KR101881219B1 - Light emitting particles for imaging, cell imaging method using same, and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a luminescent particle for non-invasive image tracking of cells transplanted in vivo using near infrared rays, a cell imaging method using the lining itself, and a method for producing the lining particle, The present invention provides a light emitting phosphor for imaging, which comprises a polymer, and the polymer contains a light emitting material. The liposomes adhere to the cells to maintain the function of the cells, exhibit excellent luminescence, and have the advantage of being able to image the cells transplanted into the living body.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a luminescent particle for imaging, a cell imaging method using the same, and a manufacturing method thereof,

The present invention relates to a light emitting particle for imaging, a cell imaging method using the same, and a method of manufacturing the same.

The conventional cell image tracking method is mainly used to detect fluorescence after staining cells with monomolecular substance or particles which exhibit fluorescence, to introduce the contrast agent into the cell, or to attach it to the cell surface, and then to use imaging techniques such as MRI and PET Method is being used.

However, MRI and other methods can accurately track and non-invasive tracking, but have cost and time-consuming limitations. Fluorescence imaging technique has been proposed. Fluorescence imaging technique has relatively low cost and excellent accessibility. However, it has difficulty in accurate tracking because of poor transparency of light, and it is sometimes necessary to use surgical technique.

Therefore, the near-infrared imaging technique having excellent tissue permeability and high accessibility is attracting attention as an ideal in vivo cell imaging technique. However, when the light emitting material is injected into the cell in the near-infrared imaging technique, the duration of the luminescence is short, The function of the cell is deteriorated. In particular, when a method of injecting a light emitting material into a cell membrane or using a luminescent gene expression method has a possibility to affect the metabolic activity of a cell or induce cancer cell formation.

Conventionally, noninvasive cell imaging using near infrared has been proposed to induce fluorescence expression through intracellular infusion and gene manipulation. In the intracellular inflow method, the introduction of monomolecular phosphors into the cells nonspecifically induces cytotoxicity due to the use of a large amount of phosphors, and there is a possibility of disturbing the cell system due to nonspecific intracellular inflow. Also, when a method of introducing the nano-particles themselves into the cells or introducing them into nonspecific cells using a targeting peptide or cell penetrating chemical is also used, there is a possibility of cytotoxicity and cell disturbance, It has also been reported that the induction of fluorescence by the gene transfection method to induce fluorescence by genetic manipulation may also disturb the cell system and induce cancer cells (Stephan, Matthias T., and Darrell J. Irvine. "Enhancing cell therapies from the outside in: cell surface engineering using synthetic nanomaterials." Nano today 6.3 (2011): 309-325.).

In addition, a method of attaching a monomolecular luminescent chemical substance to a cell surface has been proposed. However, in order to attach a monomolecular luminescent substance of a desired wavelength band (wavelength band enabling near infrared ray imaging), manipulation of a chemical substance is required, It was difficult to specifically infiltrate into the target cell. In addition, since there is a limit to the degree of cell surface adhesion, it has a disadvantage in that it is impossible to use a high concentration of a luminescent material for long-term cell tracing.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a method and apparatus for observing non-invasive cells, The purpose of that is to do.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a luminescent particle for imaging, which comprises a polymer coated with a phospholipid film, wherein the polymer comprises a luminescent material.

In one embodiment of the present invention, the phospholipid membrane comprises phosphatidyl ethanolamine (MPB-PE).

In another embodiment of the present invention, the phospholipid membrane is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2 Glycero-3-phospho- (1'-rac-glycerol) sodium salt (1,2-dioleoyl-sn-glycero-3-phospho- , DOPG), cholesterol, lecithin, and mixtures thereof.

In another embodiment of the present invention, the luminescent material is indocyanine green (ICG).

In another embodiment of the present invention, the polymer is selected from the group consisting of poly lactic-L-glycolic acid, poly lactide, polyglycolide, Poly (ethylene glycol), poly (hydroxy butyrate), poly (epsilon-caprolactone), poly (beta -malonic acid) (BLS-PGA), polycaprolactone, polyethylene glycol poly (? - malic acid), and mixtures thereof.

