KR102249366B1 - Nanoparticles for promoting angiogenic growth factors secretion, manufacturing method of conditioned medium using the nanoparticle, the conditioned medium and injection composition comprising the conditioned medium - Google Patents

Abstract

In the nanoparticles for promoting the secretion of angiogenic growth factors of the present invention, a method for preparing a conditioned medium using the same, a conditioned medium and an injection composition comprising the same, the nanoparticles of the present invention are pH-sensitve, degradable gold-iron nanoparticles. , Characterized in that it releases iron ions in the acidic pH range.

Description

Nanoparticles for promoting the secretion of angiogenic growth factors, manufacturing method of conditioned medium using the same, conditioned medium and injection composition containing the same COMPRISING THE CONDITIONED MEDIUM}

The present invention relates to nanoparticles for promoting the secretion of angiogenic growth factors, and more specifically, to prepare a conditioned medium containing a large amount of angiogenic growth factors using nanoparticles that promote the secretion of angiogenic growth factors of cells. It relates to a method, an conditioned medium prepared thereby, and an injection composition comprising the same.

Ischemic disease is one of the major causes of death in the world, and it is very difficult to recover the damage that has occurred once in ischemic disease.In particular, in the case of ischemic foot disease, there is currently no clear treatment method other than surgical removal of the affected area and regeneration is impossible. Currently, stem cells and growth factors are injected into the affected area to minimize tissue damage associated with ischemic disease.However, in the case of stem cell transplantation, which directly transplants stem cells into the human body, in the case of non-autologous tissue-derived cells In addition, the various forms of immune response that occur when cells are transplanted to the affected area have a negative effect on the treatment of the disease, as well as a high risk of additional internal damage and secondary side effects. In addition, the possibility of the transplanted stem cells growing into cancerous tissues in the body cannot be ruled out, and there is a potential risk, which limits its clinical application. In addition, even if autologous tissue-derived stem cells are used for disease treatment, when stem cells are transplanted to the affected area, most of the transplanted stem cells are due to abnormal microenvironment of the affected area (oxygen due to absence of blood vessels, decreased supply of nutrients, immune response, etc.). It cannot engraft into the affected part of the cell and dies within a short time. In addition, the existing stem cell transplantation method is to overcome such low cell viability and engraftment rate, and to obtain a therapeutic effect after stem cell transplantation in the body, stem cells are further proliferated from the outside and then transplanted in large quantities. However, since this method has limited initial yield of autologous stem cells that can be obtained from patients, and consumes a lot of cost and time in the additional external proliferation and culture process, this method puts a burden on the patient in terms of cost and provides rapid disease treatment. It is becoming a stumbling block.

On the other hand, as an alternative to stem cell transplantation, a method of treating diseases by injecting a conditioned medium, which is a stem cell culture medium, into an affected area has been proposed. Since this method of conditioned medium injection uses stem cell culture medium and does not use stem cells itself, the immune response according to the presence of autologous tissue, formation of cancer tissue due to stem cell injection, and low level of transplanted stem cells are compared to the direct stem cell injection method. It is possible to overcome the problems of survival rate and engraftment rate. However, the amount of growth factor secreted from stem cells is small compared to the injection volume (volume) of the existing culture solution, so it is difficult to approach the level of growth factor concentration that can exhibit disease treatment or tissue regeneration effect, and accordingly there is a disadvantage that the treatment effect is also lowered. .

To overcome this, it is necessary to dramatically increase the amount of growth factors secreted from stem cells per unit volume of stem cell culture medium, and to solve this problem, a specific gene in stem cells is introduced or a special cell culture method is introduced. Methods such as securing and enriching a large amount of growth factors through cell passage culture have been proposed. However, in the case of introducing foreign genes in the cell culture process, there are many obstacles to obtaining approval as a substance that can be practically applied clinically by the additional use of synthetic polymers for gene transfer used for efficient gene introduction, and the growth factor enrichment process In addition, a lot of cost and time are additionally consumed, resulting in a decrease in efficiency. In particular, when the cultivation time of stem cells is increased to secure a large number of stem cells and growth factors, it is difficult to solve additional problems that naturally occur, such as a decrease in stem cell capacity, degeneration, and abnormal growth factor emission due to frequent increase in the number of subcultures. .

