KR102560320B1 - Aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles and preparation method thereof - Google Patents

Abstract

The present invention relates to aptamer-chitosan-doxorubicin-gold-5-fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs), which are functionalized with AS1411 DNA aptamer to facilitate drug delivery, as chitosan-gold-based nanoparticles for delivering doxorubicin (Dox) and 5-fluorouracil (5-Fu) to brain tumor target sites, and a preparation method thereof.

Description

Aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles and preparation method thereof

The present invention relates to nanoparticles, and more particularly, as chitosan-gold based nanoparticles for delivering doxorubicin (Dox) and 5-fluorouracil (5-Fu) to brain tumor target sites, functionalized with AS1411 DNA aptamer to facilitate drug delivery, aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) and their preparation method It is about.

The World Health Organization (WHO) has warned that grade 4 glioblastoma multiforme (GBM) causes aggressive brain tumors that can cause serious harm to the body and cause death within a short period of time.

The glioblastoma (GBM) or glioblastoma multiforme is one of the most common brain tumors, accounting for about 12 to 15% of brain tumors, and the name of the tumor is abbreviated as glioblastoma. Although glioblastoma is the most common malignant brain cancer, it is relatively rare compared to other cancers such as colorectal or lung cancer. Glioblastoma cells are similar to astrocytes. Astrocytes maintain and nourish nerve cells and are responsible for the defense response to brain tissue damage. Genetic abnormalities in stem cells or immature astrocytes are thought to be involved in the development and malignancy of glioblastoma. Cells with these genetic abnormalities can reproduce rapidly and spread by invading surrounding brain tissue.

According to the World Health Organization (WHO), glioblastoma (GBM) can be classified as I to IV depending on the degree of malignancy, with grade I tumors being the least malignant and grade IV tumors as the most malignant with rapid growth and dangerous potential.

As a treatment for this, chemotherapy and radiation therapy have been mainly used conventionally, and chemotherapy including a first antitumor drug (temozolomide) and adjuvant drugs (lomustine, bevacizumab, procarbazine, and vincristine) is used as a single or mixed drug, or is associated with radiation therapy to treat glioblastoma (GBM) in various stages. However, factors such as drug resistance, tumor recurrence, and cytotoxicity act as factors that make treatment difficult.

In order to solve this problem, Patent Registration No. 10-1153799 (Announced on June 13, 2012) discloses a "composition for inhibiting cancer cell proliferation containing carbon nanomaterials". This relates to a composition for inhibiting cancer cell proliferation comprising a carbon nanomaterial as an active ingredient. More specifically, the carbon nanomaterial of the present invention is capable of inhibiting proliferation specifically in brain tumor cells without affecting brain nerve cells that do not proliferate after differentiation. It relates to a composition capable of inhibiting.

On the other hand, due to pathophysiological conditions, the tumor microenvironment has pH 4.5-6.8, whereas normal tissue has pH 7.4. Conventional nanoparticle-based nano drug delivery systems have a problem in that it is difficult to reach the target site because they cannot detect a unique physiological pH.

Therefore, there is a need for a nano drug delivery system capable of accurately delivering a drug to a target site by improving the drug delivery function, and the need for suitable nanoparticles for this is growing.

Registered Patent No. 10-1153799 (2012.06.13. Announcement), "Composition for Inhibiting Cancer Cell Proliferation Containing Carbon Nano Materials"

In order to solve the above problems, an object of the present invention is to provide aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) and a method for preparing the same as chitosan-gold-based nanoparticles for delivering doxorubicin (Dox) and 5-fluorouracil (5-Fu) to a target site.

In order to achieve the above object, the present invention comprises the steps of preparing a Myongwolcho extract (S10), forming gold nanoparticles (S20), preparing a solution of cetyltrimethylammonium bromide-gold nanoparticles (S30), preparing a mixture of 5-fluorouracil-gold nanoparticles (S40), forming chitosan-gold-5-fluorouracil nanoparticles (S50), and preparing chitosan-doxorubicin-gold-5-fluorouracil nanoparticles. (S60) and aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles comprising the steps of preparing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (S70). It provides a method for producing NPs.

At this time, the Myongwolcho extract manufacturing step (S10) is characterized in that by extracting Myongwolcho ( Gynura Procumbers (GP)) using an extraction solvent to prepare a Myongwolcho extract.

At this time, in the gold nanoparticle forming step (S20), gold nanoparticles (GP-AuNPs) are formed by adding AuCl to the Meyongwolcho extract prepared in the Myongwolcho extract preparation step (S10), stirring and freeze-drying, and then using the Myongwolcho extract.

At this time, in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30), the cetyltrimethylammonium bromide (CTAB) solution is mixed with the gold nanoparticles (GP-AuNPs) using the Myrtle grass extract formed in the gold nanoparticle formation step (S20) to prepare a cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution.

At this time, in the 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticle mixture preparation step (S40), the cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) are mixed with a 5-fluorouracil (5FU) solution to obtain 5 fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5 FU-CTAB-AuNPs) mixture is prepared and centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-AuNPs).

At this time, in the chitosan-gold-5-fluorouracil nanoparticle formation step (S50), chitosan solution was added to the 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticle mixture prepared in the 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticle mixture preparation step (S40) and stirred to coat chitosan to obtain chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs) It is characterized by forming.

At this time, in the chitosan-toxin Rubbiin-Gold-5 fluoro particle manufacturing stage (S60), the chitosan-gold-5 fluoro-lasyl nanoparticles obtained from the chitosan-gold-5 fluoro particle manufacturing stage (S50) added, stirred, and centrifuged to the chitosan-toxin rubbiin- It is characterized by the preparation of the Friday-5 Fluoro Lasyl Nanoparticles (CS-DOX-AU-5FU NPS).

At this time, in the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S70), EDC, NHS, and AS1411 DNA aptamer were added to the chitosan-doxorubicin-gold-5fluorouracil nanoparticles prepared in the chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S60) and stirred to aptamer-chitosan-doxorubicin-gold-5fluorouracil. It is characterized by preparing lauracil nanoparticles (Apt-CS-Dox-Au-5FU NPs).

In addition, in the present invention, gold nanoparticles are obtained by adding AuCl to the Myrtleweed extract obtained by extracting Myrtleweed and stirring to obtain gold nanoparticles, mixing Cetyltrimethylammonium bromide (CTAB) solution with the obtained gold nanoparticles (AuNPs) to form a solution of cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs), and adding 5 -Fluorouracil (5FU) solution was mixed to form 5 fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) mixture, centrifuged to obtain 5 fluorouracil-gold nanoparticles (5FU-AuNPs), chitosan solution was added to the obtained 5 fluorouracil-gold nanoparticles, stirred to coat chitosan (CS), and doxorubicin (Dox) It provides aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) obtained by adding and stirring, adding EDC, NHS and AS1411 DNA aptamer and stirring.

On the other hand, the Myongwolcho extract is characterized in that it is extracted by heating for 5 to 15 minutes at a temperature of 80 to 90 ° C. by using Meongwolcho as distilled water.

On the other hand, the gold nanoparticles (AuNPs) are characterized in that they are formed by adding AuCl to the Myongwolcho extract and stirring at 26 to 30 ° C. for 8 to 16 hours.

On the other hand, the chitosan-gold-5-fluorouracil nanoparticles (CS-Au-5Fu NPs) are characterized in that the chitosan solution formed by dissolving 10 mg of chitosan in 5-fluorouracil-gold nanoparticles in 20 mL of distilled water containing 0.5% acetic acid was added, stirred for 10 minutes and sonicated for 15 minutes to coat the chitosan, and collected by centrifugation.

The aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention are chitosan-gold-based nanoparticles for delivering doxorubicin (Dox) and 5-fluorouracil (5-Fu) to a target site, and are functionalized with aptamers and have excellent drug delivery ability to the target site.

