WO2024053985A1 - Composition for cryoprotection of exosomes including biocombatible polymer including network structure - Google Patents

Abstract

The present invention relates to a composition for cryoprotection of exosomes, the composition including a biocompatible polymer including a network structure. By using the composition for cryoprotection of exosomes of the present invention, the structural stability of exosomes can be maintained during a freezing operation, and the functional stability of exosomes can be maintained during long-term preservation following a freezing operation.

Description

Composition for cryoprotection of exosomes comprising a biocompatible polymer having a network structure

The present invention was made under project number HI20C0437020022 under the support of the Ministry of Health and Welfare of the Republic of Korea. The specialized research management organization for the project is the Korea Health Industry Development Institute, the research project name is “Advanced Medical Technology Development,” and the research project name is “Effectiveness of Stem Cell Exosomes.” “Evaluation and identification of mechanism of action”, the host organization is Sungkyunkwan University Industry-Academic Cooperation Foundation, and the research period is 2022.01.01-2022.12.31.

This invention was also made under project number 2021R1A4A1032782 under the support of the Ministry of Science and ICT of the Republic of Korea. The research management agency for the project was the National Research Foundation of Korea, the research project name was “Collective Research Support (R&D),” and the research project name was “Ischemic Disease.” “Development of stem cell-derived artificial exosome vesicles for treatment”, the host institution is Sungkyunkwan University, and the research period is 2021.06.01-2022.02.28.

In addition, this invention was made under project number 21A0503L1-11 under the support of the Ministry of Science and ICT of the Republic of Korea. The research management agency for the project was the National Research Foundation of Korea, and the research project name was “Pan-Ministry Regenerative Medicine Technology Development Project (R&D).” , the research project title is “Treatment of rheumatoid arthritis based on extracellular vesicles derived from inflammatory immune cell-targeted stem cells”, the host institution is Sungkyunkwan University, and the research period is 2022.02.01-2022.12.31.

This patent application claims priority to Korean Patent Application No. 10-2022-0113811 filed with the Korean Intellectual Property Office on September 7, 2022, the disclosure of which is incorporated herein by reference.

The present invention relates to a composition for cryoprotection of exosomes, comprising a biocompatible polymer having a network structure.

Exosomes are membrane vesicles with a size of 30-200 nm secreted from cells and are mostly present in body fluids, including blood and urine, and are cell-specific components that reflect the biological functions unique to the cell of origin (donor cell). It contains a variety of substances showing biological activity, such as phospholipids, mRNA, miRNA, and DNA, as well as various soluble proteins, extrinsic proteins, and transmembrane protein components.

The lipid bilayer of exosomes has a phospholipid bilayer structure similar to that of the cell of origin (donor cell), and is a component of substances secreted extracellularly by cells, delivering bioactive substances to recipient cells to promote cell-cell communication and cellular immunity. It is known to play the role of a signal transduction mediator that regulates cell functions such as mediation.

However, exosomes have problems with long-term storage, such as changes in physical properties and stability due to limitations of the biological material itself, and stability related to the physiological activity of exosomes may be reduced.

To date, research and development on long-term storage of exosomes has been actively conducted, and the representative long-term storage method of exosomes is ultra-low temperature freezing. This has the effect of rapidly freezing exosomes and preventing denaturation of biological materials due to temperature changes, but has the disadvantage of causing structural damage to exosomes due to the formation of water crystals within the exosomes during the freezing process. On the other hand, DMSO, a representative cryoprotectant, can penetrate into the cell membrane and replace water molecules, thereby suppressing crystal formation and preventing structural damage to exosomes caused by cryogenic freezing. However, this has the problem of potential toxicity of the organic material itself. there is.

Therefore, there is a need to develop a new cryoprotection technology that can store exosomes long-term while maintaining the stability associated with the physiological activity of exosomes.

The present inventors have made extensive research efforts to develop a technology that can store exosomes for a long period of time while maintaining the stability related to the physiological activity of exosomes. As a result, the present invention was completed by preparing a composition for cryoprotection of exosomes containing a biocompatible polymer with a network structure and confirming that the function of exosomes was stably maintained even after long-term storage.

Therefore, the purpose of the present invention is to provide a composition for cryoprotection of exosomes containing a biocompatible polymer having a network structure.

Another object of the present invention is to provide stability to exosomes during long-term storage using the composition for cryoprotection of exosomes.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

'Exosome' refers to an endoplasmic reticulum of approximately 30-200 nm in size, which has a membrane structure composed of a lipid-bilayer secreted from various cells. Inside exosomes, they contain proteins called exosomal cargo, nucleic acids (e.g., mRNA, miRNA, DNA), and a wide range of signaling factors.

