COMPLEX FOR DRUG DELIVERY AND STABILIZATION AND PREPARATION METHOD THEREOF

Abstract

A drug delivery system includes an atelocollagen-[aptamer-drug] complex which is prepared by mixing an aptamer-drug conjugate comprised of an aptamer and a drug attached to the aptamer with an atelocollagen dispersion. The aptamer is selected from the group consisting of AS1411, CRO, and ERBB2, and the drug is selected from the group consisting of Gemcitabine, Doxorubicin, and siRNA. An anticancer composition including the drug delivery system can compensate for shortcomings of existing anticancer agents and anticancer therapies.

Claims

1-7. (canceled)

8. A drug delivery system, comprising an atelocollagen-[aptamer-drug] complex which is prepared by mixing an aptamer-drug conjugate comprised of an aptamer and a drug attached to the aptamer with an atelocollagen dispersion, wherein the aptamer is selected from the group consisting of AS1411, CRO, and ERBB2, and the drug is selected from the group consisting of Gemcitabine, Doxorubicin, and siRNA, the AS1411 has the amino acid sequence of SEQ ID No:1; CRO has the amino acid sequence of SEQ ID No:2; and ERBB2 is represented by Formula 1: TABLE-US-00007 a6g66agag666gcc6gag6gcc6cgcaagggcg6aacaaFormula1 wherein 6 is 5-(NNaphthylcarboxyamide)-2-deoxyuridine.

9. The drug delivery system according to claim 8, wherein the atelocollagen dispersion is prepared by dia-filtration of an atelocollagen NaOAc/HAc solution through tangential flow filtration (TFF) in a phosphate-buffered saline (PBS) solution.

10. The drug delivery system according to claim 8, wherein the aptamer-drug conjugate is prepared by mixing the conjugate in a phosphate-buffered saline (PBS) dispersion state with an atelocollagen PBS dispersion.

11. The drug delivery system according to claim 10, wherein the complex is a sol-gel type complex including solidified atelocollagen which surrounds the aptamer-drug conjugate and is molten by body heat.

12. The drug delivery system according to claim 10, wherein the complex is a porous membrane type complex prepared by placing a sol type complex in a cell culture plate, diffusing the same to form a membrane and lyophilizing the formed membrane.

13. The drug delivery system according to claim 10, wherein the complex is a scaffold type complex prepared by injecting a sol type complex into a barrel equipped with a nozzle and then discharging the same on a plate through a 3D plotter.

14. The drug delivery system according to claim 8, wherein the aptamer-drug conjugate is a conjugate of the AS1411 and the Gemcitabine.

15. The drug delivery system according to claim 8, wherein the aptamer-drug conjugate is Gemcitabine-(Gly-Leu-Phe-Gly-Maleimidecarproyl-S(CH.sub.2).sub.6)-ttt-AS 1411, Gemcitabine-(Gly-Leu-Phe-Gly-Maleimidecarproyl-S(CH.sub.2).sub.6)-ttt-CRO, AS1411-Doxorubicin adduct, CRO-Doxorubicin adduct or ERBB2-siRNA conjugate.

16. An anticancer composition, comprising the drug delivery system according to claim 8.

17. A method of decreasing a size of tumor in a patient having cancer, comprising administering the anticancer composition of claim 16 to the patient before cancer surgery.

18. The anticancer composition according to claim 17, wherein the cancer is pancreatic cancer, breast cancer or liver cancer.

19. A method of removing residual cancer cells of a patient having cancer after cancer surgery, administering the anticancer composition of claim 16 to a patient after the cancer surgery.

20. The anticancer composition according to claim 19, wherein the cancer is pancreatic cancer, breast cancer or liver cancer.

21. The anticancer composition according to claim 16, wherein the aptamer-drug conjugate is a conjugate of the AS1411 and the.

22. A method for preparing a drug delivery system, comprising: mixing an aptamer-drug conjugate dispersion with an atelocollagen dispersion to prepare an atelocollagen-[aptamer-drug] complex, wherein the aptamer is selected from the group consisting of AS1411, CRO and ERBB2, and the drug is selected from the group consisting of Gemcitabine, Doxorubicin and siRNA, the AS1411 has the amino acid sequence of SEQ ID No:1; CRO has the amino acid sequence of SEQ ID No:2; and ERBB2 is represented by Formula 1: TABLE-US-00008 a6g66agag666gcc6gag6gcc6cgcaagggcg6aacaaFormula1 wherein 6 is 5-(NNaphthylcarboxyamide)-2-deoxyuridine.

23. The method according to claim 22, further comprising dia-filtration of atelocollagen NaOAc/HAc solution through tangential flow filtration (TFF) in a phosphate-buffered saline (PBS) solution to prepare the atelocollagen dispersion.

24. The method according to claim 22, further comprising: placing a dispersion of the atelocollagen-[aptamer-drug] complex in a cell culture plate; diffusing the same to form a membrane; and lyophilizing the formed membrane to prepare a porous membrane.

25. The method according to claim 22, further comprising: injecting a dispersion of the atelocollagen-[aptamer-drug] complex into a barrel equipped with a nozzle; and discharging the same on a plate through a 3D plotter to prepare a scaffold form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 illustrates HPLC RT of AS1411-Dox adduct after reaction of AS1411 and doxorubicin according to an embodiment of the present invention.

[0018] FIG. 2 illustrates UV-visible absorption spectrum of AS1411-Dox adduct according to an embodiment of the present invention.

[0019] FIG. 3 illustrates ERBB2 aptamer-siRNA conjugate according to an embodiment of the present invention.