1 is a flow chart showing a method for preparing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) according to the present invention.
Figure 2 is a schematic diagram showing the formation process of aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) according to the present invention.
Figure 3 is a graph of the visual spectrum analysis of Comparative Example 1 (Myeongwolcho extract, GP Aqueous extract), Example 6 (AuNPs), and Comparative Example 6 (AuCl).
Figure 4 is Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Comparative Example 2 (Dox), Comparative Example 3 (5-Fu), Comparative Example 4 (CS), a graph of visible spectrum analysis of Comparative Example 5 (AS1411 DNA aptamer).
5 is a graph showing FTIR analysis of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Example 6 (AuNPs), Comparative Example 1 (GP Aqueous extract), and Comparative Example 2 (Dox).
6 is a graph showing XRD analysis of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), and Example 6 (AuNPs).
7 is a TEM image of Example 1 (Apt-CS-Dox-Au-5FU NPs) (d), Example 3 (CS-Au-5Fu NPs) (c), Example 4 (5FU-AuNPs) (b), and Example 6 (AuNPs) (a).
8 is a graph showing zeta potential and particle size analysis (a), zeta potential analysis (b), particle size (c) after 72 hours at pH 7.4 (phosphate buffer), and particle size (d) after 72 hours at pH 5.4 (acetate buffer) of Example 1 (Apt-CS-Dox-Au-5FU NPs).
9 is a graph showing the in vitro release of 5FU from Example 1 (Apt-CS-Dox-Au-5FU NPs) in phosphate buffer (pH 7.4) and acetate buffer (pH 5.4).
10 is a graph showing the in vitro release of Dox in phosphate buffer (pH 7.4) and acetate buffer (pH 5.4) from Example 1 (Apt-CS-Dox-Au-5FU NPs).
11 is a graph showing flow cytometry for Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 2 (CS-Dox-Au-5FU NPs), and Comparative Example 2 (Dox).
Photographs showing cell uptake of nanoparticles using a fluorescence microscope of cells cultured using Example 2 (CS-Dox-Au-5FU NPs) of FIG. 12 (a) and Example 1 (Apt-CS-Dox-Au-5FU NPs) A photograph showing cell uptake of nanoparticles using a fluorescence microscope of cells cultured using a fluorescence microscope (b).
13 shows HR-TEM images of control cells (a) without nanoparticle treatment, cells treated with Example 1 (Apt-CS-Dox-Au-5FU NPs) (b, c), and cells treated with Example 2 (CS-Dox-Au-5FU NPs) (d, e).
14 shows samples of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 2 (CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Comparative Example 2 (Dox), and Comparative Example 3 (5-Fu) at various concentrations (0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 12.5, 25, 50 μg/mL), a graph showing the cell viability of a human glioblastoma cell line (LN229).
15 is a human culture treated with control group (a), comparative example 2 (Dox, b), comparative example 3 (5-Fu, c), example 4 (5FU-AuNPs, d), example 3 (CS-Au-5Fu NPs, e), example 2 (CS-Dox-Au-5FU NPs, f), and example 1 (Apt-CS-Dox-Au-5FU NPs, g), respectively. Fluorescence-assisted microscopy photograph of blastoma cell line (LN229).

The detailed description of the present invention below refers to the accompanying drawings shown as examples of embodiments in which the present invention can be practiced. These embodiments are described in detail so that those skilled in the art will be able to practice the present invention. It should be understood that the various embodiments of the present invention are different, but need not be mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be implemented in one embodiment in another embodiment without departing from the spirit and scope of the invention. Additionally, it should be understood that the location or arrangement of individual components within each described embodiment may be changed without departing from the spirit and scope of the invention.

Accordingly, the detailed description set forth below is not to be taken in a limiting sense, and the scope of the present invention, if properly described, is limited only by the appended claims, along with all equivalents as claimed by those claims. Like reference numbers in the drawings indicate the same or similar function throughout the various aspects.

The terms used in the present invention have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art or precedent, the emergence of new technologies, and the like. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

In the present invention, when a part "includes" a certain component, it means that it may further include other components, not excluding other components, unless otherwise stated.

Prior to a full-scale description, the samples and instruments used in the present invention will be briefly described. 금염화물(AuCl), 세틸트리메틸암모늄 브로마이드(CTAB), 5플루오로우라실(5-fluorouracil), 저분자량 키토산(DDA-75-85%; Mw-50-190 kDa), 나트륨 트리폴리 포스페이트(TPP), 독소루비신 하이드로클로라이드(doxorubicin hydrochloride), 세포 생존력 분석키트(WST-8 Cellomax ^TM , MediFab), 프로피듐 요오드화물(PI), 에씨듐 브로마이드(ElB), 아크리딘 오렌지(AO), 디클로로플루오레세인 디아세테이트(DCFH-DA), 로다민123 (Rh123), N-하이드록시숙신이미드(N-hydroxy succinimide, NHS) 및 카르보디이미디수화염화물(EDC*HCl)은 Sigma-Aldrich(St. Louis, MO, USA)에서 구매하였다. AS1411 DNA aptamer (5'-carboxy-C10-GGTGGTGGTGGTTGTGGTGGTGGTGG-3') with or without 5(6)Carboxyfluorescein (5'6 FAM) was designed and procured from Cosmo Genetech, Korea. Dialysis membrane (SnakeSkin ^™ , 10 kDa, molecular weight cut off) and all necessary staining kits and cell culture media were purchased from Thermo Fishers Scientific (Waltham, MA, USA).

Hereinafter, a method for preparing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) according to the present invention will be described in detail.

The method for producing aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) according to the present invention includes the steps of preparing Myongwolcho extract (S10), forming gold nanoparticles (S20), preparing cetyltrimethylammonium bromide-gold nanoparticle solution (S30), preparing 5 fluorouracil-gold nanoparticles (S40), chitosan-gold- 5 fluorouracil nanoparticle formation step (S50), chitosan-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S60), and aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S70).

More specifically, the method for producing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) according to the present invention includes Myongwolcho (Gynura procumbers (GP)) using an extraction solvent. Extract preparation step (S10); Gold nanoparticles forming step (S20); A cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) of preparing a cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution by mixing a cetyltrimethylammonium bromide (CTAB) solution with the gold nanoparticles (GP-AuNPs) using the Myeongwolcho extract formed in the gold nanoparticle formation step (S20); A mixture of 5-fluorouracil (5FU) was mixed with the cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) to prepare a 5fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) mixture, which was centrifuged to obtain 5 fluorouracil-gold nanoparticles (5 FU-AuNPs) to obtain 5 fluorouracil-gold nanoparticles manufacturing step (S40); A chitosan-gold-5 fluorouracil nanoparticle forming step (S50) of forming chitosan-gold-5 fluorouracil nanoparticles (CS-Au-5Fu NPs) by adding a chitosan solution to the 5-fluorouracil-gold nanoparticles prepared in the above 5-fluorouracil-gold nanoparticle preparation step (S40) and stirring to coat the chitosan; Chitosan-doxorubicin-gold-5 fluorouracil nanoparticles prepared by adding doxorubicin to the chitosan-gold-5 fluorouracil nanoparticles obtained in the chitosan-gold-5 fluorouracil nanoparticles preparation step (S50), stirring, and centrifuging to prepare chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (CS-Dox-Au-5FU NPs); And, EDC, NHS and AS1411 DNA aptamer are added to the chitosan-doxorubicin-gold-5 fluorouracil nanoparticles prepared in the chitosan-doxorubicin-gold-5 fluorouracil nanoparticles preparation step (S60) and stirred to aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) Aptamer-chito to prepare It includes; acid-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S70).

In order to prepare aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs), first, a Myongwolcho extract preparation step (S10) is performed.

In the Myongwolcho extract preparation step (S10), Myeongwolcho (Gynura procumbers (GP)) is extracted using an extraction solvent to prepare a Myongwolcho extract.

The Myongwolcho ( Gynura procumbers (GP)) is a climbing perennial herb of an evergreen semi-shrub of the Asteraceae family. The more specific scientific name of Myeongwolcho is ( Gynura procumbers (Lour.) Merr ). The Myongwolcho is distributed in tropical and subtropical Southeast Asian regions such as Indonesia, the Philippines, and Papua New Guinea. The stem of Myongwolcho extends up to 3 to 6 m, has a purple color when young or at low temperatures, and is green when growth is vigorous. The leaves of Myeongwolcho are egg-shaped, have petioles, are about 8 to 10 cm long, and have wavy ridges around the circumference, and young leaves are light green or dark green as they grow. The flowers of Myeongwolcho, which can sometimes be seen, are yellow and have a stinky smell, so they are removed during cultivation. As a result of verification by solidarity science based on folk remedies of the indigenous people, the Myongwolcho has been confirmed to have excellent effects on diabetes, hypertension, nephritis, etc., which are modern diseases, and is recognized as a major resource plant.

On the other hand, it is preferable that the extraction solvent in the Myongwolcho extract preparation step (S10) is distilled water.

In the manufacturing step (S10) of the Myrtle plant extract, the Myrtle plant extract is preferably extracted by heating the Myrtle plant at a temperature of 80 to 90 ° C. for 5 to 15 minutes using an extraction solvent.

It is appropriate to obtain about 0.5 to 1.5 L of the Myrtle grass extract in the Myrtle plant extract preparation step (S10).

Next, a gold nanoparticle forming step (S20) is performed.

In the gold nanoparticle forming step (S20), gold nanoparticles (GP-AuNPs) are formed by adding AuCl to the Myrghumcho extract prepared in the Myrtleweed extract preparation step (S10), stirring, and freeze-drying to form gold nanoparticles (GP-AuNPs).

In the gold nanoparticle biosynthesis step (S20), gold nanoparticles (GP-AuNPs) using the Myongwolcho extract are preferably formed by adding AuCl to the Myongwolcho extract, stirring at 26 to 30 ° C for 8 to 16 hours, and freeze-drying.

More specifically, in the gold nanoparticle biosynthesis step (S20), 232.4 mg of AuCl was added to 1 L of the Myrtle plant extract prepared in the Myrtle plant extract preparation step (S10), stirred at 28 ° C. for 12 hours, and freeze-dried to form gold nanoparticles (GP-AuNPs) using the Myrtle plant extract.

