KR20120132591A - The specific binding molecules-biodegradable nanofibers complex and method for preparing the same - Google Patents

Abstract

The present invention 1) preparing a biodegradable nanofiber by electrospinning the biodegradable polymer solution; 2) biodegradable nanofiber coating step using dopamine of step 1); And 3) mixing and binding specific binding molecules to the dopamine-coated biodegradable nanofibers of step 2), thereby providing a method for producing specific binding molecule-biodegradable nanofiber composites for cell separation. Biodegradable nanofibers bound to the specific binding molecules of the present invention can be used easily and conveniently to separate and target cells, as well as easy to use and simple binding and separation of the antibody and the target cells. In addition, since it is possible to apply in vivo, it can be conveniently used for cell separation effectively and quickly, and can be applied in vivo.

Description

Specific binding molecules-biodegradable nanofibers complex and method for preparing the same}

The present invention relates to specific binding molecule-biodegradable nanofiber composites and a method for preparing the same.

Isolation of cells is one of the most important techniques in various fields, such as biological or biomedical applications in the diagnosis or treatment of diseases, and its use is increasingly being used to isolate specific cells of interest from a mixture of different cells (McCloskey KE, et. al., Anal Chem, 2003, Dec 15; 75 (24): 6868-6874; McCloskey KE, et al., Biotechnol Prog, 2003, May-Jun; 19 (3): 899-907). Therefore, because of this importance, many research groups are currently conducting a lot of research on the technology of cell separation.

In the case of separating cells having different specific gravity, they can be separated by speed sedimentation. On the other hand, when there is little difference between cells, such as distinguishing sensitized cells from unsensitized cells, it is necessary to separate the cells by information stained with fluorescent antibodies or by direct microscopic observation. For example, a cell sorter (Kamarck, ME, Methods Enzymol . Vol. 151, p150-165, 1987) that separates and collects specific cell populations by fractionating cells according to the label strengths by labeling with fluorescent dyes. ), And recently, a cell sorting device for separating microscopically observed microparticles flowing in laminar flow generated in microchannels made using micro-processing technology (Micro Total Analysis, 98, pp.77 ~ 80, Kluwer Academic Publishers, 1998; Analytical Chemistry, 70, pp. 1909-1915, 1998).

Fluorescence activated cell sorter (FACS), one of the most widely used cell separation methods, is a device that analyzes the surface antigens of glass cells such as lymphocytes or separates cells by the presence or absence of surface antigens using laser beam. As the latex particles or cells pass through a certain sensing area, each particle or cell is quickly measured, the intensity of fluorescence of each cell is measured, and the specific cells are detected using the quantized intensity of fluorescence. Can be selectively separated. Fluorescent dyes or monoclonal antibodies can be used to isolate cells by determining the immune state of the surface and inside of the cells. However, there are drawbacks to the complex operation techniques of the device, highly skilled technicians, time consuming and expensive machines (Lancioni CL, et al., J Immunol Methods, 2009, May 15; 344 (1): 15-25).

In addition, one of the currently used magnetic activated cell sorter (magnetic activated cell sorter) is to separate the specific cells desired by the force of the magnetic field using an antibody or a corresponding material attached to the magnetic particles (magnetic particle) In this case, there is a disadvantage in that a change in the state of a cell occurs because beads necessary for cell separation bind to the cell and enter the cell.

In the case of general immune cell therapy, the patient's blood is collected, the cells are separated, and after in vitro culture, intravenous administration is put into the patient's body. However, when the body activates immune cells, most cells are trapped by the reticuloenthothelial system (RES) and the number of cells reaching the target site is very limited. The reticuloenthothelial system consists of mononuclear phagocytes, which are cells in the reticular connective tissue—liver, lung, etc., which correspond to biological immune defense against foreign bodies. Therefore, in order to overcome this point, the present invention has devised a sustained release cell release concept in which immune cells activated in vitro are implanted into a target site in the form of an implant and slowly released from the target.

Nanofibers are not only biodegradable materials that have a large surface area in a small space, are durable, very easy to handle, can be manufactured in various forms, and are easy to chemically combine various materials. It is very suitable for the above purpose because it can be manufactured. Biocompatible nanofibers have already been widely commercialized in various biomedical fields such as wound treatment, artificial skin, hemodialysis, ligament, etc. In particular, the PLGA used in the present invention is widely used in the human biomedical field because of its ability to safely hydrolyze in vivo without a specific by-product, and is a biocompatible polymer material approved by the FDA as a medical device.