[0020] FIG. 4 is photographs showing an atelocollagen-[aptamer-drug conjugate] complex (sol-gel type) according to an embodiment of the present invention.

[0021] FIG. 5 is photographs showing atelocollagen-[aptamer-drug conjugate] complexes (patch type) according to an embodiment of the present invention.

[0022] FIG. 6 is electron micrographs showing an atelocollagen-[aptamer-drug conjugate] complex (scaffold type) according to an embodiment of the present invention.

[0023] FIGS. 7A and 7B are photographs and graphs showing results of measuring in vivo stability of Cy3-AS1411-Gem conjugate and atelocollagen-[Cy3-AS1411-Gem conjugate] complex (sol-gel type).

[0024] FIG. 8 is a photograph showing atelocollagen-[aptamer-drug conjugate] complexes (patch type) according to an embodiment of the present invention.

[0025] FIGS. 9A and 9B are photographs showing test results of in vivo stability of the atelocollagen-[aptamer-drug conjugate] complexes (patch type) according to the embodiment of the present invention.

[0026] FIGS. 10A and 10B are a photograph and electron micrographs (SEM images) showing atelocollagen-[aptamer-drug conjugate] complexes (scaffold type) according to an embodiment of the present invention.

[0027] FIG. 11 is photographs showing test results of in vivo stability of the atelocollagen-[aptamer-drug conjugate] complexes (scaffold type) according to the embodiment of the present invention.

[0028] FIGS. 12A and 12B are photographs and electron micrographs (SEM images) showing atelocollagen-[Cy3-AS1411-Gem conjugate] complex (scaffold type) according to an embodiment of the present invention.

[0029] FIG. 13 is graphs illustrating efficacy examination results of AS1411, CRO, gemcitabine, AS1411-Gem conjugate and CRO-Gem conjugate to pancreatic cancer cell-lines.

[0030] FIG. 14 is graphs illustrating efficacy examination results of AS1411, CRO, gemcitabine, atelocollagen-[AS1411-Gem conjugate] and atelocollagen-[CRO-Gem conjugate] to pancreatic cancer cell-lines.

[0031] FIG. 15 illustrates comparison results of breast cancer cell-line transmission efficacies between ERBB2 aptamer-siRNA conjugate and siRNA.

[0032] FIG. 16 illustrates in vitro efficacy examination results of ERBB2 aptamer-siRNA conjugate and siRNA to breast cancer cell-lines.

[0033] FIG. 17 illustrates in vitro efficacy examination results of atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex to breast cancer cell-lines.

[0034] FIG. 18 illustrates in vitro efficacy examination results of AS1411-Dox adduct and CRO-Dox adduct to liver cancer cell-lines.

[0035] FIG. 19 illustrates in vitro efficacy examination results of atelocollagen-[AS1411-Dox adduct] complex to liver cancer cell-lines.

[0036] FIG. 20 illustrates measured results of tumor size over time after directly injecting 2.0 mg of AS1411-Gem conjugate and 0.5% atelocollagen complex to tumor in a mouse with pancreatic cancer via a syringe.

[0037] FIG. 21 illustrates measured results of tumor size over time after directly injecting atelocollagen alone to tumor in a mouse with pancreatic cancer via a syringe.

[0038] FIG. 22 is graphs illustrating a change in tumor size at different concentrations of atelocollgen-[AS1411-Gem conjugate] complex.

[0039] FIGS. 23A and 23B are a photograph and a graph illustrating results of comparing sizes of excised tumors.

[0040] FIG. 24 is photographs showing atelocollagen-[AS1411-Gem conjugate] complex (patch type) and atelocollagen-[AS1411-Gem conjugate] complex (scaffold type) implanted around the tumor.

[0041] FIGS. 25A and 25B are photographs and graphs illustrating comparison results of a change in tumor size over time after implanting the atelocollagen-[AS1411-Gem conjugate] complex (patch type, A), atelocollagen-[AS1411-Gem conjugate] complex (scaffold type, B), atelocollagen-[CRO-Gem conjugate] complex (patch type, C), and atelocollagen-[CRO-Gem conjugate] complex (scaffold type, D), respectively, around the tumors.

[0042] FIG. 26 is graphs illustrating comparison results of a change in tumor size over time after directly injecting atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex into mice with breast cancer.

[0043] FIG. 27 is a photograph showing comparison of sizes of excited tumors.

[0044] FIG. 28 is graphs illustrating comparison results of a change in tumor size over time after implanting atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (patch type, A), atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (scaffold type, B), atelocollagen-[control ERBB2 aptamer-siRNA conjugate] complex (patch type, C), and atelocollagen-[control ERBB2 aptamer-siRNA conjugate] complex (scaffold type, D), respectively, around the tumors.

[0045] FIG. 29 is graphs illustrating comparison results of a change in tumor size over time after directly injecting atelocollagen-[AS1411-Dox adduct] complex (sol-gel type) and atelocollagen-[CRO-Dox adduct] complex (sol-gel type), respectively, into mice with liver cancer.

[0046] FIG. 30 is a photograph showing comparison of sizes of excited tumors.

DETAILED DESCRIPTION

[0047] A drug stabilizing complex of the present invention may include an atelocollagen-[aptamer-L-X conjugate] complex. Herein, X denotes any substance with therapeutic effects such as anticancer effects, anti-inflammatory effects, etc., for example, drugs, toxins, siRNA, peptides, protein drugs and nano-materials or the like, and may be used in a variety of applications without limitation thereof so long as the substance has therapeutic effects.

[0048] When being conjugated as aptamer-L-X (aptamer-drug conjugate), the aptamer has a targeting function and thus delivers the drug to a target site such as a cancer cell or a site of inflammation. If the above conjugate is combined with atelocollagen, the resultant product may be stabilized inside a body thus to exhibit further improved therapeutic effects.