Next, a cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) is performed.

In the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30), the cetyltrimethylammonium bromide (CTAB) solution is mixed with the gold nanoparticles (GP-AuNPs) using the Myongwolcho extract formed in the gold nanoparticle formation step (S20) to prepare a cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution.

In the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30), it is appropriate to mix the gold nanoparticles (GP-AuNPs) using the Myeongwolcho extract and the cetyltrimethylammonium bromide (CTAB) aqueous solution in a weight ratio of 1:8.

In addition, in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30), gold nanoparticles (GP-AuNPs) and cetyltrimethylammonium bromide (CTAB) aqueous solution are mixed at 20 to 30 ° C. for 1 to 3 hours using a magnetic stirrer.

More specifically, in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30), 0.08 g of cetyltrimethylammonium bromide (CTAB) aqueous solution was mixed with 0.01 g of gold nanoparticles (GP-AuNPs) using a Myongwolcho extract formed in the gold nanoparticle formation step (S20) at 25 ° C. for 2 hours using a magnetic stirrer to obtain cetyltrimethylammonium bromide-gold nanoparticles. A solution of particles (CTAB-AuNPs) was formed.

Next, the 5 fluorouracil-gold nanoparticle manufacturing step (S40) is performed.

In the 5-fluorouracil-gold nanoparticle preparation step (S40), a 5-fluorouracil (5FU) solution was mixed with the cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) to prepare a mixture of 5fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) and centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-CTAB-AuNPs).

The 5-fluorouracil (5FU) solution in the 5FU-CTAB-AuNPs mixture preparation step (S40) is preferably prepared by dissolving 5-fluorouracil (5FU) in DMSO.

More specifically, in the 5-fluorouracil-gold nanoparticle preparation step (S40), the cetyltrimethylammonium bromide-gold nanoparticle solution prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) was mixed with a solution of 5-fluorouracil (5FU) dissolved in DMSO, and the pH was adjusted to 1 After stirring for an hour, a 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) mixture is prepared, and centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-CTAB-AuNPs).

Next, a step of forming chitosan-gold-5fluorouracil nanoparticles (S50) is performed.

In the chitosan-gold-5-fluorouracil nanoparticle forming step (S50), a chitosan solution is added to the 5-fluorouracil-gold nanoparticles prepared in the 5-fluorouracil-gold nanoparticle preparation step (S40) and stirred to coat chitosan to form chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs).

The chitosan solution in the CS-Au-5Fu NPs preparation step (S50) is preferably prepared by dissolving chitosan in distilled water containing acetic acid.

More specifically, in the chitosan-gold-5-fluorouracil nanoparticle formation step (S50), a chitosan solution formed by dissolving 10 mg of chitosan in 20 mL of distilled water containing 0.5% acetic acid was added to the 5-fluorouracil-gold nanoparticles prepared in the 5-fluorouracil-gold nanoparticle preparation step (S40), stirring for 10 minutes, and sonicating for 15 minutes to coat chitosan, and centrifugation. are collected into chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs).

Next, a step of preparing chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (S60) is performed.

In the chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S60), doxorubicin is added to the chitosan-gold-5fluorouracil nanoparticles obtained in the chitosan-gold-5fluorouracil nanoparticle preparation step (S50), followed by stirring and centrifugation to prepare chitosan-doxorubicin-gold-5fluorouracil nanoparticles (CS-Dox-Au-5FU NPs).

At this time, it is appropriate that the stirring in the chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S60) is performed for 1 to 3 hours, and the centrifugation is performed at 12000 to 18000 rpm for 20 to 40 minutes.

More specifically, in the chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S60), doxorubicin was added to the chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs) obtained in the chitosan-gold-5fluorouracil nanoparticle preparation step (S50), stirred for 2 hours, and collected by centrifugation at 15000 rpm for 30 minutes, chitosan- Doxorubicin-gold-5fluorouracil nanoparticles (CS-Dox-Au-5FU NPs) are prepared.

Next, the aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S70) is performed.

In the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S70), EDC, NHS, and AS1411 DNA aptamer were added to the chitosan-doxorubicin-gold-5fluorouracil nanoparticles prepared in the chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S60) and stirred to aptamer-chitosan-doxorubicin-gold-5fluorouracil. Prepare seal nanoparticles (Apt-CS-Dox-Au-5FU NPs).

It is appropriate that the DNA aptamer in the aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S70) is an AS1411 DNA aptamer.

The AS1411 DNA aptamer may contain or not contain 5(6)Carboxyfluorescein (5'6 FAM), and may be expressed as 5'-carboxy-C10-GGTGGTGGTGGTTGTGGTGGTGGTGG-3'. The AS1411 DNA aptamer can be purchased from Cosmo Genetech.

At this time, the EDC, NHS and AS1411 DNA aptamer may be mixed in a volume ratio of 1: 1: 0.5. In addition, the stirring in the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S70) may be performed at 2 to 6 ° C for 4` to 8 hours.

More specifically, in the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S70), 1 mL of EDC (20 μl/mL), 1 mL of NHS (20 μl/mL) and 0.5 mL of AS1 were added to the chitosan-doxorubicin-gold-5 fluorouracil nanoparticles prepared in the chitosan-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S60). 411 DNA aptamer (18.74 nm) was added and stirred at 4° C. for 6 hours to prepare aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs).

After performing the above process, aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) can be obtained.

The aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) according to the present invention are obtained by adding AuCl to the extract of Meongwolcho and stirring to obtain gold nanoparticles, and mixing the obtained gold nanoparticles (AuNPs) with cetyltrimethylammonium bromide (CTAB) solution to obtain A bromide-gold nanoparticle (CTAB-AuNPs) solution was formed, and a 5-fluorouracil (5FU) solution was mixed with the formed cetyltrimethylammonium bromide-gold nanoparticle solution to form a 5fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) mixture and centrifuged to obtain 5-fluorouracil-nanoparticles (5FU-CTAB-AuNPs) It is characterized by being obtained by adding chitosan solution to the prepared 5-fluorouracil-gold nanoparticles (5FU-AuNPs), coating chitosan by stirring, adding doxorubicin and stirring, and adding AS1411 DNA aptamer and stirring.

It is preferable that the said Mintweed extract is obtained by extracting Meisongweed with distilled water.

The extract of Meongwolcho is preferably extracted by heating Meongwolcho at a temperature of 80 to 90 ° C. for 5 to 15 minutes using an extraction solvent.

The gold nanoparticles (AuNPs) are preferably formed by adding AuCl to the extract of Myeongwolcho and stirring at 26 to 30° C. for 8 to 16 hours.

The 5-fluorouracil (5FU) solution is preferably 5-fluorouracil (5FU) dissolved in DMSO.

The chitosan solution is preferably obtained by dissolving chitosan in distilled water containing acetic acid.

In more detail, the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) of the present invention are obtained by extracting the powder of Gynura procumbers (GP) with distilled water while heating at a temperature of 85 ° C. for 10 minutes to obtain 1 L of GP-Aqueous extract, and AuCl 232.4 in 1 L of the Myeongwolcho extract. mg was added, stirred at 28 ° C for 12 hours, and lyophilized to form gold nanoparticles (GP-AuNPs) using the Myeongwolcho extract, and mixing 0.08 g of cetyltrimethylammonium bromide (CTAB) aqueous solution with 0.01 g of the gold nanoparticles (GP-AuNPs) using the Myongwolcho extract for 2 hours at 25 ° C using a magnetic stirrer to obtain cetyltrimethylammonium bromide- A solution of gold nanoparticles (CTAB-AuNPs) was formed, and a solution of 5-fluorouracil (5FU) dissolved in DMSO was mixed with the cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) solution, and the pH was adjusted to 9.0 to 9.5 and stirred for 1 hour to obtain 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU- CTAB-AuNPs) mixture was prepared, centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-CTAB-AuNPs), and a chitosan solution formed by dissolving 10 mg of chitosan in 20 mL of distilled water containing 0.5% acetic acid was added to the 5-fluorouracil-gold nanoparticles, stirred for 10 minutes and sonicated for 15 minutes to coat the chitosan, and collected by centrifugation. Tosan-gold-5 fluorouracil nanoparticles (CS-Au-5Fu NPs) were formed, and doxorubicin was added to the chitosan-gold-5 fluorouracil nanoparticles (CS-Au-5Fu NPs), stirred for 2 hours, and collected by centrifugation at 15000 rpm for 30 minutes. NPs) was prepared, and 1 mL of EDC (20 μl/mL), 1 mL of NHS (20 μl/mL), and 0.5 mL of AS1411 DNA aptamer (18.74 nm) were added to the chitosan-doxorubicin-gold-5fluorouracil nanoparticles, and stirred at 4° C. for 6 hours.