In addition, the present invention relates to a method for manufacturing a luminescent cell, comprising the steps of: (S1) preparing a luminescent cell by attaching the luminescent material to the surface of a cell through thiol-maleimide bonding; And

And injecting and observing the light-emitting cells into a subject (S2).

In one embodiment of the present invention, the cell is one or more immunocytes selected from the group consisting of human umbilical cord blood-derived mesenchymal stem cells, CD4 T cells, CD8 T cells, and dendritic cells.

In addition, the present invention provides a method for manufacturing a light emitting phosphor for image formation, comprising the steps of:

a) adding a luminescent material to the polymer;

b) mixing the polymer and lipid;

c) sonicating the mixed solution and stirring; And

d) After stirring, centrifuging to remove the organic solvent in the upper layer.

In one embodiment of the present invention, the lipid is characterized by comprising phosphatidyl ethanolamine (MPB-PE).

In another embodiment of the present invention, the lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2 Glycero-3-phospho- (1'-rac-glycerol) sodium salt (1,2-dioleoyl-sn-glycero-3-phospho- , DOPG), cholesterol, lecithin, and mixtures thereof.

In another embodiment of the present invention, the luminescent material is indocyanine green (ICG).

In another embodiment of the present invention, the polymer is selected from the group consisting of poly lactic-L-glycolic acid, poly lactide, polyglycolide, Poly (ethylene glycol), poly (hydroxy butyrate), poly (epsilon-caprolactone), poly (beta -malonic acid) (BLS-PGA), polycaprolactone, polyethylene glycol poly (? - malic acid), and mixtures thereof.

When the cell therapeutic agent is used using the luminescent particle of the present invention, the luminescent material can be traced by non-invasive cells while being contained in the luminescent material, and stays on the cell surface. Therefore, And can be monitored in real time. This is to overcome the disadvantages of MRI, which requires less fluorescence retention time and consumes a lot of cost when the conventional fluorescence tracing method is used.

FIG. 1 is a schematic diagram of a non-invasive cell imaging technique using near-infrared light.
Fig. 2 is a diagram showing the structure of the luminescent particle of the present invention, and the cell in which the luminescent particle itself is presented.
FIG. 3 is a view showing the result of confirming the shape, stability and light emitting ability of the formed light emitting phosphor.
FIG. 4 is a graph showing the results of measurement of cytotoxicity and luminescence amount according to the usage amount of the luminescent particle itself in order to confirm the appropriate amount of the luminescent particle itself.
5 is a graph showing the results of confirming whether or not the luminescent material itself is attached and the stability with time.
6 is a graph showing the results of confirming that the luminescent particle of the present invention is attached to various kinds of cells to produce luminescent cells.
FIG. 7 is a graph showing the results of confirming the ability of the human umbilical cord blood-derived mesenchymal stem cells to which the luminescent particle of the present invention is adhered, to permeate and move.
FIG. 8 is a graph showing the result of surface antigen analysis of human cord blood-derived mesenchymal stem cells to which the luminescent particle of the present invention is attached.
FIG. 9 is a graph showing the results of confirming the differentiation ability of human cord blood-derived mesenchymal stem cells to which the luminescent particle of the present invention has been adhered to a fat cell (a: dyeing result, b: relative absorbance side comparison result).
FIG. 10 is a graph showing the results of confirming the ability of human cord blood-derived mesenchymal stem cells to which the luminescent particle of the present invention is attached to maintain osteoblast differentiation ability (a: dyeing result, b: relative absorbance side comparison result).
FIG. 11 is a graph showing the results of confirming the ability of human cord blood-derived mesenchymal stem cells to which the luminescent particle of the present invention is attached to maintain chondrogenic differentiation ability.
FIG. 12 is a view showing a result obtained by injecting a stem cell into a nude mouse. FIG.

The purpose of infusing and imaging cells into a subject is to continuously monitor the progress of the treatment of patients using immune cells or stem cell therapies and thereby prevent side effects.