[0049] Amounts of atelocollagen, aptamer and an anticancer active substance X may depend upon types of aptamers, the weight of a patient, a size of tumor to be treated, or a distance between tumor and the skin, etc., which can be appropriately adjusted.

[0050] The atelocollagen-[aptamer-L-X] complex (i.e., atelocollagen-[aptamer-drug conjugate] complex) of the present invention is not particularly limited to specific aptamers, but may be produced in the form of atelocollagen-[aptamer-L-X conjugate] complex (i.e., atelocollagen-[aptamer-drug conjugate] complex) using different aptamers. In a case of producing in a form of a composite (that is, complex), improvement in stability and anticancer effects of the aptamer may be expected. The complex of the present invention may be useful in treatment of diverse cancers including pancreatic cancer, breast cancer, liver cancer, etc. depending upon types of aptamers and anticancer active materials. Of course, the complex of the present invention may be applied to other therapeutic agents such as arthritis medicines.

[0051] The composition of the present invention may be used in a variety of formulations including, for example: sol-gel type; injection products which can be directly put into cancer or arthritic sites to enable solidification by a body temperature and to allow the drug to be slowly released from the same; transplant materials such as patch, implants, etc. which can be attached to a desired site, or the like. Therefore, the composition of the present invention may be useful to, in particular, decrease a tumor size before surgery and/or remove the remaining cancer cells after surgery without side effects or repulsion during radiation therapy.

[0052] The present invention will be described in more detail by the following examples.

Example 1. Preparation of High Concentration Atelocollagen Dispersion for Medical Use

[0053] After adding 3 wt. % of a high concentration atelocollagen to NaOAc/HAc solution (1.2 g CH.sub.3COONa, 27.5 ml CH.sub.3COOH in 50 ml), the mixture was stirred and completely dissolved while maintaining pH 3.0. The solution was subjected to dia-filtration in PBS solution through tangential flow filtration (TFF), thereby preparing an atelocollagen dispersion for medical use in PBS solution.

[0054] Aptamer-Linker-Drug Conjugate Synthesis

Example 2. Synthesis of Anti-Nucleolin GRO Aptamer-Linker-Gemcitabine Conjugate [AS1411-Gem Conjugate]

[0055] By reacting maleimidecarproyl-(Gly-Phe-Leu-Gly (SEQ ID NO: 5))-gemcitabine [MC-GFLG-Gemcitabine] with HS-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 3) [HS-C6-T.sub.3-AS1411], gemcitabine-(Gly-Leu-Phe-Gly (SEQ ID NO: 6))-Mal-S-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 3) [Gemcitabine-(GLFG-MC-S-C6)-T.sub.3-AS1411, AS1411-Gem conjugate] was synthesized. In other words, RSS-C6-tttggtggtggtggttgtggtggtggtgg (SEQ ID NO: 3) [RSS-C6-T.sub.3-AS141]) was subjected to reductive reaction in the presence of DTT for about 3 hours, and the remaining DTT was removed by Centricon and replaced with an SB17 buffer solution. After putting Mal-GPLG-Gemcitabine dissolved in a small amount of DMSO into the resultant product, the mixture was shaken overnight. Purification was performed through reverse-phase HPLC (Waters-Xbridge OST C18 1050 mm, 65, TEAE/ACN buffer), thereby yielding Gemcitabine-(GLFG-MC-S-C6)-T.sub.3-AS1411 [AS1411-Gem conjugate]. Through ESI-MS, a molecular weight of Gemcitabine-(GLFG-MC-S-C6-T.sub.3-AS1411 [AS1411-Gem conjugate] was determined. C.sub.334H.sub.422F.sub.2N.sub.117O.sub.198P.sub.29S [Cal. MW=10211.90 Obs. MW=10211.87]

Example 3. Synthesis of Anti-Nucleolin CRO Aptamer-Linker-Gemcitabine Conjugate [CRO-Gem Conjugates]

[0056] By reacting maleimidecarproyl-(Gly-Phe-Leu-Gly (SEQ ID NO: 5))-gemcitabine [MC-GFLG-Gemcitabine] with HS-C6-tttcctcctcctccttctcctcctcctcc (SEQ ID NO: 4) [HS-C6-T.sub.3-CRO], gemcitabine-(Gly-Leu-Phe-Gly (SEQ ID NO: 6))-Mal-S-C6-tttcctcctcctccttctcctcctcctcc (SEQ ID NO: 4) [Gemcitabine-(GLFG-MC-S-C6)-T.sub.3-CRO, CRO-Gem conjugate] was synthesized. In other words, RSS-C6-tttcctcctcctccttctcctcctcctcc (SEQ ID NO: 4) [RSS-C6-T.sub.3-CRO] was subjected to reductive reaction in the presence of DTT for about 3 hours, and the remaining DTT was removed by Centricon and replaced with an SB17 buffer solution. After putting Mal-GPLG-Gemcitabine dissolved in a small amount of DMSO into the resultant product, the mixture was shaken overnight. Purification was performed through reverse-phase HPLC (Waters-Xbridge OST C18 1050 mm, 65, TEAE/ACN buffer), thereby yielding gemcitabine-(GLFG-MC-S-C6)-T.sub.3-CRO [CRO-Gem conjugate]. Through ESI-MS, a molecular weight of gemcitabine-(GLFG-MC-S-C6)-T.sub.3-CRO [CRO-Gem conjugate] was determined. C.sub.317H.sub.422F.sub.2N.sub.83O.sub.198P.sub.29S [Cal. MW=9531.49 Obs. MW=9532.09].