Hereinafter, detailed effects of the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs) according to the present invention are confirmed through the following Examples, Comparative Examples and Experimental Examples.

Example One. Aptamer -Chitosan- doxorubicin -gold- 5 Fluorouracil nanoparticles ( Apt - CS -Manufacture of Dox-Au-5FU NPs)

Nanoparticles of Example 1 were prepared according to the following preparation method.

Myeongwolcho extract preparation step (S10): Myeongwolcho (Gynura procumbers (GP)) powder was extracted while heating at a temperature of 85 ° C. for 10 minutes using distilled water to obtain 1 L of Myongwolcho extract (GP-Aqueous extract).

Gold nanoparticles forming step (S20): 232.4 mg of AuCl was added to 1L of the Myrghumcho extract prepared in the Myrghumcho extract preparation step (S10), stirred at 28 ° C for 12 hours, and lyophilized to form gold nanoparticles (GP-AuNPs) using the Myongwolcho extract.

Preparation step of cetyltrimethylammonium bromide-gold nanoparticle solution (S30): Mix 0.08g of an aqueous cetyltrimethylammonium bromide (CTAB) solution with 0.01g of gold nanoparticles (GP-AuNPs) using the extract of Myeongwolcho formed in the gold nanoparticle formation step (S20) using a magnetic stirrer at 25 ° C. for 2 hours to obtain cetyltrimethylammonium bromide-gold nanoparticles (CTAB- AuNPs) solution was formed.

5 Fluorouracil-gold nanoparticle preparation step (S40): Mix the cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) with a 5-fluorouracil (5FU) solution in DMSO, adjust the pH to 9.0 to 9.5, and stir for 1 hour Thus, a mixture of 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) was prepared and centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-AuNPs).

Forming chitosan-gold-5-fluorouracil nanoparticles (S50): To the 5-fluorouracil-gold nanoparticles (5FU-AuNPs) prepared in the 5-fluorouracil-gold nanoparticle mixture preparation step (S40), a chitosan solution formed by dissolving 10 mg of chitosan in 20 mL of distilled water containing 0.5% acetic acid was added, stirred for 10 minutes, and sonicated for 15 minutes to coat chitosan. and collected by centrifugation to form chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs).

Chitosan-Doxorubicin-Au-5 Fluorouracil Nanoparticle Preparation Step (S60): Doxorubicin was added to the chitosan-Au-5 Fluorouracil Nanoparticles (CS-Au-5Fu NPs) obtained in the Chitosan-Au-5 Fluorouracil Nanoparticle Preparation Step (S50), stirred for 2 hours, centrifuged at 15000 rpm for 30 minutes, and collected by chitosan-doxorubicin -Au-5 fluorouracil nanoparticles (CS-Dox-Au-5FU NPs) were prepared.

Aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S70): 1 mL of EDC (20 μl/mL), 1 mL of NHS (20 μl/mL) and 0.5 mL of AS1411 DNA were added to the chitosan-doxorubicin-gold-5 fluorouracil nanoparticles prepared in the chitosan-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S60). Tamer (18.74 nm) was added and stirred at 4° C. for 6 hours to prepare aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs).

Example 2. Chitosan- doxorubicin -gold- 5 Fluorouracil nanoparticles ( CS - Dox - Au - 5FU Preparation of NPs)

Nanoparticles of Comparative Example 1 were prepared according to the following manufacturing method.

Myeongwolcho extract preparation step (S10): Myeongwolcho (Gynura procumbers (GP)) powder was extracted while heating at a temperature of 85 ° C. for 10 minutes using distilled water to obtain 1 L of Myongwolcho extract (GP-Aqueous extract).

Gold nanoparticles forming step (S20): 232.4 mg of AuCl was added to 1L of the Myrghumcho extract prepared in the Myrghumcho extract preparation step (S10), stirred at 28 ° C for 12 hours, and lyophilized to form gold nanoparticles (GP-AuNPs) using the Myongwolcho extract.

Preparation step of cetyltrimethylammonium bromide-gold nanoparticle solution (S30): Mix 0.08g of an aqueous cetyltrimethylammonium bromide (CTAB) solution with 0.01g of gold nanoparticles (GP-AuNPs) using the extract of Myeongwolcho formed in the gold nanoparticle formation step (S20) using a magnetic stirrer at 25 ° C. for 2 hours to obtain cetyltrimethylammonium bromide-gold nanoparticles (CTAB- AuNPs) solution was formed.

5 Fluorouracil-gold nanoparticle preparation step (S40): Mix the cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) with a 5-fluorouracil (5FU) solution in DMSO, adjust the pH to 9.0 to 9.5, and stir for 1 hour Thus, a mixture of 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) was prepared and centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-AuNPs).

Forming chitosan-gold-5-fluorouracil nanoparticles (S50): To the 5-fluorouracil-gold nanoparticles (5FU-AuNPs) prepared in the 5-fluorouracil-gold nanoparticle mixture preparation step (S40), a chitosan solution formed by dissolving 10 mg of chitosan in 20 mL of distilled water containing 0.5% acetic acid was added, stirred for 10 minutes, and sonicated for 15 minutes to coat chitosan. and collected by centrifugation to form chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs).

Chitosan-Doxorubicin-Au-5 Fluorouracil Nanoparticle Preparation Step (S60): Doxorubicin was added to the chitosan-Au-5 Fluorouracil Nanoparticles (CS-Au-5Fu NPs) obtained in the Chitosan-Au-5 Fluorouracil Nanoparticle Preparation Step (S50), stirred for 2 hours, centrifuged at 15000 rpm for 30 minutes, and collected by chitosan-doxorubicin -Au-5 fluorouracil nanoparticles (CS-Dox-Au-5FU NPs) were prepared.

Example 3. Chitosan-Gold- 5 Fluorouracil nanoparticles ( CS - Au - 5Fu NPs ) manufacture of

Nanoparticles of Comparative Example 2 were prepared according to the following manufacturing method.

Myeongwolcho extract preparation step (S10): Myeongwolcho (Gynura procumbers (GP)) powder was extracted while heating at a temperature of 85 ° C. for 10 minutes using distilled water to obtain 1 L of Myongwolcho extract (GP-Aqueous extract).

Gold nanoparticles forming step (S20): 232.4 mg of AuCl was added to 1L of the Myrghumcho extract prepared in the Myrghumcho extract preparation step (S10), stirred at 28 ° C for 12 hours, and lyophilized to form gold nanoparticles (GP-AuNPs) using the Myongwolcho extract.

Preparation step of cetyltrimethylammonium bromide-gold nanoparticle solution (S30): Mix 0.08g of an aqueous cetyltrimethylammonium bromide (CTAB) solution with 0.01g of gold nanoparticles (GP-AuNPs) using the extract of Myeongwolcho formed in the gold nanoparticle formation step (S20) using a magnetic stirrer at 25 ° C. for 2 hours to obtain cetyltrimethylammonium bromide-gold nanoparticles (CTAB- AuNPs) solution was formed.

5 Fluorouracil-gold nanoparticle preparation step (S40): Mix the cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) with a 5-fluorouracil (5FU) solution in DMSO, adjust the pH to 9.0 to 9.5, and stir for 1 hour Thus, a mixture of 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) was prepared and centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-AuNPs).

Forming chitosan-gold-5-fluorouracil nanoparticles (S50): To the 5-fluorouracil-gold nanoparticles (5FU-AuNPs) prepared in the 5-fluorouracil-gold nanoparticle mixture preparation step (S40), a chitosan solution formed by dissolving 10 mg of chitosan in 20 mL of distilled water containing 0.5% acetic acid was added, stirred for 10 minutes, and sonicated for 15 minutes to coat chitosan. and collected by centrifugation to form chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs).

Example 4. 5 Fluorouracil - of gold nanoparticles (5FU-AuNPs) manufacturing

Nanoparticles of Comparative Example 3 were prepared according to the following manufacturing method.

Myeongwolcho extract preparation step (S10): Myeongwolcho (Gynura procumbers (GP)) powder was extracted while heating at a temperature of 85 ° C. for 10 minutes using distilled water to obtain 1 L of Myongwolcho extract (GP-Aqueous extract).

Gold nanoparticles forming step (S20): 232.4 mg of AuCl was added to 1L of the Myrghumcho extract prepared in the Myrghumcho extract preparation step (S10), stirred at 28 ° C for 12 hours, and lyophilized to form gold nanoparticles (GP-AuNPs) using the Myongwolcho extract.

Preparation step of cetyltrimethylammonium bromide-gold nanoparticle solution (S30): Mix 0.08g of an aqueous cetyltrimethylammonium bromide (CTAB) solution with 0.01g of gold nanoparticles (GP-AuNPs) using the extract of Myeongwolcho formed in the gold nanoparticle formation step (S20) using a magnetic stirrer at 25 ° C. for 2 hours to obtain cetyltrimethylammonium bromide-gold nanoparticles (CTAB- AuNPs) solution was formed.