As shown in Fig. 1, there is proposed a method of introducing a fluorescent substance, a nano-particle itself or the like into a cell, a method of expressing fluorescence in a cell through gene manipulation, and a method of imaging a monomolecular luminescent chemical substance attached to a cell surface However, technical limitations have been found in that the fluorescence retaining ability is deteriorated, the functionalities of the cell therapeutic agent are lowered due to the imaging, and imaging causes side effects.

Accordingly, the present inventors have intensively studied to construct a system capable of long-term cell tracing while minimizing cytotoxicity and maintaining therapeutic efficacy of a cell therapeutic agent. As a result, they have found that when a luminescent material is incorporated into a polymer using a multi- It was confirmed that the luminescent particle prepared by capturing the polymer on the phospholipid membrane retains the function of the cell therapeutic agent while maintaining long-term fluorescence, thereby completing the present invention.

That is, the present invention captures the light emitting material in the polymer nanoparticle itself coated with the phospholipid monolayer in order to minimize the problem of the duration of the conventional light emission and to minimize the cytotoxicity due to the use of the high concentration of the light emitting material. In order to solve the problem of maintaining the function of the cells, the nanoparticles were adhered to the cell surface, not to the cells.

Accordingly, the present invention includes a polymer coated with a phospholipid membrane,

Wherein the polymer comprises a light emitting material.

Also, a step (S1) of attaching the above-mentioned lipid to the surface of the cell through thiol-maleimide bonding to prepare a luminescent cell; And

And injecting and observing the light-emitting cells into a subject (S2).

In addition, there is provided a method of manufacturing a light emitting phosphor for image formation comprising the steps of:

a) adding a luminescent material to the polymer;

b) mixing the polymer and lipid;

c) sonicating the mixed solution and stirring; And

d) After stirring, centrifuging to remove the organic solvent in the upper layer

In one embodiment of the present invention, the phospholipid membrane of the present invention is coated with a polymer and the polymer contains a luminescent material. The luminescent material itself is not attached to the cell but is attached to the cell in the form of a pore , It is possible to solve the problem that the fluorescence retention period of the prior art is short, which affects the cell functionality or causes side effects.

The phospholipid membrane contains phosphatidyl ethanolamine (MPB-PE). Phosphatidylethanolamine is contained in the phospholipid membrane, so that the maleimide group is present on the surface of the phospholipid membrane. And thiol-maleimide bonds with the thiol group so that the liposome can attach to the cell surface.

The phospholipid membrane may contain other kinds of lipids besides phosphatidylethanolamine. The lipid may be 1,2-dioleoyl-sn (sn-glycero-3-phosphocholine) (1'-rac-glycerol) sodium salt (1,2-dioleoyl-sn-glycero-3 (1'-rac-glycerol) sodium salt, DOPG), cholesterol, lecithin, or a mixture thereof.

It is preferable that the luminescent material uses indocyanine green (ICG) in consideration of the wavelength of near infrared rays.

The polymer may be at least one selected from the group consisting of poly lactic-L-glycolic acid (PLGA), poly lactide, polyglycolide, polygamargutanoic acid (BLS-PGA) Poly (ethylene glycol), poly (hydroxy butyrate), poly (ε-caprolactone), poly (β-malic acid )), Or a mixture thereof. Poly lactic-L-glycolic acid (PLGA) may be used as in the embodiment of the present invention, but the present invention is not limited thereto.

The constituent components of the above-mentioned phospholipid membrane preferably include 30 to 50% by weight of DOPC, 1 to 20% by weight of DOPG and 40 to 60% by weight of MPB-PE, more preferably DOPC 40 wt%, DOPG 10 wt%, and MPB-PE 50 wt%.

In another embodiment of the present invention, the type of cell to which the lining is attached may be an immune cell such as a human umbilical cord blood-derived mesenchymal stem cell, a CD4 T cell, a CD8 T cell, or a dendritic cell, It is not limited.