Therefore, there is a need for more therapeutic agents and research techniques that can effectively regenerate blood vessels.

An object of the present invention is to provide nanoparticles for promoting the secretion of angiogenic growth factors.

Another object of the present invention is to provide a method for preparing a conditioned medium containing a large amount of angiogenic growth factors using the nanoparticles.

Another object of the present invention is to provide a conditioned medium containing a large amount of angiogenic growth factors formed using the nanoparticles.

Another object of the present invention is to provide a composition for injection comprising the conditioned medium as an active ingredient.

Nanoparticles for promoting angiogenesis growth factor secretion for an object of the present invention are pH-sensitve, degradable gold-iron nanoparticles, and are characterized in that they release iron ions in an acidic pH range.

In one embodiment, the acidic pH range may be from pH 4 to pH 5.

In one embodiment, the angiogenic growth factor may include at least one of fibroblast growth factor 2 (FGF2) and Vascular endothelial growth factor (VEGF).

A method of preparing an conditioned medium for another object of the present invention includes adding pH-sensitive degradable gold-iron nanoparticles that release iron ions in an acidic pH range to a cell culture medium and culturing cells.

In one embodiment, the gold-iron nanoparticles may be added to the cell culture medium at a concentration of 3 μg/ml or less.

In one embodiment, the gold-iron nanoparticles may be introduced into the cell to promote the secretion of angiogenic growth factor of the cell.

In this case, the angiogenic growth factor may include at least one of a fibroblast growth factor and a vascular endothelial growth factor.

In one embodiment, the cells may be human adipose-derived stem cells.

The conditioned medium for another object of the present invention includes a cell culture medium cultured from a cell culture medium containing pH-sensitive degradable gold-iron nanoparticles that release iron ions in an acidic pH range.

Injectable compositions for other purposes of the present invention include as an active ingredient a conditioned medium containing a cell culture medium cultured from a cell culture medium containing pH-sensitive degradable gold-iron nanoparticles that release iron ions in an acidic pH range. .

In one embodiment, the injection composition may be an injection composition for ischemic disease.

According to the nanoparticles for promoting the secretion of angiogenic growth factors of the present invention, a method for preparing a conditioned medium using the same, an conditioned medium, and an injection composition comprising the same, the present invention provides gold-iron nanoparticles that release iron ions in an acidic pH range. Provided, the nanoparticles of the present invention can be introduced into the cell to promote the secretion of angiogenesis growth factor in the cell. At this time, the nanoparticles of the present invention can be introduced into the cell by the cell encapsulation function, thereby preventing apoptosis caused by direct and instantaneous introduction of excess ions into the cell, and increasing cell viability. In addition, since the amount of angiogenic growth factor released by stem cells is improved under the influence of the iron ions emitted by the nanoparticles of the present invention, a high concentration of angiogenic growth factor can be formed in the cell culture medium (adjusted medium), and accordingly, the present invention Since the conditioned medium of the present invention excludes serum input during cell culture and can enhance the secretion of angiogenic growth factors within a short period of time, practical disease treatment is possible. Thus, it is possible to provide an injection composition for ischemic diseases comprising the modulated medium of the present invention as an active ingredient, and the injection composition of the present invention can exhibit excellent effects.