On the other hand, antibodies are widely used for the detection, isolation and measurement of biological substances using antibodies specific for cell surface antigens because of their specificity to antigens. Accordingly, the present inventors have fabricated PLGA-based nanofibers as biotransplantable cell isolation scaffolds and have successfully attached dopamine to attach specific antibodies, and use this system to specifically identify CD4 T cells present in lymph nodes. It succeeded in separating. Such a cell separation system has completed the present invention by confirming that the biodegradable nanofibers are not only able to be applied later in vivo, but also easy to use and rapid.

In order to achieve the above object, the present invention comprises the steps of 1) preparing biodegradable nanofibers by electrospinning a biodegradable polymer solution; 2) biodegradable nanofiber coating step using dopamine of step 1); And 3) mixing and binding specific binding molecules to the dopamine-coated biodegradable nanofibers of step 2), thereby providing a method for producing specific binding molecule-biodegradable nanofiber composites for cell separation.

The present invention also provides a specific binding molecule-biodegradable nanofiber composite prepared according to the above method.

In addition, the present invention comprises the steps of: 1) culturing by mixing the cells with the specific binding molecule-biodegradable nanofiber complex; And 2) separating the cells bound to the complex from the mixture of step 1), and providing a cell separation method using biodegradable nanofibers having specific binding molecules bound thereto.

Biodegradable nanofibers bound to the specific binding molecules of the present invention can be used easily and conveniently to separate and target cells, as well as easy to use and simple binding and separation of the antibody and the target cells. In addition, since it is possible to apply in vivo, it can be conveniently used for cell separation effectively and quickly, and can be applied in vivo.

As described above, the biodegradable nanofibers to which the specific binding molecules of the present invention are bound are faster and simpler to use than conventional cell separation methods, and thus can be easily used as well as to separate target cells specifically and efficiently. It can be usefully used in the development or production of cell separation apparatus and separation method for separating specific cells desired from mixed cells.

1 is a diagram showing the porosity and pore size control and the diameter control of nanofibers according to the concentration.
Figure 2 is a diagram showing the evaluation of biocompatibility for each nanofiber produced by electrospinning.
3 is a diagram of the development of functionalized coating technology using biodegradable nanofibers.
4 is a picture of a dopamine-coated cell-friendly biodegradable nanofiber support.
FIG. 5 is a graph showing the amount of bound antibody upon dopamine-coated cell-friendly biodegradable nanofiber support at various concentrations (0-40 ug).
6 is a diagram showing cell separation according to various antibody concentrations (0 to 40 ug).

Example 1 Preparation of Materials

PLGA. (RESOMER ^® LG 857 S) was used and dopamine (3,4-Dihydroxyphenethylamine hydrochloride, 3-Hydroxytyramine hydrochloride) was purchased from Sigma and Aldrich.

Seven-week-old C57BL / 6 mice were purchased from Narabiotech (Seoul, Korea) and bred in Korea University animal lab. All mice used in the experiments were 7-10 weeks old. All procedures were conducted with the approval of Korea University Experimental Animal Operation Committee (KUIACUC-2009-105). Anti-mouse CD3 (145-2C11), CD4 (GK1.5, RM4-4) and NK1.1 (PK136) monoclonal antibodies (with or without fluorescein isothiocyanate (FITC) bound or bound) mAbs), phycoerythrin (PE), or allophycocyanin (APC) were purchased from eBioscience (San Diego, Calif.). Anti-mouse CD19 (6D5) mAb coupled to Peridinin Chlorophyll Protein Complex (PerCP) was purchased from BioLegend (San Diego, Calif.). All other reagents were purchased from Sigma and Aldrich at the highest commercially available grades.

Example 2 Preparation of PLGA Nanofibers by Electrospinning

Dissolve PLGA at 2 wt% in HFP (Wako Chemical, Ltd., 1,1,1,3,3,3-Hexafluoro-2-methyl-2-propanol). Wrap cylinder shape collector (diameter, r = 9cm) with aluminum foil and fix Syringe containing PLGA solution to apply voltage of 17.5kv.

Example 3 Preparation of Dopamine-Coated Biodegradable Nanofibers

Fix the fiber in the square dish by swelling the PLGA fiber dried on EtOH. Then wash with DW. Remove DW and immerse for 5 minutes with Tris-HCl (10 mM, pH 8.5). Make a solution of 40 mg of dopa in 20 ml of Tris-HCl. The PLGA fibers are immersed in a solution prepared for 4 h and coated.