In another embodiment of the present invention, the stem cells to which the bone cells are attached maintain the ability to differentiate into other cells, and they can differentiate into various cells such as adipocytes, osteoblasts, cartilage cells, and the like.

After the cells with the liposome attached thereto are injected into a subject, the movement of the cells and the therapeutic effect thereof can be monitored by near infrared ray photography.

The cells to which the liposome of the present invention is adhered can be administered intravenously, subcutaneously, intraperitoneally or locally according to a desired method, and the dose is appropriately determined depending on the body weight, age, sex, health condition, diet, The range varies depending on the method, excretion rate and severity of the disease.

The subject is a mammal including a human or non-human, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cows, sheep, goats, pigs, rabbits and the like.

Hereinafter, embodiments of the present invention will be described in detail. It should be noted, however, that the following examples are illustrative of the present invention and that the present invention is not limited to the following examples.

[Example]

Example 1. Preparation of luminescent molecular sieve

In order to prepare the luminescent particle based on the polymer and the phospholipid membrane, PLGA (Poly lactic-L-glycolic acid) 50:50 was dissolved in chloroform at 1 mg / ml. DOPC was dissolved in chloroform at 25 mg / ml, DOPG at 6 mg / ml and MPB-PE at 10 mg / ml, and then 52 μl (1.3 mg), 55 μl (0.3 mg) and 208 μl were respectively mixed.

300 μl of the PLGA solution was added to the mixed lipid, and 385 μl of chloroform was added thereto to make a total of 1 ml. Thereafter, 200 μl of ICG (indocyanine green) dissolved in water was added at a concentration of 1.25 mg / ml.

The final mixture of ICG was dispersed for 2 minutes by ultrasonic dispersion method. The dispersed solution was dropped into 6 ml of water, ultrasonically dispersed again, and stirred at 800 rpm for 16 hours. When stirring was completed, the mixture was centrifuged at 3000 g for 5 minutes and washed 2 times to prepare luminescent particles. The schematic diagram of the produced luminescent particle itself is shown in Fig. 2 (a).

Example 2: Cell surface attachment method of luminescent particle

The luminescent particles prepared in Example 1 were dispersed in PBS (Phosphate Buffer Saline) at a concentration of 20 mg / ml. Cell suspension was prepared by dispersing in PBS at 4 × 10 ⁶ / ml irrespective of cell type.

The luminescent particle was added to each 100 μl of the cell suspension, and the total reaction volume was adjusted to 1 ml using PBS. Thereafter, the mixture was reacted together with slight stirring at 37 DEG C for 30 minutes. After completion of the reaction, the mixture was centrifuged at 2500 rpm and 4 ° C for 5 minutes. 900 μl of the supernatant was discarded, 900 μl of SH-PEG dispersed in PBS at 1.11 mg / ml was added, Respectively. After the reaction was completed, centrifugation was carried out at 2500 rpm at 4 ° C for 5 minutes. The supernatant was removed, and centrifugation was carried out two more times in the same manner for washing.

FIG. 2 (b) is a schematic diagram showing that the luminescent particle containing the ICG produced is attached to the cell surface. As shown in FIG. 2 (b), it is expected that near-infrared imaging can be performed because the luminescent particle is attached to the cell membrane of the cell by thiol-maleimide chemical bonding and the luminescent particle captures ICG.

Example 3: Preparation of the luminescent particle and form confirmation

3.1. Confirmation of appearance using scanning electron microscope and transmission electron microscope

The appearance was confirmed using a scanning electron microscope (JEOL, JSM6700F). As shown in Fig. 3 (a), it can be confirmed that the luminescent particle itself is well formed into a spherical shape.

Further, the external appearance was confirmed by using a transmission electron microscope (JEOL, JEM-3010). As shown in Fig. 3 (b), it can be confirmed that the light emitting mouth itself is well formed into a spherical shape.

3.2. Confirm stability by changing size of mouth according to time

From the results of the observation through the scanning electron microscope and the transmission electron microscope, the diameter of the mouthpiece was confirmed with time.