1 is a schematic diagram for explaining the promotion of secretion of cell angiogenesis growth factor by the gold-iron nanoparticles of the present invention.
2 is a view for explaining a method of manufacturing gold-iron nanoparticles according to an embodiment of the present invention.
3 is a view showing a TEM image of the gold-iron nanoparticles of the present invention.
4 is a view for explaining whether the gold-iron nanoparticles of the present invention are introduced into cells.
5 is a view for explaining the cytotoxicity of the gold-iron nanoparticles of the present invention.
6 is a view for explaining the expression level of angiogenic growth factor gene expressed in human adipose-derived stem cells after treatment with gold-iron nanoparticles of the present invention.
7 is a view for explaining the lower extremity ischemia induction model of the present invention.
8 shows a graph of the results 4 weeks after injection of the conditioned medium treated with the gold-iron nanoparticles of the present invention into an immunodeficient mouse lower extremity ischemia induction model.
9 is a diagram showing the quantification and immunostaining data of ^{CD31 +} blood vessels in leg tissue at 4 weeks of treatment after induction of lower limb ischemia in immunodeficient mice.
^{FIG. 10 is a diagram showing smooth muscle alpha actin +} blood vessel quantification and immunostaining data at 4 weeks of treatment after induction of lower limb ischemia in immunodeficient mice.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the present invention, various modifications may be made and various forms may be applied, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, it should be understood to include all changes, equivalents, or substitutes included in the spirit and scope of the present invention. In describing each drawing, similar reference numerals have been used for similar elements.

The terms used in the present application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate the presence of features, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features or steps. It is to be understood that it does not preclude the possibility of addition or presence of, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein including technical or scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.

The nanoparticles for promoting the secretion of angiogenic growth factors of the present invention are pH-sensitive and degradable gold-iron nanoparticles.

The gold-iron nanoparticles of the present invention are nano-sized particles that can be decomposed by pH, and iron is ionized from the nanoparticles in an acidic pH range to release iron ions. In this case, the acidic pH range may be from 4 to 5.

The gold-iron nanoparticles of the present invention may be added to a cell culture medium to promote the secretion of angiogenic growth factors of cells.

1 is a schematic diagram for explaining the promotion of secretion of cell angiogenesis growth factor by the gold-iron nanoparticles of the present invention.

Referring to FIG. 1, since the gold-iron nanoparticles of the present invention have a size of a nano-unit, when they are added to a cell culture medium, they naturally flow into the cells by the process of inclusion of the cells. Inclusion is a process in which cells introduce foreign substances into cells through the formation of vesicles called inclusions, and the gold-iron nanoparticles of the present invention move in a state trapped in the inclusions. At this time, the gold-iron nanoparticles of the present invention, which are pH-sensitive decomposable nanoparticles that are decomposed in the acidic pH range by the low pH environment (pH 4-5) of the inclusion, are ionized into iron ions, and the iron ions are released to the outside of the inclusion. And provided within the cell. Iron ions released into cells stimulate mitochondria in cells to induce angiogenesis growth factor secretion. That is, the cell angiogenesis growth factor secretion ability is improved by the gold-iron nanoparticles of the present invention.

In general, when metal ions in a solution state are directly treated on normal cells and stem cells, the metal ions are treated directly to the cells by the aqueous solution itself. Is consumed and can eventually lead to cell death. In addition, an excessive amount of metal ions that are introduced instantaneously may induce damage to organelles such as mitochondria, which are energy generating organs in the cell, thereby inducing the death of additional cells.

However, in the case of the gold-iron nanoparticles of the present invention, as described above, since they are introduced into the cell through the cell encapsulation function, the energy consumption of the cell is very low, and a precise amount of particles is delivered through the encapsulation, thereby instantaneous By preventing excessive ion inflow, it does not induce cell death by preventing damage to organelles such as mitochondria, and it is possible to regulate cell behavior such as increased secretion of angiogenic growth factors. That is, the gold-iron nanoparticles of the present invention can provide metal ions into the cell without being harmless to the cell, and thus can easily control cell behavior such as increased secretion of angiogenic growth factors.

On the other hand, in the nanoparticles of the present invention, gold has stability even at a low pH compared to iron, which is a transition metal, so that unlike iron ions, it is not released into cells, and remains in the inner envelope, so that the nanoparticles of the present invention are introduced into the cell. At the same time, it can give additional functions such as genes, drugs, and bio-imaging.

2 is a view for explaining a method of manufacturing gold-iron nanoparticles according to an embodiment of the present invention.