Example 4: Preparation of Antibody-Biodegradable Nanofiber Complexes

<4-1> Fixation of Antibodies to Dopamine-Coated Biodegradable Nanofibers

In order to bind the antibody to the dopamine-coated biodegradable nanofibers, the electrospun biodegradable nanofibers prepared in Example 2 were coated with biodegradable nanofibers using dopamine as in Example 4. The biodegradable nanofibers were checked for coating degree over time through an electron microscope (FIG. 4). Wetting dry fiber (with dopamine coating 4h) with EtOH and fabricating a fiber sheet to size. After sterilizing the polydopamine coated nanofiber, wetting with Tris-HCl buffer again. Prepare antibody solution by treating antibody with Tris-HCl buffer. Immerse the antibody solution in the sheet.

<4-2> Determination of Content of Antibodies Immobilized on Dopamine-Coated Biodegradable Nanofibers

The antibody content of the antibody-immobilized biodegradable nanofibers prepared in Example <4-1> was measured using a BCA protein assay reagent kit and absorbance at 562 using a spectrophotometer.

As a result, as shown in Figure 3, as the concentration of the antibody was shown to increase in proportion to the content of the bound antibody (Fig. 5).

Example 5: Confirmation of the efficiency of the antibody immobilized on the biodegradable nanofibers

<5-1> Preparation and Separation of Cells

Peripheral {Submental, Mandibular, Superficial Cervical, Axillary, Lateral Axillary, Inguinal, Popliteal} lymph nodes were extracted from C57BL / 6 mice. . Single cell suspensions were filtered through 70 μm nylon nets (BD biosciences) and cells were washed with phosphate buffered saline containing 5% fetal bovine serum (FBS) (Lonza Walkersville, MD USA).

<5-2> Confirmation of the efficiency of the antibody immobilized on the biodegradable nanofibers

To determine how efficiently anti-mouse CD4 antibodies bound to nanofibers can bind to CD4 + T cells, only 20 ug of CD4 + T cells can specifically bind to dopami coated nanofibers. After binding the CD4 (clone GK1.5) antibody, the antibody was immobilized with a mixed single cell (T, B or NK cell) obtained by homogenizing the lymph nodes of the mouse as in Example <6-1>. After binding to the biodegradable nanofibers, the cells remaining unbound were analyzed using a fluorescence activated cell sorter (FACS). That is, the cells prepared as described above were incubated for 4 to 20 minutes with biodegradable nanofibers coated with dopamine to fix CD4 antibodies. Samples were taken from the samples and FACS staining was performed on cells not bound to biodegradable nanofibers.

As a result, FACS staining of the mixed single cells obtained from the lymph nodes confirmed the population of CD4 + and CD8 + cells. CD4 + was about 33%, CD8 + was about 29%, CD4 + was 5%, CD8 + was 64% appeared (FIG. 6A). Therefore, the separation efficiency of the desired CD4 + T cells appeared 85% (Fig. 6b).

Claims (6)

1) electrospinning the biodegradable polymer solution to obtain biodegradable nanofibers;
2) coating dopamine on the biodegradable nanofibers to obtain dopamine-coated biodegradable nanofibers; And
3) binding specific binding molecules to the dopamine-coated biodegradable nanofibers; a method for producing a specific binding molecule-biodegradable nanofiber composite for cell separation comprising a. The method of claim 1,
The biodegradable polymers include polyglycolic acid (PGA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), and polyethylene glycol , PEG) A method for producing a specific binding molecule-biodegradable nanofiber composite for cell separation, characterized in that any one selected from. The method of claim 1,
Step 2) is a specific binding molecule for cell separation, characterized in that the dopamine-coated biodegradable nanofibers are obtained by supporting the biodegradable nanofibers in a mixed solution of dopamine dissolved in tris hydrochloric acid buffer solution for 3-4 hours. -Preparation method of biodegradable nanofiber composite. The method of claim 1,
The specific binding molecule is a method for producing a specific binding molecule-biodegradable nanofiber complex for cell separation, characterized in that the antibody. Method for producing a specific binding molecule-biodegradable nanofiber composite prepared according to any one of claims 1 to 4. A cell separation method using a specific binding molecule-biodegradable nanofiber complex prepared according to any one of claims 1 to 4,
a) mixing and culturing cells in a specific binding molecule-biodegradable nanofiber complex prepared according to any one of claims 1 to 4; And
b) separating the cells bound to the specific binding molecule-biodegradable nanofiber complex after the mixed culture.

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