The results are shown in FIG. 3 (c), and it can be seen that the size is maintained at a similar level for 15 days after the formation of the grain itself.

3.3. Cell Tracking of Near-IR Imaging Technique of Luminescent Cells

Cells to which the luminescent molecules were attached were dispersed in PBS at a specific concentration for near infrared imaging in vitro. At the time of imaging, the portion to be photographed with an 808 nm laser was illuminated and the image was imaged with a camera equipped with an 850 nm filter. The intensity of near-infrared light on the image was adjusted using Pylon software. The results are shown in FIG. 3 (d), and it can be seen that the particles prepared by the manufacturing method of the present invention have sufficient luminosity (left: PBS, right: luminescent nanoparticle itself).

Example 4. Confirmation of optimum amount of luminescent particle

Experiments were carried out to determine the optimum ratio of optimal use between cells and luminescent particles. The number of cells was fixed at 4 × 10 ⁵ and the amount of the liposome was controlled by the variable. In other words, 0mg to 8mg of the mouth was used, and the cytotoxicity of each dose was examined by WST-1 assay, and the amount of fluorescence generated in the cells was measured by flow cytometry.

(HMSC) and CD4 + T cells were used as the types of cells. The results were shown in FIGS. 4 (a) and 4 (b) for hMSC, (d). As a result, it was confirmed that when the amount of the liposomes was 8 mg or more, it was saturated.

Example 5. Confirmation of adhesion of luminescent molecules to cell surface and confirmation of stability of luminescent cells with time

The adhesion of the luminescent molecules to the cell surface was confirmed by confocal microscopy and fluorescence microscopy. In order to confirm the cell surface adhesion of the luminescent molecules using a microscope, human cord blood-derived mesenchymal stem cells, CD4 + T cells, and EL4 cells, which are lymphoma cells, were used.

The adhesion of the human umbilical cord blood-derived mesenchymal stem cells and the stability with time were confirmed in FIG. As shown in FIG. 5 (a), it was confirmed that the luminescent organs were adhered well by a confocal microscope (ZEISS, LSM 510). As shown in FIG. 5 (b), flow cytometry (Miltenyi Biotec, MACSQuant  Analyzer 10). As a result, it was confirmed that the luminescent particles were well attached to the human umbilical cord blood-derived mesenchymal stem cells. In addition, it was confirmed in Fig. 5 (c) that the human cord blood-derived mesenchymal stem cells to which the luminescent particle was attached maintained a high survival rate up to 96 hours, indicating that the luminescent particle of the present invention did not cause toxicity .

FIG. 6 shows that the luminescent particle of the present invention adheres to various kinds of cells (a: human umbilical cord blood-derived stem cells, b: human umbilical cord blood-derived stem cells not attached to a slide glass, c: EL4 cells, d: CD4 + T cells).

Example 6 Confirmation of Functionality of Luminescent Cells

6.1. Identification of the migration ability of stem cells

In order to confirm that the function of the cell can be maintained even when the luminescent particle itself adheres to the cell surface, the ability of the human umbilical cord blood-derived stem cell to move is confirmed.

Human cord blood-derived mesenchymal stem cells (hMSCs) and human cord blood-derived mesenchymal stem cells (NP attached hMSCs) with luminescent pores were cultured for 16 hours at 37 ° C. in a medium lacking FBS (Fetal Bovine Serum) Lt; / RTI > After 16 hours, 4 × 10 ⁴ cells were stored in a 200 μl medium without FBS in the upper wells, using 8.0 μm pored 24 transwell units (Corning, United States). The lower wells were filled with 600 μl of culture medium containing 30% FBS and 8.0 μm pored 24 transwell units were stored at 37 ° C. for 12 hours. The upper wells were stained with 2.5 μM calcein blue and AM solution for 1 hour and the fluorescence intensity was detected at 360 nm / 449 nm (Excitation / Emission) by a fluorescence intensity analyzer (Molecular Devices, Spectramax M5) And the lower surface were compared.