Referring to FIG. 2, in the method for producing gold-iron nanoparticles of the present invention, a gold precursor solution and an iron precursor solution are respectively added dropwise to the polymer solution while stirring the polymer solution to which a reducing agent is added, thereby adding gold-iron nanoparticles. And forming.

At this time, as an example, the polymer solution may be a polyvinylpyrrolidone (PVP) solution, and the reducing agent may be sodium borohydride (NaBH ₄ ).

When a gold precursor solution and an iron precursor solution are added dropwise while stirring the polymer solution, nanoparticles containing gold and iron are formed. For example, the gold precursor may be gold (III) chloride hydrate (Gold (III) chloride hydrate, HAuCl ₄ -xH ₂ O), and the iron precursor is iron (III) chloride, FeCl ₃ ) Can be.

The gold-iron nanoparticles including gold and iron formed in the step of forming the gold-iron nanoparticles may be separated from the solution by centrifuging the solution.

A more detailed description of this will be described in more detail with reference to Examples below.

The method for preparing a conditioned medium of the present invention includes the steps of adding the gold-iron nanoparticles of the present invention described above to a cell culture medium, and culturing the cells.

The cells may refer to stem cells, and for example, may be human adipose-derived mesenchymal stem cells. The cell culture medium used in the stem cell culture process contains growth factors secreted by stem cells. At this time, in the case of culturing the cells in the cell culture medium to which the gold-iron nanoparticles are added according to the present invention, as described above, the gold-iron nanoparticles are introduced into the cell by the cell inclusion function. Iron ions are released from the nanoparticles by the low pH environment. The released iron ions promote the secretion of angiogenic growth factors of cells, thereby forming a conditioned medium containing a large amount of angiogenic growth factors. At this time, the neoplastic growth factor includes at least one of fibroblast growth factor 2 (FGF2) and Vascular endothelial growth factor (VEGF).

Meanwhile, the gold-iron nanoparticles may be added to the cell culture medium at a concentration of 3 μg/ml or less. When the gold-iron nanoparticles are added in excess of 3 μg/ml per unit volume of the cell culture medium, toxicity may be caused to cells and thus cells may be killed.

According to the present invention, by adding the gold-iron nanoparticles of the present invention, which can increase the secretion of angiogenic growth factors of (stem) cells, to the stem cell culture medium, metal ions are provided into the cells by cell encapsulation. While it is possible to prevent death, it is possible to increase the secretion amount of angiogenic growth factors from stem cells by iron ions released from gold-iron nanoparticles at the same time. Accordingly, the concentration of angiogenic growth factor in the stem cell conditioned medium can be dramatically increased, and therefore, the conditioned medium of the present invention can exhibit excellent therapeutic effects for diseases requiring angiogenesis.

That is, since the conditioned medium according to the present invention contains a large amount of angiogenic growth factors in the conditioned medium due to increased secretion of angiogenic growth factors from stem cells, the conventional stem cell transplantation method has a low survival rate of stem cells transplanted to the affected area and Stem cell-modulated medium injection, suggested as an alternative to overcome the problem of the treatment effect due to engraftment rate and cancer tissue changes, and potential risks, and the concentration of angiogenic growth factors in the adjusted medium itself is low, resulting in a practical treatment effect. Unlike difficult, it is possible to effectively induce angiogenesis through the conditioned medium of the present invention, and accordingly, the conditioned medium according to the present invention as an active ingredient can be used as a therapeutic agent for treating ischemic disease. I can. For example, the conditioned medium of the present invention can be used as an injection composition for ischemic diseases.

Hereinafter, for a more specific example, the gold-iron nanoparticles of the present invention, a method of synthesizing the same, and the preparation of a conditioned medium using the gold-iron nanoparticles of the present invention, and effects thereof in ischemic diseases will be described. .

Preparation of gold-iron nanoparticles

(1) material

Gold(III) chloride hydrate (HAuCl ₄ -xH ₂ O, 99.995%), iron (III) chloride (Iron(III) chloride, FeCl ₃ , 98%), poly(vinyl pyrroly) Don) (Poly (vinyl pyrrolidone), PVP, MW = 55,000 Da), sodium borohydride (NaBH ₄ ) were purchased from Aldrich and used without further purification. _{PVP, NaBH 4} , HAuCl ₄ , FeCl ₃ solutions were prepared based on deionized water.