5 Fluorouracil-gold nanoparticle preparation step (S40): Mix the cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) with a 5-fluorouracil (5FU) solution in DMSO, adjust the pH to 9.0 to 9.5, and stir for 1 hour Thus, a mixture of 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) was prepared and centrifuged to obtain 5-fluorouracil-gold nanoparticles (5FU-AuNPs).

Example 5. cetyltrimethylammonium bromide- gold nanoparticles ( CTAB - AuNPs ) preparation of the solution

Nanoparticles of Comparative Example 4 were prepared according to the following manufacturing method.

Myeongwolcho extract preparation step (S10): Myeongwolcho (Gynura procumbers (GP)) powder was extracted while heating at a temperature of 85 ° C. for 10 minutes using distilled water to obtain 1 L of Myongwolcho extract (GP-Aqueous extract).

Gold nanoparticles forming step (S20): 232.4 mg of AuCl was added to 1L of the Myrghumcho extract prepared in the Myrghumcho extract preparation step (S10), stirred at 28 ° C for 12 hours, and lyophilized to form gold nanoparticles (GP-AuNPs) using the Myongwolcho extract.

Preparation step of cetyltrimethylammonium bromide-gold nanoparticle solution (S30): Mix 0.08g of an aqueous cetyltrimethylammonium bromide (CTAB) solution with 0.01g of gold nanoparticles (GP-AuNPs) using the extract of Myeongwolcho formed in the gold nanoparticle formation step (S20) using a magnetic stirrer at 25 ° C. for 2 hours to obtain cetyltrimethylammonium bromide-gold nanoparticles (CTAB- AuNPs) solution was formed.

Example 6. new moon Gold nanoparticles using extract ( AuNPs ) manufacture of

Nanoparticles of Comparative Example 5 were prepared according to the following manufacturing method.

Myeongwolcho extract preparation step (S10): Myeongwolcho (Gynura procumbers (GP)) powder was extracted while heating at a temperature of 85 ° C. for 10 minutes using distilled water to obtain 1 L of Myongwolcho extract (GP-Aqueous extract).

Gold nanoparticles forming step (S20): 232.4 mg of AuCl was added to 1L of the Myrghumcho extract prepared in the Myrghumcho extract preparation step (S10), stirred at 28 ° C for 12 hours, and lyophilized to form gold nanoparticles (GP-AuNPs) using the Myongwolcho extract.

comparative example One. new moon extract ( GP - Aqueous extract ) manufacture of

The Myrtle Leaf extract of Comparative Example 6 was prepared according to the following manufacturing method.

Myeongwolcho extract preparation step (S10): Myeongwolcho (Gynura procumbers (GP)) powder was extracted while heating at a temperature of 85 ° C. for 10 minutes using distilled water to obtain 1 L of Myongwolcho extract (GP-Aqueous extract).

Comparative Examples 2 to 6. Doxorubicin (Dox), 5-fluorouracil (5-Fu), chitosan (Cs) and AS1411 DNA aptamer

For comparison, doxorubicin (Comparative Example 2), 5-fluorouracil (Comparative Example 3), chitosan (Comparative Example 4), AS1411 DNA aptamer (Comparative Example 5), and AuCl (Comparative Example 6) were prepared.

Hereinafter, experimental examples are performed with respect to Examples 1 to 6 and Comparative Examples 1 to 6. Although there is a mismatch in the order of names on the drawings, they correspond to the same material if they contain the same components.

Experimental example 1. Characterization of nanoparticles ( UV visible spectrum)

An experiment was conducted to see if gold nanoparticles were formed using Gynura procumbers (GP).

First, shown in Figure 3 is a graph for the visual spectrum analysis of Comparative Example 1 (Myeongwolcho extract, GP Aqueous extract), Example 6 (AuNPs), and Comparative Example 6 (AuCl).

As shown in FIG. 3, in the case of the biosynthesized Example 6 (AuNPs), the maximum absorption spectrum was shown at 540 nm by the localized surface plasmon resonance (LSPR) effect (size, shape, and color (yellow to red-blue) of AuNPs).

And, shown in FIG. 4 is Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Comparative Example 2 (Dox), Comparative Example 3 (5-Fu), Comparative Example 4 (CS), and Comparative Example 5 (AS1411 DNA aptamer).

As shown in FIG. 4, the maximum absorption spectrum of Comparative Example 3 (5-Fu) was 266 nm, the maximum absorption spectrum of Comparative Example 2 (Dox) was 480 nm, and the maximum absorption spectrum of Comparative Example 4 (CS) was 202-210 nm. In the case of Comparative Example 5 (AS1411 DNA aptamer), the maximum absorption peak was shown at 260 nm.

In addition, Example 4 (5FU-AuNPs) showed absorption peaks at 530 nm and 266 nm, indicating the presence of 5FU in the AuNPs.

In addition, in the case of Example 3 (CS-Au-5Fu NPs), the absorbance increased with the presence of all functional molecules.

On the other hand, in the case of the aptamer-functionalized Example 1 (Apt-CS-Dox-Au-5FU NPs), a Feuthe absorption peak was shown at 260 nm, and a broad peak from 480 nm to 530 nm, indicating successful loading of 5Fu and Dox in the final nanoparticles.

Experimental example 2. Characterization of nanoparticles ( FTIR analyze)

FTIR analysis was performed on Comparative Example 1 (Myeongwolcho extract, GP Aqueous extract), Example 6 (AuNPs), Comparative Example 3 (5-Fu), Comparative Example 2 (Dox), Example 4 (5FU-AuNPs), Example 3 (CS-Au-5Fu NPs), and Example 1 (Apt-CS-Dox-Au-5FU NPs).

The results are as shown in FIG. 5 . 5 is a graph showing FTIR analysis of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Example 6 (AuNPs), Comparative Example 1 (GP Aqueous extract), and Comparative Example 2 (Dox).

Comparative Example 1 (Myeongwolcho extract, GP Aqueous extract) showed peaks at 3278 cm ^-1 , 1629 cm ^-1 , 1350 cm ^-1 , 1047 cm ^-1 and 832 cm ^-1 . Example 6 (AuNPs) showed a middle sharp peak at 3575cm ^-1 assigned to OH stretching due to coordination of Au and water molecules. Comparative Example 1 (myongwolcho extract, GP Aqueous extract) showed a broad absorption peak at 3278 cm ^-1 .

The bands at 2901 cm ⁻¹ and 1571 cm ⁻¹ of Example 6 (AuNPs) appeared due to CH and C=H stretching of alkanes and cyclic alkenes. The peak at 1422 cm ⁻¹ of Example 6 (AuNPs) represents OH bending. The peaks at 775 cm ^-1 and 618 cm ^-1 of Example 6 (AuNPs) indicate strong CH bending.

The spectrum of Comparative Example 3 (5-Fu) showed peaks at 3134 cm ⁻¹ , 3068 cm ⁻¹ , and 2832 cm ⁻¹ , indicating NH stretching of aliphatic primary amines and amine salts. The bands at 1673 cm ^-1 and 1432 cm ^-1 represent C = N stretching and OH bending, respectively. The peak at 1248 cm ⁻¹ indicates CN stretching.

Comparative Example 2 (Dox) shows a band at 3429 cm ^-1 indicating OH intermolecular bonding, while peaks at 1682 cm ^-1 and 1433 cm ^-1 are due to C=O stretching and OH bending, respectively. In addition, peaks were shown at 1249 cm ^-1 and 1114 cm ^-1 corresponding to CO stretching and CO stretching, respectively.

Example 4 (5FU-AuNPs) showed major absorption bands at 3094 cm ^-1 , 2916 cm ^-1 and 2814 cm ^-1 corresponding to 5-FU. The GP-Au NPs exhibited peaks completely shifted at 3575 cm ^-1 and 2901 cm ^-1 , and appeared at 3094 cm ^-1 , 2916 cm ^-1 , and 2814 cm ^-1 . A new broad peak appeared at 1673 cm ^-1 at a shift of 5FU at 1669 cm ^-1 (C = O stretch). The 1248 cm ^-1 peak (CF stretching) shifted to 1230 cm ^-1 , 1011 cm ^-1 , 746 cm ^-1 and 541 cm ^-1 corresponding to GP-Au NPs and 5FU.

Example 3 (CS-Au-5Fu NPs) showed a peak at 2868 cm ^-1 corresponding to OH stretching and at 3431 cm ^-1 corresponding to CH stretching. The peaks at 1637 cm ⁻¹ (C=O stretching) and 1540 cm ⁻¹ (NH bending) correspond to amide I and amide II of CS. This indicates that CS was substantially coated with Au-5FU NPs.

Example 1 (Apt-CS-Dox-Au-5FU NPs) did not show significant peak shift due to the minimal concentration of dox and the aptamer functionalized surface rich in nitrogen base.

Experimental example 3. Characterization of nanoparticles ( XRD analyze)

XRD analysis was performed on Example 6 (AuNPs), Example 4 (5FU-AuNPs), Example 3 (CS-Au-5Fu NPs), and Example 1 (Apt-CS-Dox-Au-5FU NPs).