As can be seen from FIG. 7 (a), it was confirmed that the cells migrated at a level similar to that of human cord blood-derived mesenchymal stem cells (hMSC) and human cord blood-derived mesenchymal stem cells (NP attached hMSC) 7 (b), the human cord blood-derived mesenchymal stem cells (NP attached hMSCs) to which the luminescent particles were attached exhibited better migration ability. Thus, even when the luminescent particles of the present invention were attached But did not affect the maintenance of stem cell function.

6.2. Surface antigen analysis

In addition, surface antigen analysis was performed to confirm the function maintenance of the human cord blood-derived mesenchymal stem cells to which the luminescent molecules were attached.

The human umbilical cord blood-derived mesenchymal stem cells to which the luminescent molecules were attached and the untreated human umbilical cord blood-derived mesenchymal stem cells which were the control group were well dispersed into one cell, and the monoclonal mouse-derived anti-human fluorescent substance- CD34-FITC, CD44-PE, CD45-FITC, CD73-PE, CD105-FITC, CD133-PE. Unreacted antibodies were removed by centrifugation and the reacted cells were finally dispersed in 4% formaldehyde solution and analyzed on a flow cytometer (BD Biosciences, FACS Calibur system).

The results are shown in FIG. 8. As a control, human cord blood-derived mesenchymal stem cells (Control) were used and compared with human cord blood-derived mesenchymal stem cells (LESC) CD34, CD45, and CD133, surface antigens that should not be expressed in normal mesenchymal stem cells, were not expressed or expressed at a minimal level in both the control and the human umbilical cord blood-derived mesenchymal stem cells to which the luminescent particle was attached. In addition, surface antigens CD44, CD73, and CD105 expressed in normal mesenchymal stem cells were well expressed in both the control and mesenchymal stem cells derived from human cord blood. In other words, there was no significant difference between the human umbilical cord blood-derived mesenchymal stem cells and the human cord blood-derived mesenchymal stem cells to which the luminescent molecules were attached.

6.3. Confirmation of stem cell differentiation ability maintenance

In order to confirm whether the human umbilical cord blood-derived mesenchymal stem cells to which the luminescent molecules are attached are maintained in their differentiation ability, untreated human umbilical cord blood-derived mesenchymal stem cells (native hUCB-MSC) Differentiation was induced in human cord blood derived mesenchymal stem cells (LESC).

To differentiate into osteoblasts and adipocytes, 1 × 10 ⁵ cells of each group were dispensed into 6-well plates. After the cells reached 70-80% confluency, the cells were treated with osteoblast differentiation induction medium (DMEM containing 10% FBS, 100 nM dexamethasone, 50 μM ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate) (DMEM supplemented with 10% FBS, 200 μM indomethacin, 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine, and 0.5 μg / ml insulin) (Sigma-Aldrich, St. Louis, USA) , And the medium was changed every 2-3 days. After 2.5 weeks of inducing differentiation, the cells were stained to see if they were differentiated into osteoblasts or adipocytes.

Oil Red O staining was performed to detect fat droplets in differentiated cells for adipocyte differentiation. Cells were fixed in 10% formalin for 1 hour before staining and washed with 60% isopropanol. Then, Oil Red O staining was carried out for 10 minutes, and the result of the staining was shown in Fig. 9a. As shown in Figure 9a, the fat droplet was visualized through Oil Red O staining. In order to confirm adipocyte differentiation, the stained cells were measured for absorbance at a wavelength of 500 nm (absorbance was measured using an EL800 microplate reader (BIO-TEK Instruments, Winooski, VT)). The high absorbance indicates that the differentiation has proceeded, and the degree of adipocyte differentiation of the control and LESC cells was confirmed by relative absorbance measurement. As a result, as shown in FIG. 9B, the degree of differentiation of the control and LESC cells was similar.