(2) synthesis

In order to synthesize gold-iron nanoparticles according to an embodiment of the present invention, poly(vinyl pyrrolidone) (Poly(vinyl pyrrolidone), PVP, MW = 55,000 Da) 1.11% ( w/v) 9 mL was prepared, and 1 mL of sodium hydroxide (NaOH, 98%) 0.4% (w/v) was added. Then, gold (III) chloride hydrite (HAuCl ₄ ) 0.4% (w/v) and iron (III) chloride (Iron(III) chloride, FeCl ₃ , 98%) 0.2% (w/v) were stirred A total of 1 mL each was added dropwise. Then, the reaction was performed at room temperature for 15 minutes, and the synthesized nanoparticles were obtained through a centrifuge (8000 rpm, 15 minutes). The synthesized nanoparticles were washed twice with DI water and acetone, respectively, and redispersed in deionized water.

TEM image of gold-iron nanoparticles

The TEM image of the gold-iron nanoparticles of the present invention prepared using a transmission electron microscope (JEM-1010, JEOL Ltd.) was confirmed, and its energy dispersive spectroscopy (EDS) was confirmed. The results are shown in FIG. 3.

3 is a view showing a TEM image of the gold-iron nanoparticles of the present invention.

3A shows a TEM image of the gold-iron nanoparticles of the present invention, and B shows the result of EDS analysis thereof.

Referring to FIG. 3, it can be seen that nano-sized gold-iron nanoparticles made of gold and iron were prepared according to the present invention.

Influx of gold-iron nanoparticles into cells

Human adipose-derived stem cells were cultured for 12 hours in a 16 mL culture medium having a concentration of 3 μg/ml of gold-iron nanoparticles in ^{8 X 10 5 cells/dish, and Kanopsky} After standing at 4° C. for 4 hours using (Karnovsky's) fixation, it was washed with cold 0.05 M cacodylate buffer. Then, the cells were fixed in 1% osmium tetraoxide at 4° C. for 2 hours, and washed twice with cold distilled water. Subsequently, the sample was placed overnight at 4° C. in 0.5% uranyl acetate. Then, dehydration is sequentially performed using ethanol (30, 50, 70, 80, 90, 95, 100%), rinsed with propylene oxide, and then 24 at 70°C in Spurr's resin. Leave it for hours. Subsequently, a thin portion with a thickness of 100 nm was obtained using an ultramicrotome (Leica), placed on a 200-mesh copper grid, and observed using a transmission electron microscope (JEM-1010, JEOL Ltd.) I did. The results are shown in FIG. 4.

FIG. 4 is a diagram for explaining whether gold-iron nanoparticles of the present invention are introduced into cells, and shows a TEM image of cells treated with gold-iron nanoparticles.

Referring to FIG. 4, it can be seen that the gold-iron nanoparticles of the present invention are present in the cells when the gold-iron nanoparticles of the present invention are added and then cultured (the center image of FIG. 4 (red arrow)). Reference). That is, when the gold-iron nanoparticles of the present invention are cultivated together with the cells, it can be confirmed that the gold-iron nanoparticles are introduced into the cells and the nanoparticles are present in the cells.

Cytotoxicity of gold-iron nanoparticles

To confirm the cytotoxicity of the gold-iron nanoparticles of the present invention, 1.5 X 10 ⁴ human adipose-derived mesenchymal stem cells were treated with gold-iron nanoparticles by concentration, and then the cytotoxicity was calculated after 24 hours. kit-8, Dojindo Molecular Technologies, Inc., Rockville, MD, USA). The CCK-8 assay is a method of examining intracellular dehydrogenase activity using a formazan dye, and the number of living cells is proportional to the amount of formazan dye.