The results are as shown in FIG. 6 . 6 is a graph showing XRD analysis of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), and Example 6 (AuNPs).

Example 6 (AuNPs) showed crystal planes closely matching standard gold (04-0784) at 38.23° (111), 44.42° (200), 64.61° (220) and 77.64° (311).

Example 4 (5FU-AuNPs) exhibited the same crystal peaks as AuNPs except for the 16.66°, 31.54° and 33.54° peaks responsible for the cationic surfactant CTAB used in the formulation of Example 4 (5FU-AuNPs).

Example 3 (CS-Au-5Fu NPs) showed a related crystal peak of CS at 20.10 ° along with all crystal peaks of AuNPs.

Example 1 (Apt-CS-Dox-Au-5FU NPs) showed a small shift of the CS crystal peak at 19.92° together with all the Au crystal peaks. It is essential that the functionalization of AS1411 DNA aptamer on Example 1 (Apt-CS-Dox-Au-5FU NPs) did not show any significant changes in the respective crystal properties of Au and CS.

Experimental example 4. Characterization of nanoparticles ( TEM and zeta potential analysis)

The smaller the nanoparticle size, the more independent the antitumor treatment. The nanoparticle size of Example 1 (Apt-CS-Dox-Au-5FU NPs) was confirmed through TEM.

A photograph of the TEM analysis is as shown in FIG. 7 . 7 is a TEM image of Example 1 (Apt-CS-Dox-Au-5FU NPs) (d), Example 3 (CS-Au-5Fu NPs) (c), Example 4 (5FU-AuNPs) (b), and Example 6 (AuNPs) (a).

As shown in (a) of FIG. 7, in the case of Example 6 (AuNPs), monodisperse nanoparticles exhibited spherical and elliptical shapes and had a size of less than 50 nm.

In addition, as shown in (b) of FIG. 7, in the case of Example 4 (5FU-AuNPs), although spheres and triangles were shown, the size of the particles was relatively larger than that of Example 6 (AuNPs).

In addition, as shown in (c) of FIG. 7, Example 3 (CS-Au-5Fu NPs) showed spherical, elliptical, and hexagonal shapes, and the particle size was similar to that of Example 4 (5FU-AuNPs).

In addition, as shown in (d) of FIG. 7, in the case of Example 1 (Apt-CS-Dox-Au-5FU NPs), spherical and elliptical shapes were exhibited, and the size was less than 30 nm, indicating small nanoparticles.

Meanwhile, zeta potential analysis was performed on Example 1 (Apt-CS-Dox-Au-5FU NPs). The results are shown in FIG. 8, and FIG. 8 is a graph showing the zeta potential and particle size analysis (a), zeta potential analysis (b), particle size after 72 hours at pH 7.4 (phosphate buffer) (c), and particle size (d) after 72 hours at pH 5.4 (acetate buffer) of Example 1 (Apt-CS-Dox-Au-5FU NPs).

The higher the therapeutic efficiency of the drug delivery system, the better the stability and the smaller the nanoparticle size. Example 1 (Apt-CS-Dox-Au-5FU NPs) showed good stability with a zeta potential of 16.26, as shown in (a) to (b) of FIG. 8, which is expected to be advantageous for targeting negatively charged surface receptor proteins in tumor or cancer cells. Meanwhile, the hydrodynamic particle size stability of Example 1 (Apt-CS-Dox-Au-5FU NPs) was measured at different pHs (5.4 and 7.4) for 72 hours. Example 1 (Apt-CS-Dox-Au-5FU NPs) at pH 7.4 showed a hydrodynamic size of 237.2 nm, and Example 1 (Apt-CS-Dox-Au-5FU NPs) at pH 5.4 showed a hydrodynamic size of 422.1 nm. The results indicate that low pH plays an important role in increasing the hydrodynamic size of chitosan-based nanoparticles by enabling the swelling behavior of Example 1 (Apt-CS-Dox-Au-5FU NPs) due to chitosan.

Experimental example 5. Analysis of drug encapsulation and loading efficiency

Highly encapsulated drug nanoparticles are considered optimal nanoparticles for efficient tumor treatment.

First, analysis of the average particle size, polydispersity index (PDI), and average zeta potential of various nanoparticles prepared in Examples of the present invention was performed. The results of analyzing the average particle size, polydispersity index (PDI) and average zeta potential of various nanoparticles prepared in Examples of the present invention are shown in Table 1 below.

Average particle size (nm) Polydispersity
index (PDI) Average zeta potential Example 6
(AuNPs) 67.22 ± 0.92 0.5 ± 0.01 -39.0 Example 4
(5FU-AuNPs) 113.03 ± 8.23 0.46 ± 0.18 -7.98 Example 3
(CS-Au-5Fu NPs) 170.53 ± 3.07 0.257 ± 0.03 25.2 ± 0.1 Example 1
(Apt-CS-Dox-Au-5FU NPs) 196.2 ± 2.89 0.255 ± 0.01 16.26 ± 0.51

On the other hand, unloaded 5FU and Dox were measured from the supernatant obtained by centrifugation at each step of the nanoparticle preparation. The concentration of the released drug was measured using HPLC (Agilent Technologies 1260 series, DAD detector with UV-vis absorbance, detection at λ=233nm(Dox) and 266nm(5FU), column C18 (1.8 μm, 2.1 Х 150 mm). The mobile phase was acetonitrile and 10 mM potassium phosphate at a ratio of 22.75:77.25%. A buffer solution (pH 6.0) was used and an isocratic flow rate of 1.2 mL/min was applied. Detailed HPLC analysis was performed according to a previous report (Taylor et al.). In addition, 5FU and Dox encapsulation efficiency (DEE) and drug loading efficiency (DLE) were calculated using Equations 1 and 2 below, all experiments were performed in triplicate, and data were presented as mean ± SD.

The drug encapsulation efficiency (DEE) and drug loading efficiency (DLE) of the nanoparticles prepared in Examples of the present invention are shown in Table 2 below.

Au NPs: 5FU ratio (w/w) % of DEE % of DLE 1:0.062 85.89 ± 0.92 6.37 ± 0.16 1:0.125 79.99 ± 2.16 8.83 ± 0.35 1:0.25 74.59 ± 1.30 11.45 ± 0.27 1:0.5 66.34 ± 2.76 11.85 ± 0.53 Dox:CS - Au - 5FU NPs ratio (w/w) % of DEE % of DLE 0.0078:1 96.88 ± 1.56 1.35 ± 0.15 0.0156:1 94.35 ± 0.46 2.52 ± 0.23 0.0312:1 90.71 ± 0.75 4.32 ± 0.22 0.0624:1 86.79 ± 1.72 7.8 ± 0.12 0.125:1 83.67 ± 1.26 14.10 ± 0.64

Table 2 shows the % DEE and DLE efficiencies at various concentrations of 5FU and Dox.

To improve the 5FU drug loading on the negatively charged gold nanoparticles, cationic CTAB was used as an interlinker at a constant concentration. Various concentrations of 5FU (0.062 ~ 0.5%) were loaded at a constant weight ratio of gold nanoparticles (1%). DEE for CTAB-gold nanoparticles decreased when the concentration of 5FU was increased. On the other hand, AuNPs: 5FU ratio (1: 0.5%) showed a DEE of 66.34% and a DLE of 11.85%, indicating the highest efficiency. On the other hand, DLE did not increase significantly when the concentration of 5FU was increased above the optimum (0.25%).

Therefore, the ratio of AuNPs-5FU (1 : 0.25%) with 74.59% DLE and 11.45% DLE was found to be the optimal concentration. Accordingly, in the 5-fluorouracil-gold nanoparticle preparation step (S40), when preparing a mixture of 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs) by mixing cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) with a 5-fluorouracil (5FU) solution, CTAB-AuNPs and 5FU are 1: 0.25% A mixture in proportion would be most appropriate.

On the other hand, for the loading of positively charged Dox, CS-Au-5FU NPs were combined in Dox at an appropriate concentration (0.725 mg/mL), in a 1:0.5 v/v ratio. Then, various concentrations of Dox were loaded onto the CS-Au-5FU NPs. Among them, when mixed at a weight ratio of 0.125: 1 (Dox: CS-Au-5FU NPs), they exhibited a DEE (%) of 83.67 and a DLE (%) of 14.10 due to the high bonding surface area and bonding of CS. This indicates that Example 3 (CS-Au-5Fu NPs) containing chitosan is more efficient in terms of encapsulation and loading efficiency of 5FU and Dox, compared to Example 6 (AuNPs).

That is, in the production method of the present invention, by performing the chitosan-gold-5fluorouracil nanoparticle forming step (S50), the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles according to the present invention suggest that higher efficiency can be exhibited in the encapsulation and loading efficiency of doxorubicin.