Example 4. Synthesis of Anti-Nucleolin GRO Aptamer-Doxorubicin Adduct [AS1411-Dox Adduct]

[0057] By reaction AS1411 with doxorubicin-HCl (adramycin-HCl), AS1411-Dox adduct was synthesized. In other words, 20 M of GRO, 150 M of doxorubicin-HCl and 0.37% formaldehyde were put into 0.5 ml of buffer solution (20 mM sodium phosphate, 150 mM NaCl, 0.5 mM EDTA; pH 7.0) and subjected to reaction while shaking overnight at 10 C. After the reaction, separation/purification were performed through reverse-phase HPLC (Waters-Xbridge OST C18 1050 mm, 0.1 M TEAE/ACN buffer, 260 nm, 490 nm). After the separation/purification, the resultant product was concentrated and desalted by Centricon (Millipore, MW 3000 cut).

[0058] AS1411-Dox adduct was identified by HPLC retention time, UV-Vis spectra and ESI-MS (FIG. 1 and FIG. 2).

Example 5. Synthesis of CRO Aptamer-Doxorubicin Adduct [CRO-Dox Adduct]

[0059] By reacting a control aptamer with doxorubicin-HCl (Adramycin-HCl), a control-Dox adduct was synthesized. In other words, 20 M of control aptamer, 150 M of doxorubicin-HCl and 0.37% formaldehyde were put into 0.5 ml buffer (20 mM sodium phosphate, 150 mM NaCl, 0.5 mM EDTA; pH 7.0) and subjected to reaction while shaking overnight at 10 C. After the reaction, separation/purification were performed through reverse-phase HPLC (Waters-Xbridge OST C18 1050 mm, 0.1 M TEAE/ACN buffer, 260 nm, 490 nm). After the separation/purification, the resultant product was concentrated and desalted by Centricon (Millipore, MW 3000 cut).

[0060] CRO-Dox adduct was identified through HPLC retention time, UV-Vis spectra and ESI-MS.

Example 6. Synthesis of ERBB2 Aptamer-siRNA Conjugate

[0061] From ERBB2 aptamer [a6g66agag666gcc6gag6gcc6cgcaagggcg6aacaa (SEQ ID NO: 7), =NapdU [5-(N-naphthylcarboxyamide)-2-deoxyuridine]] described in Patent Application Nos. KR10-1279584 and U.S. Pat. No. 9,309,515B2 filed in the name of the present inventors, ERBB2 aptamer-siRNA conjugate was synthesized. The siRNA targeting Bcl-2 in a cell was synthesized so as to have a maleimide group at the terminal part thereof. Further, the terminal of the ERBB2 aptamer was synthesized by modifying it into a thiol group. The thiolated aptamer and maleimide-siRNA were reacted with 8 mM sulfo-SMPB, and the ERBB2 aptamer was activated by a reaction with 10 mM TCEP dissolved in PBS at 37 C. for 1 hour. The above two materials were prepared after purification through Centri-Sep column. These materials were fully mixed, followed by reaction at 37 C. for 2 hours. Finally, these two materials, which have completed conjugation, were subjected to PAGE purification and quantification by a UV spectrophotometer (FIG. 3)

[0062] Preparation of Atelocollagen-[Aptamer-Drug Conjugate] Complex

Example 7. Preparation of Ateolocollagen-[Aptamer-Drug Conjugate] Complex Composition (Sol-Gel Type)

[0063] The aptamer-drug conjugate prepared through synthesis was put into PBS, and allowed to be completely dissolved using a mixer at room temperature. The reagent prepared as described above was mixed with a high concentration atelocollagen dispersion at an appropriate concentration of 0.5 to 3.0%, and then processed by a rotator cuff device at room temperature for 30 minutes to prepare a mixture solution. The atelocollagen-[aptamer-drug conjucate] complex (sol-gel type) is in a liquid state (sol) at a low temperature. On the other hand, this complex is solidified to form a gel at 37 C. when being directly injected into tumors in vivo, so that the atelocollagen surrounding the aptamer-drug conjugate (sol-gel type) is gradually molten and the drug may be slowly released to serve as an effective tumor therapeutic agent (FIG. 4).

[0064] 1) Pancreatic cancer: Atelocollagen-[AS1411-Gem conjugate] complex (sol-gel type)

[0065] 2) Breast cancer: Atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (sol-gel type)

[0066] 3) Liver cancer: Atelocollagen-[AS1411-Dox adduct] complex (sol-gel type)

Example 8. Preparation of Atelocollagen-[Aptamer-Drug Conjugate] Complex Composition (Patch Type)

[0067] A mixture solution was prepared using the high concentration collagen dispersion prepared above and the synthesized aptamer-drug conjugate in the same manner as the formation of a sol-gel type product. After putting the prepared mixture solution into a 48-well cell culture plate and diffusing the same to form a uniform film with a thickness of 0.5 mM, the film was frozen at minus () 80 C. The frozen sample was lyophilized in a lyophilizer maintaining at 70 C. for 30 hours, thereby yielding a porous membrane (FIG. 5).

Example 9. Preparation of Atelocollagen-[Aptamer-Drug Conjugate] Complex Composition (Scaffold Type)

[0068] Using an aptamer-drug conjugate synthesized with an atelocollagen dispersion as a bio-compatible and biodegradable natural polymer, an atelocollagen-[aptamer-drug conjugate] complex having a three-dimensional (3D) structure (scaffold type) was prepared. The aptamer-drug conjugate generated by degradation of atelocollagen was combined with cancer cells so that the conjugate could selectively act on the cancer cells. In the preparation, a solid-free form fabrication process was adopted in 3D plotter. Further, the preparation method included: introducing the atelocollagen-[aptamer-drug conjugate] complex mixture solution to a barrel equipped with a precise nozzle (diameter: 300 m); and fixing the barrel to a plate at 37 C. using a 3D plotter at a discharge pressure of 30050 kPa with a floating rate of 5 mm/s, thereby fabricating 3 layers of 3D atelocollagen-[aptamer-drug conjugate] complex (scaffold type).