For confirmation of osteoblast differentiation, Alizarin Red S staining specific to calcium was performed to detect alkaline phosphate activity. Cells were washed with PBS and fixed at 4 ° C for 1 h with 70% ice-cold ethanol. After washing three times with distilled water, cells were stained for 10 minutes at room temperature using 40 mM Alizarin Red S (Sigma-Aldrich, St. Louis, USA). The result of staining was shown in FIG. As shown in Fig. 10A, osteoblast cells were induced and mineral deposition appeared. In addition, the relative absorbance was measured at a wavelength of 405 nm with respect to the staining result. The results are shown in FIG. 10B, and it was confirmed that the absorbance of LESC was higher than that of the stem cells of the control group. It is possible to maintain the differentiation ability even if it is attached, and it may increase the differentiation ability.

For chondrocyte differentiation, 5 × 10 ⁵ cells of each group were placed in a 15-mL polypropylene tube and 1 mL of chondrocyte differentiation medium (Lonza, Wakersville, MD) was added and cultured for 3 weeks. The resulting round pellets were fixed on paraffin and cut to produce a 3-μm-sized section. The sample was stained with toluidine blue to detect glycosaminoglycan, and the result of staining was shown in FIG. As shown in Fig. 11, it can be seen that it is differentiated into cartilage cells.

As a result of confirming the differentiation ability of the control cells and the LESC, it was confirmed that the three lines could be differentiated into adipocytes, osteoblasts and chondrocytes as shown in FIGS. 9, 10 and 11. That is, it was confirmed that even if the luminescent particle itself is attached, the ability to differentiate mesenchymal stem cells can be maintained.

Example 7. Non-invasive imaging

Human umbilical cord blood-derived mesenchymal stem cells with a luminescent particle on their surface were injected into 6-week-old female nude mice (Balb / c nude) and tested. 8 × 10 ⁵ cells were dispersed in 50 μl of PBS and injected intramuscularly into the thighs of nude mice. Thereafter, the cells were injected with the cells at a time point from the time when the cells were injected using the laser light source of 808 nm and the filter of 850 nm. The exposure and contrast values of the light sources were adjusted using Pylon software and the values were always the same.

In the non-clinical trial, the luminal stem cells were injected into the nude mouse through intramuscular injection, and the result of imaging was shown in Fig. As a result, we confirmed that noninvasive imaging, which does not require surgery or dissection, is possible.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the embodiments described above are in all respects illustrative and not restrictive.

Claims (14)

delete delete delete delete delete A polymer coated with a phospholipid membrane,
(S1) a step of attaching a luminescent material for imaging to a surface of a cell through thiol-maleimide bonding, wherein the polymer comprises a luminescent material; And
(S2) injecting the light-emitting cells into an object other than a human, and observing the light-emitting cells,
The phospholipid membrane contained 61.0 wt% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl (1'-rac-glycerol) sodium salt (1,2-dioleoyl-sn-glycero-3-phospho- (1'-rac-glycerol) sodium salt, DOPG) And 24.4 wt% of phosphatidyl ethanolamine (MPB-PE), and the luminescent material is indocyanine green (ICG).
The method according to claim 6,
Wherein said cell is at least one immune cell selected from the group consisting of human umbilical cord blood-derived mesenchymal stem cells, CD4 T cells, CD8 T cells, and dendritic cells.
delete delete delete delete delete A polymer coated with a phospholipid membrane,
Wherein the polymer comprises a light emitting material, wherein the light emitting particle for imaging is attached to a cell surface through a thiol-maleimide bond,
The phospholipid membrane contained 61.0 wt% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl (1'-rac-glycerol) sodium salt (1,2-dioleoyl-sn-glycero-3-phospho- (1'-rac-glycerol) sodium salt, DOPG) 14.6 wt.% Of phosphatidylethanolamine and 24.4 wt.% Of phosphatidyl ethanolamine (MPB-PE), and the luminescent material is indocyanine green (ICG).
14. The method of claim 13,
Wherein said cell is at least one immunocyte selected from the group consisting of human umbilical cord blood-derived mesenchymal stem cells, CD4 T cells, CD8 T cells, and dendritic cells.

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