Experimental example 6. in vitro Confirm drug release

The in vitro release of 5FU and Dox from Example 1 (Apt-CS-Dox-Au-5FU NPs) was measured in phosphate buffer (pH 7.4) and acetate buffer (pH 5.4). Nanoparticles (10 mg) were suspended in 5 mL of each buffer and transferred to a dialysis bag (12 kDa). Then, the 5FU and Dox-loaded nanoparticles were incubated in 50 mL of phosphate buffer (pH 7.4) and acetate buffer (pH 5.4) at 37° C. under agitation at 50 rpm. The dissolution medium was equilibrated by taking 5 mL of dissolution medium every hour and adding an equal volume of fresh buffer. Drug (5FU and Dox) concentrations at each interval were measured by the HPLC method mentioned in section (2.6). Experiments were performed in duplicate and data are presented as mean ± SD. In addition, to explore the mechanism of 5FU and Dox release in Example 1 (Apt-CS-Dox-Au-5FU NPs), a kinetic drug release model (Zero order, First order, Korsmeyer-Peppas, Hixson-Crowell and Higuchi) was used according to previous reports (Danyuo et al., 2019; Sathiyaseelan & Kalaichelvan, 2018).

The results are as shown in FIGS. 9 and 10 . Figure 9 is a graph showing the in vitro release of 5FU in phosphate buffer (pH7.4) and acetate buffer (pH5.4) from Example 1 (Apt-CS-Dox-Au-5FU NPs), Figure 10 is a test of Dox in phosphate buffer (pH7.4) and acetate buffer (pH5.4) from Example 1 (Apt-CS-Dox-Au-5FU NPs) It is a graph showing the release in the tube.

The in vitro drug release of 5FU and Dox from Apt-Dox-CS-Au-5FU NPs was tested at two different pHs (7.4 and 5.4) using HPLC, and as shown in Figures 9 and 10, the amount of drug release was observed higher at pH 5.4 than at pH 7.4. This is due to the fact that CS plays an important role in pH-dependent drug release due to acidic pH-induced protonation of -NH2 groups in CS. That is, the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention can actively release drugs containing 5FU and Dox at a specific pH.

That is, the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention release less drug at the normal tissue pH of 7.4 to prevent wastage of the drug in the normal tissue, and smoothly release the drug at the pH of the tumor microenvironment of 5.4, which is characterized by excellent drug delivery ability to the target site.

Experimental example 7. Confirmation of cellular uptake of nanoparticles

In order to confirm the cellular uptake of each nanoparticle, the cellular uptake of the nanoparticles was confirmed using flow cytometry, the cellular uptake of the nanoparticles was confirmed using a fluorescence microscope, and the cellular uptake of the nanoparticles was confirmed using HR-TEM.

The Glioblastoma brain tumor cell line (LN229) used to confirm cellular uptake of the nanoparticles was procured from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco's modified DMEM combined with fetal bovine serum (10%) and 1% antibiotics (penicillin and streptomycin). LN229 cells were cultured in a humidified incubator at 37°C and 5% CO ₂ environment.

Experimental example 7-1. flow cytometry Confirmation of cellular uptake of nanoparticles using

To elucidate the cellular uptake capacity of the Apt-Dox-CS-Au-5FU NPs, the cellular uptake of the nanoparticles was measured by flow cytometry. LN229 cells were cultured in T-25 cell culture flasks and cultured for 24 hours under the conditions mentioned above. Then, LN229 cells were treated with Comparative Example 2 (Dox), Example 2 (CS-Dox-Au-5FU NPs), and Example 1 (Apt-CS-Dox-Au-5FU NPs) for 4 hours in a 5% CO ₂ , 37°C incubator. Next, the medium was removed, the cells were washed with PBS (pH7.4), and the trypsinized cells were collected by centrifugation at 2000 rpm for 5 minutes. Collected LN229 cells were washed with cold PBS, resuspended and measured with a BD FACSCalibur equipped with a 488 nm laser in the FL2 channel.

The results are as shown in FIG. 11 . 11 is a graph showing flow cytometry for Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 2 (CS-Dox-Au-5FU NPs), and Comparative Example 2 (Dox).

Experimental example 7-2. Confirmation of cellular uptake of nanoparticles using a fluorescence microscope

The cellular uptake efficiency of nanoparticles is determined by their fluorescence intensity. Cell uptake of LN229 of Example 1 (Apt-CS-Dox-Au-5FU NPs) and Example 2 (CS-Dox-Au-5FU NPs) was observed using a fluorescence microscope (Olympus, CKX53 culture microscope, Japan).

LN229 cells (density of 1.9×10 5 ) were seeded in a 6-well plate and cultured for 24 hours at 37° C. in a 5% CO2 incubator. Then, the cells were cultured for 4 hours with Example 1 (Apt-CS-Dox-Au-5FU NPs) and Example 2 (CS-Dox-Au-5FU NPs). After culturing, the culture medium was removed, washed with PBS, and observed under a fluorescence microscope.

The results are as shown in FIG. 12 . Figure 12a is a photograph showing the cellular uptake of nanoparticles using a fluorescence microscope of cells cultured using Example 2 (CS-Dox-Au-5FU NPs), b of Example 1 (Apt-CS-Dox-Au-5FU NPs) is a photograph showing the cellular uptake of nanoparticles using a fluorescence microscope of cells cultured using a fluorescence microscope.

As shown in FIG. 12, Example 1 (Apt-CS-Dox-Au-5FU NPs) was more absorbed into cells than Example 2 (CS-Dox-Au-5FU NPs) due to overexpression of nucleolin. In addition, Example 1 (Apt-CS-Dox-Au-5FU NPs) was successfully absorbed into cells as confirmed through green fluorescence emission, and Example 2 (CS-Dox-Au-5FU NPs) showed little fluorescence emission under fluorescent conditions.

Therefore, it is suggested that the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention can be absorbed by the brain tumor cell line (LN229) within 4 hours.

Experimental example 7-3. HR - TEM Confirmation of cellular uptake of nanoparticles using

To analyze nanoparticle cell internalization, cells were incubated with Example 1 (Apt-CS-Dox-Au-5FU NPs) and Example 2 (CS-Dox-Au-5FU NPs) for 4 hours under the above-mentioned conditions. On the other hand, a control group that was not treated with anything was also prepared. After incubation, LN229 cells were trypsinized, collected by centrifugation, washed in cold PBS, and fixed in formaldehyde (4%) for 20 min. Cells were then observed under HR-TEM to measure nanoparticle cellular internalization.

The results are as shown in FIG. 13 . 13 shows HR-TEM images of control cells without nanoparticle treatment (a), cells treated with Example 1 (Apt-CS-Dox-Au-5FU NPs) (b, c), and cells treated with Example 2 (CS-Dox-Au-5FU NPs) (d, e).

As shown in FIG. 13, in the case of cells treated with Example 1 (Apt-CS-Dox-Au-5FU NPs), it was confirmed that more nanoparticles were internalized in the cells than in cells treated with Example 2 (CS-Dox-Au-5FU NPs). Through this, when treated with Example 1 (Apt-CS-Dox-Au-5FU NPs), the drug was found in the brain tumor cell line (LN2 29) can be confirmed that it is more easily absorbed.

Experimental example 8. Evaluation of cytotoxicity against human glioblastoma cell line (LN229)

The in vitro cytotoxicity of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 2 (CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Comparative Example 2 (Dox), and Comparative Example 3 (5-Fu) against the human glioblastoma cell line (LN229) was evaluated.

100 μL of LN229 cells (density 1.9 × 10 5 ) were added to each well of a 96-well plate and maintained in a 5% CO2, 37°C incubator. Samples of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 2 (CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Comparative Example 2 (Dox), and Comparative Example 3 (5-Fu) were mixed at various concentrations (0.25, 0.5, 0.75, 1, 2. 5, 5, 7.5, 10, 12.5, 25, 50 μg/mL) and treated the cells for 24 hours. After incubation, WST solution (10 μL/well) was added and incubated for 4 hours. The percentage of cell viability was calculated by reading the plate at 450 nm with a multi-well plate reader. Experiments were performed in triplicate and data are presented as mean ± SD.

The results are as shown in FIG. 14 . 14 shows samples of Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 2 (CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Comparative Example 2 (Dox), and Comparative Example 3 (5-Fu) at various concentrations (0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 12.5, 25, 50μg/mL) is a graph showing the cell viability of the human glioblastoma cell line (LN229). As shown in FIG. 14, when treated with Example 1 (Apt-CS-Dox-Au-5FU NPs), cell viability was low at all concentrations, and as a result, it was confirmed that the killing effect on the human glioblastoma cell line (LN229) that causes tumors was excellent.

Meanwhile, changes in apoptosis of nanoparticle-treated cells were evaluated by various fluorescent staining techniques. Live and dead cell stages were observed by acridine orange-ethidium bromide (AO/EB) and propidium iodide (PI) staining, loss of mitochondrial membrane potential (Δψm). It was measured through rhodamine 123 (Rh123) staining, and the production of reactive oxygen species was studied through DCFH-DA staining. The cell staining analysis was observed under a fluorescence-assisted microscope (Olympus, CKX53 culture microscope, Japan).