[0069] In addition, a cell carrier in a grid structure was formed to increase pore efficiency, and the atelocollagen was prepared in different concentrations (0.5, 1.5, and 3.0 wt. %), thus to produce 3D atelocollagen-[aptamer-drug conjugate] complex (scaffold type) that can control a mechanical strength or degradation rate of the cell carrier. For cross-linkage between the atelocollagen-[aptamer-drug conjugate] complex (scaffold type) and the cell carrier, genipin was used. More particularly, the above two substances were cross-linked in 1% genipin solution under a condition of 20 C. for 24 hours (FIG. 6).

[0070] In Vivo Stability Verification

Example 10. In Vivo Stability Experiment of Atelocollagen-[Aptamer-Drug Conjugate] Complex Composition (Sol-Gel Type)

[0071]

TABLE-US-00001 TABLE 1 Number Treat Treat concentration 1 Negative 2.0% Ateloollagen control 2 Positive 10 M Cy3-AS1411-Gem Conjugate control 3 Sample (1) 10 M Cy3-AS1411-Gem Conjugate & 2.0% Atelocollagen 4 Sample (2) 10 M Cy3-AS1411-Gem Conjugate & 0.3% Atelocollagen

[0072] Cy3-labelled AS1411-Gem conjugate [Cy3-AS1411-Gem conjugate] and atelocollagen-[Cy3-AS1411-Gem conjugate] complex (sol-gel type) were prepared at specific concentrations as shown in the above table. Each of the pre-treated Cy3-AS1411-Gem conjugate and atelocollagen-[Cy3-AS1411-Gem conjugate] complex (sol-gel type) was injected into the subcutaneous layer of each nude mouse. Thereafter, according to a method for measuring a brightness level of fluorescence by date, it was observed how much Cy3-AS1411-Gem conjugate is maintained in a stable state. For this purpose, IVIS, which is a fluorescence measurement device, was used to measure the fluorescent level at intervals of 3 to 4 days. Through analysis of the obtained results, it could be seen that, when Cy3-AS1411-Gem conjugate alone was injected into an animal, the conjugate was substantially spread from a body site into which the above conjugate was introduced to the whole body of the animal, and the fluorescence disappeared within 1 day. On the other hand, when the atelocollagen-[Cy3-AS1411-Gem conjugate] complex (so-gel type) and the atelocollagen were injected together, it was observed that the conjugate at a low concentration was slowly spread over several days whereas the Cy3-AS1411-Gem conjugate at a high concentration still remained in a considerable amount on the injection site for maximum 35 days. Accordingly, in a case of a complex of the Cy3-AS1411-Gem conjugate and the atelocollagen, it was demonstrated that the complex could quite stably remain in vivo, compared to administration of the conjugate alone (FIG. 7A and FIG. 7B).

Example 11. In Vivo Stability Experiment of Atelocollagen-[Aptamer-Drug Conjugate] Complex Composition (Patch Type)

[0073] Cy3-labelled AS1411-Gem conjugate [Cy3-AS1411-Gem conjugate] and atelocollagen-[Cy3-AS1411-Gem conjugate] were lyophilized to form sheets. The sheets were fabricated in a size of 5 mm and 8 mm using each of atelocollagens at concentrations of 0.5%, 1.5% and 3.0% (FIG. 8).

[0074] After transplanting the prepared sheets on the outer skin layer of each mouse, degradation rates were visually measured. The sheets at 0.5% and 1% concentrations were observed to be molten and absorbed within 5 days. Further, the sheet at 1.5% concentration partially remained after 5 days and was completely molten on day 8. Further, the sheet at 3% concentration was observed to remain without being completely molten even after 8 days (FIGS. 9A and 9B).

Example 12. In Vivo Experiment of Atelocollagen-[Aptamer-Drug Conjugate] Complex Composition (Scaffold Type)

[0075] Cell carriers were fabricated in a 3D grid structure using atelocollagen by means of a 3D plotter. More particularly, the cell carriers were fabricated in sizes of 552 mm.sup.3 and 10102 mm.sup.3 using the atelocollagen at concentrations of 0.5%, 1.5% and 3.0%. Further, in order to control a degradation rate in vivo, the cell carriers were cross-linked in a 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride solution (EDC, Sigma Chemical Co., St. Louis, Mo., USA) for 24 hours. Otherwise, the cell carriers were cross-linked using genipin as a bio-compatible material to form a scaffold. The scaffold obtained as described above was washed with distilled water using an ultrasonic mill, followed by lyophilization at 50 C. (FIG. 10A and FIG. 10B).

[0076] The cross-linked scaffold was transplanted on the outer skin layer of a mouse in the same manner as the method performed above, and was observed over time. It was confirmed that no substantial degradation was observed even at a time of 8 days passed after transplantation. Therefore, it was demonstrated that the degradation rate was prominently reduced due to the cross-linkage (FIG. 11).

[0077] Cy3-AS1411-Gem conjugate was carried on the scaffold to be observed by an electron microscope after (FIGS. 12A and 12B).

