2 CCTGGTGGCAACTCTAAG/iSp18/ SEQ ID GGCTCACATTGGCTACAG/iSp18/ NO: 21 CTATCCGATCGAGGCATG/iSp18/ CATACTGAGAGCGTTCCG S3 CATGCCTCGATCGGATAG/iSp18/ SEQ ID GCGTATCTGAACTGCGAC/iSp18/ NO: 22 GCAAGTGCTGCGTCATAC/iSp18/ CCACCGAATGGTGTATCG S4 GGCATTGTACCGTAACCG/iSp18/ SEQ ID CGGTCAGCTTAGACATCG/iSp18/ NO: 23 CGCAAGACGTTAGTGTCC/iSp18/ GTATGACGCAGCACTTGC S5 GTCATGGAATCACGGTCC/iSp18/ SEQ ID CGGTTACGGTACAATGCC/iSp18/ NO: 24 GTCGCAGTTCAGATACGC/iSp18/ CTGTAGCCAATGTGAGCC S6 GGACACTAACGTCTTGCC/iSp18/ SEQ ID CGTCCGAGCATAGGATTC/iSp18/ NO:25 CGGAACGCTCTCAGTATG/iSp18/ CGATACACCATTCGGTGG

(52) TABLE-US-00005 TABLE 5 Struc- ture Sequence (5′ to 3′) Octa- S1 CGATGTCTAAGCTGACCG/iSp18/ SEQ ID hedron GGACCGTGATTCCATGAC/iSp18/ NO: 26 (L-Od) CTTAGAGTTGCCACCAGG S2 GTCATGGAATCACGGTCC/iSp18/ SEQ ID GGCTCACATTGGCTACAG/iSp18/ NO: 27 CTATCCGATCGAGGCATG S3 CTGTAGCCAATGTGAGCC/iSp18/ SEQ ID GCGTATCTGAACTGCGAC/iSp18/ NO: 28 GCAAGTGCTGCGTCATAC S4 GTCGCAGTTCAGATACGC/iSp18/ SEQ. ID CGGTCAGCTTAGACATCG/iSp18/ NO: 29 CGCAAGACGTTAGTGTCC S5 GAATCCTATGCTCGGACG/iSp18/ SEQ ID CATACTGAGAGCGTTCCG/iSp18/ NO: 30 CCTGGTGGCAACTCTAAG S6 CCACCGAATGGTGTATCG/iSp18/ SEQ ID CGTCCGAGCATAGGATTC/iSp18/ NO: 31 CATGCCTCGATCGGATAG S7 GGCATTGTACCGTAACCG/iSp18/ SEQ ID CGATACACCATTCGGTGG/iSp18/ NO: 32 GTATGACGCAGCACTTGC S8 CGGAACGCTCTCAGTATG/iSp18/ SEQ ID CGGTTACGGTACAATGCC/iSp18/ NO: 33 GGACACTAACGTCTTGCG

(53) The strands constituting the structure were mixed so as to have a concentration of 1 μM based on each strand. In this case, a TM buffer (10 mM Tris-HCl, 5 mM MgCl.sub.2, pH 8.0) was used as a buffer. The mixture was denatured through heating at 95° C. by using a RT-PCR machine, and was slowly cooled at 4° C. to be annealed.

(54) As a result, it was confirmed by a non-denaturing PAGE that the resulting four (L-Td, L-TP, L-Cb, and L-Od) structures had been formed, and the results are each shown in FIGS. 6a to 6d (FIG. 6a: L-Td, FIG. 6b: L-TP, FIG. 6c: L-Cb, and FIG. 6d: L-Od).

(55) (2) Labeling with Fluorescent Dye (Red Fluorescence)

(56) A red fluorescent dye (SYTO® 62 Red Fluorescent Nucleic Acid Stain, S11344, life technologies) having binding properties with DNA was mixed so as to have a final concentration of 1 μM. Herein, the DNA structure and the dye were bonded to each other at a concentration ratio of 1:1. The red fluorescent dye used in the present experiment has properties which do not show fluorescence when being present alone, but show fluorescence while being bonded to DNA.

(57) 2. Evaluation of In Vivo Distribution

(58) (1) Preparation of Animal Tumor Model

(59) A balb/c nude mouse (5 weeks old, male) was used as an experimental animal group, and a tumor was formed by subcutaneously inoculating SCC7 cells (1.0×10.sup.6 cells suspended in the culture medium) into the left thigh of the mouse. When the tumor volume became 50 mm.sup.3 or more, the tumor was used for the experiment.

(60) (2) In Vivo and Ex Vivo Imaging

(61) A sample of the L-DNA nanostructure (a final concentration of 1 μM, 200 μL) labeled with fluorescence prepared in 1 was injected into the caudal vein of the mouse tumor model (I. V. injection). When compared to the case before the sample was injected, a change in in vivo distribution of the nanostructure was observed by using an IVIS imaging system apparatus at immediately after injection (0 min), 5, 10, 15, 20, 25, and 30 minutes, and 1, 2, 3, 4, 5, 6, 7, 8, 24, and 48 hours (filter set: Ex=640 nm, Em=680 nm), and the results are each shown in FIGS. 7a to 7f (FIG. 7a: Free dye, FIG. 7b: L-Td, FIG. 7c: L-TP, FIG. 7d: L-Cb, FIG. 7e: L-Od, and FIG. 7f: Comparison of in vivo images of four structures on the same scale).

(62) Based on the in vivo imaging results, ex vivo images were observed by sacrificing the mouse at the time when the fluorescent intensity of each structure in the tumor was highest and removing 6 organs of brain, heart, lung, liver, kidney, and spleen and tumor, and are each shown in FIGS. 8a to 8f (FIG. 8a: Free dye, FIG. 8b: L-Td, FIG. 8c: L-TP, FIG. 8d: L-Cb, FIG. 8e: L-Od, and FIG. 8f: Comparison of ex vivo images of four structures on the same scale).