Specifically, gold-iron alloy nanoparticles of various concentrations were treated on cells grown in 24-well plates for 24 hours and washed three times with PBS. Then, after replacing with a new cell culture solution, the CCK-8 solution was added to each well and then stored in an incubator for 2 hours. Subsequently, the absorbance at 450 nm was measured with a plate reader to measure the percentage (%) of living cells, and the cytotoxicity was confirmed through this. The results are shown in FIG. 5.

5 is a view for explaining the cytotoxicity of the gold-iron nanoparticles of the present invention.

In Figure 5, *p value means <0.05 compared to the untreated group.

Referring to FIG. 5, the cells were statistically compared with cells not treated with gold-iron nanoparticles (0 μg/ml) until treatment with gold-iron nanoparticles at a concentration of 3 μg/ml per unit volume of cell culture solution. It can be seen that there is no difference in toxicity. That is, it can be seen that the gold-iron nanoparticles of the present invention do not exhibit cytotoxicity in human adipose-derived stem cells up to a concentration of 3 μg/ml per unit volume of cell culture medium.

Expression of angiogenic growth factors of gold-iron nanoparticles

1.0 X 10 ⁵ human adipose-derived stem cells were treated with up to 3 μg/ml gold-iron nanoparticles and incubated for 12 hours, followed by qRT-PCR (Quantitative real-time polymerase chain reaction). The mRNA expressions of Fibroblast growth factor 2 (FGF2) and Vascular endothelial growth factor (VEGF) were compared.

Specifically, samples treated with gold-iron nanoparticles at various concentrations were lysed through a TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted through chloroform (Sigma, St. Louis, USA) and concentrated through isopropanol (Sigma). Remove the supernatant, rinse the RNA pellet, rinse in 75% (v/v) ethanol and dry in air, then 0.1% (v/v) diethyl pyrocarbonate-treated water (Sigma). ).

Subsequently, for qRT-PCR, SsoAdvancedTM Universal SYBR Green Supermix kit (Bio-Rad, Hercules, CA, USA) and CFX ConnectTM real-time PCR detection system (Bio-Rad) were used. Primers for qRT-PCR are as follows: human GAPDH (forward 5'-GTC GGA GTC AAC GGA TTT GG-3', reverse 5'-GGG TGG AAT CAA TTG GAA CAT-3'), human VEGF (forward 5'-GAG GGC AGA ATC ATC ACG AAG T-3', reverse 5'-CAC CAG GGT CTC GAT TGG AT-3'), human FGF2 (forward 5'-GAC GGC AGA GTT GAC GG-3', reverse 5'-CTC TCT CTT CTG CTT GAA GTT-3'). The qRT-PCR results are shown in FIG. 6.

6 is a view for explaining the expression level of angiogenic growth factor gene expressed in human adipose-derived stem cells after treatment with gold-iron nanoparticles of the present invention.

In Figure 6, *p value means <0.05 compared to the untreated group.

6, after the gold-iron nanoparticle treatment of the present invention, all the expression levels of angiogenic growth factor genes expressed in human adipose-derived stem cells were increased, and in particular, nanoparticles at a concentration of 3 μg/ml were treated for 12 hours. It can be seen that the highest expression of the angiogenic growth factor gene was found in the cell experimental group.

That is, it can be seen that the secretion of angiogenic growth factors is promoted in cells cultured by adding the gold-iron nanoparticles of the present invention.

Manufacture of adjusted medium

(1) cell culture

Human adipose-derived mesenchymal stem cells were purchased and used from Lonza (Bazel, Switzerland), and 10% (v/v) Fetal bovine serum (FBS), Gibco BRL, Gaithersburg, MD, USA) and 1% (v/v) A mixed solution of Dulbecco's Modified Eagle Medium (Gibco BRL) to which penicillin/streptomycin (Gibco BRL) was added was used as a culture solution. Cell culture was carried out in a constant temperature incubator at 37° C. and 95% humidity-maintained environment containing 5% (v/v) CO _{2 in} air, and the cell culture solution was changed every 3 days. Cells with a cell passage number of 8 or less were used in the experiment, and were discarded when the number of passages exceeded 8.