[0078] In Vitro Efficacy Validation

Example 13. In Vitro Efficacy of Aptamer-Drug Conjugate to Pancreatic Cancer

[0079] WST-1 assay for pancreatic cancer cell lines, i.e., BxPC3, Miapaca-2, Panc-1 and capan-1 was performed. Cell inhibition efficacy validation of AS1411-Gem conjugate to the pancreatic cancer cell lines was performed in vitro through WST-1 assay. First, a control aptamer CRO in a naked form without being combined with an anticancer agent, AS1411 having a cancer cell-targeting function, as well as CRO-Gem conjugate and AS1411-Gem conjugate which are combined with an anticancer agent effective to pancreatic cancer, i.e., gemcitabine, respectively, were prepared (Table 2).

TABLE-US-00002 TABLE2 Name SEQUENCE MW AS1411 TTTGGTGGTGGTGGTTGTGGTGGTGGTGG 9185.01 (SEQIDNO:3) CRO TTTCCTCCTCCTCCTTCTCCTCCTCCTCC 8504.50 (SEQIDNO:4) AS1411- (Gem).sub.n-L-GGTGGTGGTGGTTGTGGTGG 8576.61 Gem TGGTGG(SEQIDNO:1) conjugate CRO-Gem (Gem).sub.n-L-CCTCCTCCTCCTTCTCCTCC 7896.10 conjugate TCCTCC(SEQIDNO:2)

[0080] Gem=gemcitabine, n=1 to 10

[0081] In comparison of cell viability and proliferation of the cells through WST-1 assay, it was confirmed that AS1411 aptamer has much better efficacy than CRO as the control aptamer, and further showed more excellent efficacy than the case of treatment using the original anticancer agent, that is, gemcitabine alone.

[0082] Experimental procedure: Each of the pancreatic cancer cell lines, that is, BPC3, Miapaca-2, Panc-1 and capan-1 (ATCC, RPMI+10% FBS), was seeded on a 96-well plate in a cell number of 1 to 210.sup.4 cells/well, which was defined by a cell test method to determine an appropriate cell concentration, followed by growing for 1 day. The aptamer and aptamer-drug conjugate were dissolved in PBS, respectively, and were immediately treated in each well at concentrations from 0.1 M up to 100 M. The treated cells were incubated in a 5% CO.sub.2 incubator for 72 hours, treated with 10 l of a reagent solution for WST-1 assay (WST-1, Roche), respectively, followed by measuring the absorbance at 440 nm using an ELISA reader (FIG. 13).

Example 14. In Vitro Efficacy of Atelocollagen-[Aptamer-Drug Conjugate] Complex to Pancreatic Cancer

[0083] WST-1 assay for the pancreatic cancer cell lines, i.e., Miapaca-2 and MCF-7 was performed. Cell inhibition efficacy validation of the atelocollagen-[AS1411-Gem conjugate] complex to the pancreatic cancer cell lines was performed in vitro through WST-1 assay. First, a control aptamer CRO in a naked form without being combined with an anticancer agent, AS1411 having a cancer cell-targeting function, as well as atelocollagen-[AS1411-Gem conjugate] complex and atelocollagen-[CRO-Gem conjugate] complex which are combined with an anticancer agent effective to pancreatic cancer, i.e., gemcitabine, respectively, were prepared.

[0084] In comparison of cell viability and proliferation of the cells through WST-1 assay, it was confirmed that the atelocollagen-[AS1411-Gem conjugate] complex has much better efficacy than the atelocollagen-[CRO-Gem conjugate] complex, and further showed more excellent efficacy than the case of treatment using the original anticancer agent, that is, gemcitabine alone.

[0085] Experimental procedure: Each of the pancreatic cancer cell lines, that is, Miapaca-2 and MCF-7 (ATCC, RPMI+10% FBS), was seeded on a 96-well plate in a cell number of 1 to 210.sup.4 cells/well, which was defined by a cell test method to determine an appropriate cell concentration, followed by growing for 1 day. The aptamer and atelocollagen-[Aptamer-drug conjugate] complex were dissolved in PBS, respectively, and were immediately treated in each well at concentrations from 0.1 M up to 100 M. The treated cells were incubated in a 5% CO.sub.2 incubator for 72 hours, treated with 10 l of a reagent solution for WST-1 assay (WST-1 Roche), respectively, followed by measuring the absorbance at 440 nm using an ELISA reader (FIG. 14).

Example 15. In Vitro Efficacy of ERBB2 Aptamer-Drug Conjugate to Breast Cancer

[0086] In order to identify targeting to a breast cancer target cell, ERBB2 aptamer-linker-siRNA-Cy3, which was modified with Cy3 at siRNA, and siRNA-Cy3 as a control group were prepared. ERBB2 over-expressed MCF7 cell lines were treated with these two synthesized materials, respectively, followed by observation through a fluorescence microscope. In a case of the siRNA control groups including untreated one and the other only combined with Cy3, the cells did not show fluorescence. On the other hand, the cells exhibited fluorescent in the test group combined with ERBB2 aptamer only. By analyzing such results, it could be found that siRNA might be accurately delivered to target cells through targeting by aptamer (FIG. 15)

[0087] In addition, WST-1 assay for the breast cancer cell lines, i.e., BT-474 and MCF-7, was performed. Cell inhibition efficacy validation of ERBB2 aptamer to the breast cancer cell lines was performed in vitro through WST-1 assay. After treating the breast cancer cell lines with a control ERBB2 aptamer without activity, as well as ERBB2 aptamer-siRNA conjugate wherein an effective anticancer agent having excellent anticancer efficacy, that is, siRNA is bound to ERBB2 target aptamer having a cancer cell targeting function, respectively, WST-1 assay was performed. In comparison of cell viability and proliferation of the cells, it was demonstrated that the ERBB2 aptamer-siRNA conjugate has much better efficacy than the ERBB2 aptamer, thereby verifying excellent efficacy in the breast cancer cell line (FIG. 16).