(63) As a result of the experiment, it could be confirmed that all the four L-DNA nanostructures used in the experiment showed cancer tissue selectivity.

(64) Among them, the best cancer tissue selectivity was found in L-Td, and the next best selectivity was found in this order of L-TP, L-Cb, and L-Od. In the tissue other than the cancer tissue, it was found that L-structures were usually found from the kidney.

(65) III. Evaluation of Selective Delivery of Drug to Cancer Tissue In Vivo

(66) (1) Optimal Binding Ratio Using Job Plot

(67) In order to confirm the optimal binding ratio of Td and doxorubicin (DOX), the Job's plot method was used as in FIG. 9 (see analytical chemistry, 1971, 43, 1265, FIG. 9a: L-Td55 and FIG. 9b: L-Td92). Td and DOX were prepared at a concentration of 1 μM, respectively, in the case of L-Td55 and at a concentration of 1 μM and 3 μM, respectively, in the case of L-Td92, mixed at each volume ratio (see the following Table 6, and the total volume was fixed at 100 μl), and then incubated at normal temperature for 1 hour.

(68) TABLE-US-00006 TABLE 6 Sample Td 10 9 8 7 6 5 4 3 2 1 0 (Td-DOX Complex) DOX 0 1 2 3 4 5 6 7 8 9 10 Total: 100 μl Control TM buffer 10 9 8 7 6 5 4 3 2 1 0 (free DOX) DOX 0 1 2 3 4 5 6 7 8 9 10 Total: 100 μl

(69) And then, the samples were scanned at a wavelength of 200 to 800 nm by using an UV-visible spectrophotometer, and free DOX to which a TM buffer was added instead of the Td sample was used as a control. At 480 nm where the highest absorbance value of DOX was shown, it was judged that the binding ratio used in the sample in which the difference in absorbance values of free DOX and Dox loaded Td was highest formed a composite best, and at a ratio thus determined, DOX was loaded into Td.

(70) (2) Establishment of Xenograft Tumor Model

(71) A balb/c nude mouse (5 weeks old, male) was used as an experimental animal group, and a tumor was formed by subcutaneously inoculating SCC7 cells (1.0×10.sup.6 cells suspended in the culture medium) into the left thigh of the mouse. When the tumor volume became approximately 50 mm.sup.3, the tumor was used for the experiment.

(72) (3) Cancer Tissue Selective DOX and Therapy

(73) For the selective therapy of cancer tissue, DOX was each loaded into L-Td55 and L-Td92, which are a carrier having cancer tissue selectivity. A sample was prepared by mixing the nanostructure with DOX at the optimal binding ratio obtained through the Job plot method of the slide #1 (L-Td55: DOX=1:24, L-Td92: DOX=1:48).

(74) The therapy experiment was performed by classifying the mice into total six groups (PBS, L-Td55, L-Td92, free DOX, DOX@L-Td55, and DOX@L-Td92), and 7 mice per group were used. Before the therapy was performed each time, the tumor volume and the mouse body weight were measured. In this case, the tumor volume was calculated by a method of (minor axis.sup.2*major axis)/2. Each sample was injected into the caudal vein of the mice completely subjected to measurement (see Slide #2 for concentration), the therapy was performed once every three days, and the therapy was performed total 6 times. On day 18 which was 3 days after the last 6th therapy, the final tumor weight was measured by measuring the tumor volume and the mouse body weight, and then measuring the mouse body weight to remove the tumor, and the results are shown in FIGS. 11 and 12 (FIG. 11: Photographs of external parts of mice and a photograph of tumors removed, FIG. 12a: Change in volume of tumor, FIG. 12b: Change in body weight, FIG. 12c: Number of survived m ice, and FIG. 12d: Final tumor weight).

(75) (4) Tissue Fragment Experiment (Histological Analysis)

(76) The therapy was finished, and 6 organs of brain, heart, lung, liver, kidney, and spleen and tumor were removed by sacrificing the survived mouse for each group, thereby observing the presence of organ damage and the tumor state. All the organs and tumor were fixed in 4% formaldehyde (4° C., overnight), and then embedded in paraffin after the tissue dehydration process. The paraffin block thus prepared was cut into a thickness of 5 μm, stained with hematoxylin and eosin, and observed through optical microscopy, and the results are shown in FIG. 13.

(77) As a result of the experiment, it was confirmed that tumors were increased 4 to 5 times in the case of DOX@L55 and DOX@L92, while tumor volumes were increased up to about 40 times for the other groups, and thus the growth in tumor was delayed when DOX was loaded into the carrier.

(78) Due to the tumor growth rate of the tumor bearing mice using SCC7 known to be rapidly growing, it was judged that an increase in tumor volume had been observed in all the cases. When a tumor model is established using human-derived cells, a much better therapeutic effect is expected.

(79) For the control which was not subjected to therapy, the L55 treatment group, the L92 treatment group, and the free DOX treatment group showing slight therapeutic effects, the animal groups of individual 1 to 3 mice died during the therapy period.

(80) When the graph of change in body weight is observed, it is judged that the increase in body weight is due to an increase in tumor volume. When the weights of tumors obtained through ex vivo imaging were compared with each other, the tumor weights of the groups other than DOX@L55 or DOX@L92 exhibited values about 5 times higher than the tumor weights of DOX@L55 or DOX@L92.

(81) When the tissue fragments were observed, no part particularly damaged by DOX had been found in the tissues other than cancer tissues.