(2) Preparation of adjustment medium

^{Cultivate 0.8 X 10 6} human adipose-derived mesenchymal stem cells in a 150 mm cell culture dish, and use 16 mL of a serum-free medium containing the gold-iron nanoparticles of the present invention at a concentration of 3 μg/ml. Incubated for hours. In order to obtain a conditioned medium, the cells before serum-free culture were washed three times with phosphate-buffered saline (PBS), Gibco BRL, and then replaced with a serum-free medium. After culturing the cells for two days, the conditioned medium was collected to prepare an conditioned medium according to an embodiment of the present invention.

In vitro and in vivo experiments of conditioned medium

In order to confirm the effect of disease treatment through angiogenesis through injection of the conditioned medium according to the present invention, in vitro and in vivo experiments were performed. In the present invention, all animal experiments were conducted by a certified experimental method after inspection by the Animal Experimental Ethics Committee of Sungkyunkwan University, and animal experiments and animal breeding were carried out in accordance with the procedures in the SPF facility (IACUC number: SKKUIACUC-17-5- 3-4).

(1) lower limb ischemia mouse modeling

Four-week-old female immunodeficient mice (BALB/c-nu, 20-25 g body weight, Orient, Seongnam, Gyeonggi, Korea) were added to xylazine (10 mg/kg) and ketamine (100 mg/kg). kg), and then the femoral artery and surrounding blood vessels were tightly tied with 6-0 silk suture (silk suture, AILEE, Busan, Korea). Subsequently, the external bone artery and its upstream arteries were tied, and then modeled by resecting from the branch point of the external bone artery to the end divided into the saphenous vein and the popliteal artery. The index for quantitative analysis of the lower limb ischemia induction model is as shown in FIG. 7.

7 is a view for explaining the lower extremity ischemia induction model of the present invention.

In FIG. 7, the left image represents limb salvage, the middle image represents toe necrosis, and the right image represents limb loss.

(2) Treatment and treatment of lower limb ischemia modeling mice

Induction of lower extremity ischemia in mice modeled with lower extremity ischemia through arterial incision (No treatment (NT)), injection of conditioned medium after induction of lower extremity ischemia (Normal conditioned medium (NC)), and gold- It was set as a group of conditioned medium injections (AuFe NPs conditioned medium (AC)) collected from cells treated with iron nanoparticles. The NT group in which only lower extremity ischemia was induced without any treatment was set as a negative control, and conditioned media collected from cells treated with gold-iron nanoparticles and conditioned media collected through normal cell culture induces lower extremity ischemia at 200 µl daily. It was injected for 4 days into the site (cranial tibialis muscle of the central thigh). The results are shown in FIG. 8.

8 shows a graph of the results 4 weeks after injection of the conditioned medium treated with the gold-iron nanoparticles of the present invention into an immunodeficient mouse lower limb ischemia induction model.

In Fig. 8, NT represents an untreated group, NC represents a general conditioned medium, and AC represents an conditioned medium according to the present invention.

Referring to Figure 8, in the experimental group (AC) in which the adjusted medium added with gold-iron nanoparticles was injected, the existing conditioned medium (human adipose-derived stem cell culture solution collected without the use of nanoparticles, NC) or a negative control (treatment It can be seen that it shows a remarkably increased appearance change compared to the non-ischemic induction mouse, NT).

That is, it can be seen that the addition of the gold-iron nanoparticles according to the present invention induces the secretion of angiogenic growth factors of stem cells, and thus exhibited an excellent effect on lower limb ischemia modeling mice.