Example 16. In Vitro Efficacy of Atelocollagen-[Aptamer-Drug Conjugate] Complex to Breast Cancer

[0088] Cell inhibition efficacy validation of atelcollagen-[ERBB2 aptamer-siRNA conjugate] complex to breast cancer cell lines, i.e., BT-474 and MCF-7, was performed in vitro through WST-1 assay. After treating the breast cancer cell lines with naked ERBB2, atelocollagen-[control aptamer-siRNA conjugate] complex without activity, as well as atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex wherein an effective anticancer agent having excellent anticancer efficacy, that is, siRNA is bound to ERBB2 target aptamer having a cancer cell targeting function, respectively, WST-1 assay was performed. In comparison of cell viability and proliferation of the cells, it was confirmed that the ERBB2 aptamer-siRNA conjugate has much better efficacy than the control group, that is, the atelocollagen-[control aptamer-siRNA conjugate] complex aptamer, thereby verifying excellent efficacy in the breast cancer cell line (FIG. 17).

Example 17. In Vitro Efficacy of AS1411-Dox Adduct to Liver Cancer

[0089] In the same manner as executed above, cell inhibition efficacy validation of AS1411-Dox adduct as an aptamer-drug conjugate as well as a control-Dox adduct as a control aptamer to breast cancer cell lines, i.e., HepG2 and Huh-7, was performed in vitro through WST-1 assay. From the result, it was confirmed that the AS1411-Dox adduct has much better efficacy than the control-Dox adduct, thereby verifying active performance of the composition (FIG. 18).

Example 18. In Vitro Efficacy of Atelocollagen-[Aptamer-Drug Conjugate] Complex to Liver Cancer

[0090] In the same manner as described in Example 17, the liver cell lines were prepared, and the prepared breast cancer cell lines were treated with atelocollagen-[AS1411-Dox adduct] complex having anticancer effects, atelocollagen-[CRO-Dox-adduct] complex without a targeting function and atelocollagen-Dox, respectively, followed by performing the efficacy validation through WST-1 assay. As predicted, the results showed that the atelocollagen-[AS1411-Dox adduct] complex having a targeting function has prominent efficacy, as compared to the control group, that is, the atelocollagen-[CAO-Dox-adduct] complex. Further, in a case of administration of Dox alone as well as collagen, cell inhibition due to non-selective cell permeation was observed (FIG. 19).

[0091] In Vivo Efficacy Validation

Example 19. Efficacy Validation of Atelocollagen-[AS1411-Gem Conjugate] Complex (Sol-Gel Type) in Pancreatic Cancer Animal Model

[0092] Preparation of animal model: 6 week-old Balb/c nude female mice were prepared as in vivo tumor models. An average weight of the mice used in the experiments was 20 g. Human-derived pancreatic cancer cell lines, that is, capan-1 cells were purchased from ATCC Co., and proliferated. By subcutaneous injection of the proliferated capan-1 cells into the mice in an amount of as many as 510.sup.7 cells to form a tumor. By observing every day in 2 to 3 weeks after the injection, the growth of tumor was visually checked and a size of the tumor was measured using a caliper.

[0093] Efficacy Validation of the complex: After the tumor has grown to an appropriate size, each of the atelocollagen-[AS1411-Gem conjugate] complexes (sol-gel type) as shown in Table 3 below, was directly injected to a site of the grown tumor in each mouse through a syringe (Table 3).

TABLE-US-00003 TABLE 3 Composition of atelocollagen- Number of [AS1411-Gem Conjugate] Complex Treat surviving (Sol-Gel type) number mice AS1411-Gem Conjugate 2.0 mg 4 4 AS1411-Gem Conjugate 1.5 mg 4 4 AS1411-Gem Conjugate 1.0 mg 4 4 AS1411-Gem Conjugate 0.5 mg 4 4

[0094] Further, in order to determine anticancer effects of the atelocollagen-[AS1411-Gem conjugate] complexes (sol-gel type), a change in the tumor size and a change in the weight of the implant were observed over a predetermined period of time (FIG. 20 and FIG. 21).

[0095] As compared to a control group, that is, a case of injecting 0.5% atelocollagen alone, the above experimental group exhibited prominent cancer-inhibitory efficacy, and it was observed that anticancer effects was increased with higher concentration (FIG. 22).

[0096] At a time of 30 days passed after the transplantation of the complex, the tumors were excited from each group. All excited tumors were subjected to measurement of weight and photographing, followed by comparing the actual tumor sizes to one another (FIGS. 23A and 23B).

Example 20. Efficacy Validation of Atelocollagen-[AS1411-Gem Conjugate] Complex (Patch Type) and Atelocollagen-[AS1411-Gem Conjugate] Complex (Scaffold Type) in Pancreatic Cancer Animal Model

[0097]

TABLE-US-00004 TABLE 4 Number of Treat surviving Treat concentration number mice Atelocollagen-[AS1411-Gem 3 3 Conjugate] Complex (patch type) Atelocollagen-[AS1411-Gem 3 3 Conjugate] Complex (scaffold type) Atelocollagen-[CRO-Gem Conjugate] 3 3 Complex (patch type) Atelocollagen-[CRO-Gem Conjugate] 3 3 Complex (scaffold type) DPBS 0.5% Atelocollagen 3 3

[0098] Tumor animal models were prepared in the same manner as described in Example 19, and then, the atelocollagen-[AS1411-Gem conjugate] complex (patch type) and atelocollagen-[AS1411-Gem conjugate] complex (scaffold type) prepared by the same method as described in Examples 8 and 9 were transplanted around the tumors in the tumor animal models, respectively (FIG. 24).