Figure 15 shows the IC of LN229 cells treated with drugs (Comparative Example 2 (Dox), Comparative Example 3 (5-Fu)) and drug-loaded nanoparticles (Example 1 (Apt-CS-Dox-Au-5FU NPs), Example 2 (CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs)) It is a fluorescence-assisted micrograph showing cell fluorescence staining analysis for 50 concentration.

More specifically, FIG. 15 shows Control (a), Comparative Example 2 (Dox, b), Comparative Example 3 (5-Fu, c), Example 4 (5FU-AuNPs, d), Example 3 (CS-Au-5Fu NPs, e), Example 2 (CS-Dox-Au-5FU NPs, f), Example 1 (Apt-CS-Dox-Au-5FU NPs, g), respectively. It is a fluorescence-assisted microscope photograph of the treated human glioblastoma cell line (LN229).

conclusion.

The characteristics and effects of the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention were confirmed through the above Examples, Comparative Examples and Experimental Examples.

Through Experimental Examples 1 to 3, it was confirmed that gold nanoparticles (AuNPs) could be synthesized using Gynura procumbers (GP) according to the production method of the present invention, and it was also confirmed that successful loading of 5Fu and doxorubicin (Dox) was achieved in the final nanoparticles.

Through Experimental Example 4, it was confirmed that the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention have an advantageous hydrodynamic size for targeting negatively charged surface receptor proteins in cancer cells.

Through Experimental Example 5, the method for preparing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention is prepared by mixing 5-fluorouracil (5FU) solution with cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) in the 5-fluorouracil-gold nanoparticle preparation step (S40) to obtain 5-fluorouracil- When preparing the mixture of cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs), it was confirmed that it was appropriate to mix CTAB-AuNPs and 5FU at a ratio of 1: 0.25%. In terms of encapsulation and loading efficiency, it was confirmed that higher efficiency can be exhibited.

Through Experimental Example 6, it was confirmed that the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention released relatively little drug at the normal tissue pH of 7.4 to prevent wastage of the drug in the normal tissue, and to smoothly release the drug at the tumor microenvironment pH of 5.4 to have excellent drug delivery capability to the target site.

Through Experimental Example 7, it was confirmed that the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention can be more easily absorbed by the brain tumor cell line (LN229) than the chitosan-doxorubicin-gold-5fluorouracil nanoparticles (CS-Dox-Au-5FU NPs).

Through Experimental Example 8, when treated with aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) according to the present invention, Example 2 (CS-Dox-Au-5FU NPs), Example 3 (CS-Au-5Fu NPs), Example 4 (5FU-AuNPs), Comparative Example 2 (Dox), Comparison at all concentrations Compared to Example 3 (5-Fu), the cell viability was lower, and as a result, it was confirmed that the killing effect on the brain tumor cell line (LN229) was excellent.

Therefore, the present invention is a novel pharmaceutical nanoparticle for treating brain tumors, which has excellent encapsulation and loading efficiency of doxorubicin, has excellent drug delivery ability to the target site by smoothly releasing the drug at pH 5.4 of brain tumor tissue, can easily penetrate into brain tumor cell line (LN229), and has excellent cell killing effect on brain tumor cell line (LN229), aptamer-chitosan-doxorubicin-gold- It is stated that 5 fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) and a manufacturing method thereof were developed.

Although the present invention has been described with the accompanying drawings, this is only one example of various embodiments including the gist of the present invention, and the purpose is to enable those skilled in the art to easily practice, and it is clear that the present invention is not limited to the above-described embodiments. Therefore, the protection scope of the present invention should be construed by the following claims, and all technical ideas within the equivalent scope thereof by change, substitution, substitution, etc. within the scope not departing from the gist of the present invention will be included in the rights of the present invention. In addition, it is clear that some configurations in the drawings are provided exaggerated or reduced than the actual ones to more clearly explain the configuration.

Claims (12)

Myeongwolcho extract preparation step (S10), gold nanoparticle formation step (S20), cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30), 5 fluorouracil-gold nanoparticle mixture preparation step (S40), chitosan-gold-5 fluorouracil nanoparticle formation step (S50), chitosan-doxorubicin-gold-5 fluorouracil nanoparticle preparation step (S60), and aptamer- A method for producing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) comprising a step (S70) of preparing chitosan-doxorubicin-gold-5fluorouracil nanoparticles. According to claim 1,
The Myeongwolcho extract preparation step (S10) is an aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs), characterized in that for producing a Myeongwolcho extract by extracting Myeongwolcho ( Gynura Procumbers (GP)) using an extraction solvent. According to claim 2,
In the gold nanoparticle forming step (S20), gold nanoparticles (GP-AuNPs) are formed by adding AuCl to the Myrghumcho extract prepared in the Myeongwolcho extract preparation step (S10), stirring, and freeze-drying. According to claim 3,
In the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30), cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) solution is prepared by mixing the cetyltrimethylammonium bromide (CTAB) solution with the gold nanoparticles (GP-AuNPs) using the Myrtleweed extract formed in the gold nanoparticle formation step (S20) Aptamer-chitosan-doxorubicin-gold, characterized in that Method for preparing 5-fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs). According to claim 4,
In the 5-fluorouracil-gold nanoparticle mixture preparation step (S40), a 5-fluorouracil (5FU) solution was mixed with the cetyltrimethylammonium bromide-gold nanoparticles (CTAB-AuNPs) prepared in the cetyltrimethylammonium bromide-gold nanoparticle solution preparation step (S30) to obtain a 5fluorouracil-cetyltrimethylammonium bromide-gold nanoparticle (5FU-CTAB-AuNPs) mixture Preparation of aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs) characterized by obtaining 5 fluorouracil-gold nanoparticles (5FU-AuNPs) by centrifugation. Method for producing. According to claim 5,
In the chitosan-gold-5-fluorouracil nanoparticle forming step (S50), chitosan solution was added to the 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticle mixture prepared in the 5-fluorouracil-cetyltrimethylammonium bromide-gold nanoparticle mixture preparation step (S40) and stirred to coat chitosan to form chitosan-gold-5fluorouracil nanoparticles (CS-Au-5Fu NPs) A method for producing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs), characterized in that. According to claim 6,
In the chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S60), doxorubicin is added to the chitosan-gold-5fluorouracil nanoparticles obtained in the chitosan-gold-5fluorouracil nanoparticle preparation step (S50), stirred and centrifuged to obtain chitosan-doxorubicin-gold-5fluorouracil nanoparticles (CS-Dox-Au-5FU NPs). - Manufacturing method of chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs). According to claim 7,
In the aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S70), EDC, NHS, and AS1411 DNA aptamer were added to the chitosan-doxorubicin-gold-5fluorouracil nanoparticles prepared in the chitosan-doxorubicin-gold-5fluorouracil nanoparticle preparation step (S60) and stirred to aptamer-chitosan-doxorubicin-gold-5fluorouracil. A method for producing aptamer-chitosan-doxorubicin-gold-5fluorouracil nanoparticles (Apt-CS-Dox-Au-5FU NPs), characterized in that for producing seal nanoparticles (Apt-CS-Dox-Au-5FU NPs). AuCl was added to the Myrtleweed extract obtained by extracting Myongwolcho and stirred to obtain gold nanoparticles, and a cetyltrimethylammonium bromide (CTAB) solution was mixed with the obtained gold nanoparticles (AuNPs) to form a cetyltrimethylammonium bromide-gold nanoparticle (CTAB-AuNPs) solution, and 5-fluorouracil was added to the formed cetyltrimethylammonium bromide-gold nanoparticle solution (5FU) The dissolved solution was mixed to form a mixture of 5 fluorouracil-cetyltrimethylammonium bromide-gold nanoparticles (5FU-CTAB-AuNPs), and centrifuged to obtain 5 fluorouracil-gold nanoparticles (5FU-AuNPs). Add chitosan solution to the obtained 5 fluorouracil-gold nanoparticles, stir to coat chitosan (CS), and add doxorubicin (Dox) to stir, Aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs), characterized in that obtained by adding and stirring EDC, NHS and AS1411 DNA aptamer. According to claim 9,
The Aptamer-Chitosan-Doxorubicin-Gold-5 Fluorouracil Nanoparticles (APt-CS-Dox-Au-5FU NPs). According to claim 9,
The gold nanoparticles (AuNPs) are aptamer-chitosan-doxorubicin-gold-5 fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs). According to claim 9,
Chitosan-gold-5-fluorouracil nanoparticles (CS-Au-5Fu NPs) are obtained by adding a chitosan solution formed by dissolving 10 mg of chitosan in 20 mL of distilled water containing 0.5% acetic acid to 5-fluorouracil-gold nanoparticles, stirring for 10 minutes and sonicating for 15 minutes to coat chitosan, and collected by centrifugation Aptamer-chitosan-doxorubicin -Au-5fluorouracil nanoparticles (APt-CS-Dox-Au-5FU NPs).