(3) Leg tissue acquisition and tissue staining of lower extremity ischemia modeling mice

Lower limb ischemia modeling mice were sampled according to the post anesthesia procedure on day 28. The collected legs were frozen in an O.C.T compound (optical cutting temperature compound, SciGen Scientific, Gardenas, CA, USA). After that, samples were obtained and stained by the following method after flakes were sliced to a thickness of 10 μm: The slices were stained with anti-CD31 antibody (Abcam, Cambridge, UK) and anti-SM alpha-actin antibody (Abcam), for visualization. Fluorescein isothiocyanate-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA) was used. Stained tissues were stained with 4,6-diamidino-2-phenylindole (DAPI) and observed using a fluorescence microscope (Ti-U, Nikon, Tokyo, Japan). Became. The results are shown in Figs. 9 and 10,

^{FIG. 9 shows leg tissue CD31 +} blood vessel quantification and immunostaining data at 4 weeks of treatment after induction of immunodeficiency mouse ischemia, and FIG. 10 shows smooth muscle alpha actin of leg tissue at 4 weeks of treatment after induction of immunodeficiency mouse ischemia. ^{+ It} is a diagram showing blood vessel quantification and immunostaining data.

In FIGS. 9 and 10, blue indicates the cell nucleus, and green indicates vascular factors (CD31 and smooth muscle alpha actin, respectively, in FIGS. 9 and 10).

9 and 10, the conditioned medium to which the gold-iron nanoparticles of the present invention were added, CD31 ⁺ and smooth muscle alpha actin ⁺ phosphorus microvessels were statistically significant compared to the control group (NC) and the negative control group (NT). It can be seen that the number of microvessels found in the ischemic tissue increased from the immunostaining result. This is a result consistent with that confirmed with reference to FIG. 8, and means that the conditioned medium prepared by adding the gold-iron nanoparticles of the present invention has an excellent angiogenic effect compared to the use of the conventional conditioned medium.

Therefore, according to the review above, the amount of angiogenic growth factor released from the cells by introducing the degradable gold-iron nanoparticles prepared according to the present invention into mesenchymal stem cells, a type of human adult adipose-derived stem cells. It can be seen that it can be dramatically enhanced without the use of additional external genes, drugs or compounds. In addition, by grafting the gold-iron nanoparticles according to the present invention to the existing cell culture method, a culture medium (adjusted medium) having an improved concentration of angiogenic growth factors in a cell culture medium can be prepared, and the present invention comprising a high concentration angiogenic growth factor It can be seen that angiogenesis is increased in the mouse lower extremity ischemia model in the case of injecting the conditioned medium of ischemia at the stage of tissue formation through gene and protein expression, and that the damage (natural cutting) of the final ischemic disease site is greatly alleviated. I can confirm. That is, the conditioned medium according to the present invention can exhibit excellent angiogenesis effects while overcoming the limitation of the treatment concentration of growth factors and the problem of remaining in the body of non-degradable nanoparticles in the treatment of injecting the existing cell culture medium (the conditioned medium) into the affected area. It can be seen that the cell-based modulated medium can be effectively applied in the field of application of the disease treatment effect.

Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art will be able to variously modify and change the present invention without departing from the spirit and scope of the present invention described in the following claims. You will understand that you can.

Claims (11)

delete delete delete Adding pH-sensitive degradable gold-iron nanoparticles that release iron ions in the acidic pH range to the cell culture medium; And
Comprising the step of culturing the cells,
Manufacturing method of conditioned medium.
The method of claim 4,
The gold-iron nanoparticles are added to the cell culture medium at a concentration of 3 μg/ml or less,
Method of manufacturing conditioned medium.
The method of claim 4,
The gold-iron nanoparticles are introduced into the cells to promote the secretion of angiogenic growth factors of the cells,
Method of manufacturing conditioned medium.
The method of claim 6,
The angiogenic growth factor is characterized in that it comprises at least one of fibroblast growth factor and vascular endothelial growth factor,
Method of manufacturing conditioned medium.
The method of claim 4,
The cells are human adipose-derived stem cells, characterized in that,
Method of manufacturing conditioned medium.
Comprising a cell culture medium cultured from a cell culture medium containing pH-sensitive degradable gold-iron nanoparticles that release iron ions in an acidic pH range,
Adjustment medium.
Injectable composition for the treatment of ischemic diseases, comprising as an active ingredient an conditioned medium containing a cell culture medium cultured from a cell culture medium containing pH-sensitive degradable gold-iron nanoparticles that release iron ions in the acidic pH range . delete

Images