[0099] In order to determine anticancer effects of the transplanted atelocollagen-[AS1411-Gem conjugate] complex (patch type) and atelocollagen-[AS1411-Gem conjugate] complex (scaffold type), a change in the tumor size and a change in the weight of the implant were observed over a predetermined period of time. From the results, it was confirmed that the atelocollagen-[AS1411-Gem conjugate] complex (scaffold type) has much better anticancer efficacy than the atelocollagen-[AS1411-Gem conjugate] complex (patch type) (FIGS. 25A and 25B).

Example 21. Efficacy Validation of Atelocollagen-[ERBB2 Aptamer-siRNA Conjugate] Complex (Sol-Gel Type) in Breast Cancer Animal Model

[0100] Preparation of animal model: 6 week-old Balb/c nude female mice were prepared as in vivo tumor models. An average weight of the mice used in the experiments was 20 g. A human-derived breast cancer cell lines, that is, BT-474 cells were purchased from ATCC Co., and proliferated. By subcutaneous injection of the proliferated BT-474 cells into the mice in an amount of as many as 110.sup.7 cells to form a tumor. By observing every day in 2 to 3 weeks after the injection, the growth of tumor was visually checked and a size of the tumor was measured using a caliper.

TABLE-US-00005 TABLE 5 Treat Number of Treat concentration number survival Atelocollagen-[ERBB2 aptamer-siRNA 3 3 Conjugate] Complex (Sol-Gel type) ERBB2 aptamer-siRNA Conjugate 2 mg & 0.5% Atelocollagen DPBS 0.5% Atelocollagen 3 3

[0101] Efficacy validation of atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (sol-gel type): After the tumor has grown to an appropriate size, the atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (sol-gel type) as shown in Table 5 above, was directly injected to a site of the grown tumor in each mouse through a syringe. Further, in order to determine anticancer effects of the atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (sol-gel type), a change in the tumor size and a change in the weight of the implant were observed over a predetermined period of time. Prominent cancer-inhibitory efficacy was observed, as compared to the control group, that is, a case of injecting 0.5% atelocollagen alone (FIG. 26).

[0102] At a time of 30 days passed after the injection of atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (sol-gel type), the tumors were excited from each group. All excited tumors were subjected to photographing, followed by comparing the actual tumor sizes to one another (FIG. 27).

Example 22. Efficacy Validation of Atelocollagen-[ERBB2 Aptamer-siRNA Conjugate] Complex (Patch Type) and Atelocollagen-[ERBB2 Aptamer-siRNA Conjugate] Complex (Scaffold Type) in Breast Cancer Animal Model

[0103] Tumor animal models were prepared in the same manner as described in Example 21, and then, the atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (patch type) and atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (scaffold type) prepared by the same method as described in Examples 7 and 8 were transplanted around the tumors in the tumor animal models, respectively.

[0104] In order to determine anticancer effects of the transplanted atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (patch type) and atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (scaffold type), a change in the tumor size and a change in the weight of the implant were observed over a predetermined period of time. From the results, it was confirmed that the atelocollagen-[ERBB2 aptamer-siRNA conjugate] complex (scaffold type) has much better anticancer efficacy than the atelocollagen-[ERBB2-Gem conjugate] complex (patch type) (FIG. 28).

Example 23. Efficacy Validation of Atelocollagen-[AS1411-Dox Adduct] Complex (Sol-Gel Type) in Liver Cancer Animal Model

[0105] Preparation of animal model: 6 week-old Balb/c nude female mice were prepared as in vivo tumor models. An average weight of the mice used in the experiments was 20 g. A human-derived liver cancer cell lines, that is, Huh-7 cells were purchased from ATCC Co., and proliferated. By subcutaneous injection of the proliferated Huh-7 cells into the mice in an amount of as many as 510.sup.6 cells to form a tumor. By observing every day in 2 to 3 weeks after the injection, the growth of tumor was visually checked and a size of the tumor was measured using a caliper.

TABLE-US-00006 TABLE 6 Number of Treat surviving Treat concentration number mice Atelocollagen-[AS1411-Dox Adduct] 3 3 Complex AS1411-Dox Adduct 2 mg & 0.5% Atelocollagen Atelocollagen-[CRO-Dox Adduct] 3 3 Complex CRO-Dox Adduct 2 mg & 0.5% Atelocollagen DPBS 0.5% Atelocollagen 3 3

[0106] Efficacy validation of atelocollagen-[AS1411-Dox adduct] complex (sol-gel type): After the tumor has grown to an appropriate size, atelocollagen-[AS1411-Dox adduct] complex (sol-gel type) as shown in Table 6 above, was directly injected to a site of the grown tumor in each mouse through a syringe. Further, in order to determine anticancer effects of the complex, a change in the tumor size and a change in the weight of the implant were observed over a predetermined period of time. As compared to the control group, that is, a case of injecting 0.5% atelocollagen alone, as well as atelocollagen-[CRO-Dox adduct] complex (sol-gel type) without activity, it was observed that the atelocollagen-[AS1411-Dox adduct] complex (sol-gel type) has prominent cancer-inhibitory efficacy (FIG. 29).

[0107] At a time of 30 days passed after the transplantation of the atelocollagen-[AS11-Dox adduct] complex (sol-gel type), the tumors were excited from each group. All excited tumors were subjected to photographing, followed by comparing the actual tumor sizes to one another (FIG. 30).

[0108] The complex of the present invention is stable in a body and applicable to anticancer therapeutic agents and anti-inflammatory therapeutic agents.