The exosome is an intercellular signaling mediator secreted by cells, and various cell signals transmitted through it affect cell behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. It is known to control In particular, it can be used as a new concept bio material applicable to various diseases because it regulates the functions of cells involved in anti-aging, tissue regeneration, and various diseases depending on the characteristics of the derived cells.

However, exosomes have problems in that the overall physical properties and stability of exosomes may change depending on the storage period and method, and the activity of the effective physiologically active substances of exosomes may be reduced. Therefore, in order to effectively use the specific genetic material and physiologically active factors contained in exosomes, not only can ultra-low temperature freezing, a representative long-term storage method of exosomes, be applied, but also DMSO, which has the potential for toxicity, can be used as a cryoprotectant. Structural damage to exosomes caused by ultra-low temperature freezing can be prevented, and ultra-low temperature freezing can also be applied.

Therefore, according to one aspect of the present invention, the present invention provides a composition for cryoprotection of exosomes, comprising a biocompatible polymer having a network structure.

The present inventors not only maintain the structural stability of exosomes during the freezing process by using a composition for cryoprotection of exosomes containing biocompatible polymers, but also maintain the functional stability of exosomes when stored for a long time after freeze-drying. It was confirmed that it could be maintained.

In particular, when using the composition of the present invention, not only can the problem of structural damage to exosomes caused by ultra-low temperature freezing in the existing long-term storage method described above be overcome, but also the use of DMSO, a cryoprotectant with organic toxicity, is eliminated. I confirmed that it can be done.

As used herein, the term 'biocompatible polymer' refers to a large molecular substance with a molecular weight of approximately 10,000 Da or more that is not toxic to the human body when administered, and has biological functionality using excellent biocompatibility and the material's inherent biological activity. It can be used as a material.

In one embodiment of the present invention, the biocompatible polymer is hyaluronic acid, collagen, alginate, starch, chitosan, gelatin, and dextran. ), cellulose, alginic acid, chondroitin sulfate, and heparin, but is not limited thereto.

In a specific embodiment of the present invention, the biocompatible polymer may be hyaluronic acid.

In this specification, the term 'hyaluronic acid (HA)' is one of glucosamine glycans, and the repeating unit consisting of N-acetyl glucosamine and glucuronic acid is linear. It refers to a connected biopolymer material. Hyaluronic acid is present in high concentrations in the extracellular matrix, connective tissue and skin, and in particular, maintains moisture in intercellular spaces, maintains cells based on forming a jelly-like matrix within tissues, maintains tissue lubricity and flexibility, and maintains tissue lubricity and flexibility. It has many functions, including resistance to external forces such as mechanical disturbance and prevention of cell infection. In addition, it is known to be involved in the storage and diffusion of cell growth factors and nutrients, and to be synthesized by keratinocytes and fibroblasts.

In one embodiment of the present invention, the composition for cryoprotection of exosomes may include 0.01 to 1.0 wt% (w/v) of hyaluronic acid polymer. Specifically, the freeze protection composition contains 0.01 to 1.0 wt% (w/v), 0.01 to 0.08 wt% (w/v), 0.01 to 0.06 wt% (w/v), 0.01 to 0.04 wt% of hyaluronic acid polymer. (w/v), 0.02 to 1.0 wt%(w/v), 0.02 to 0.08 wt%(w/v), 0.02 to 0.06 wt%(w/v), 0.02 to 0.04 wt%(w/v), 0.04 to 1.0 wt% (w/v), 0.04 to 0.08 wt% (w/v), 0.04 to 0.06 wt% (w/v), 0.06 to 1.0 wt% (w/v), 0.06 to 0.08 wt% ( w/v) or 0.08 to 1.0 wt% (w/v).

More specifically, the hyaluronic acid polymer may be included in an amount of 0.05 wt% (w/v). However, when preparing the composition for exosome cryoprotection of the present invention, it is not limited thereto, as long as it contains hyaluronic acid polymer within the above-mentioned range.

In one embodiment of the present invention, the exosome may be a stem cell-derived exosome.

The “stem cell” refers to an undifferentiated cell with self-replication ability and the ability to differentiate into two or more different types of cells, that is, stemness. These stem cells are largely divided into embryonic stem cells, adult stem cells, gametes, and cancer stem cells, which can be produced using embryos. Stem cells are cells extracted from the embryonic development process and refer to the cell mass stage before forming specific organs less than 14 days after fertilization. Recently, embryonic stem cells are also manufactured from normal cells through dedifferentiation. Therefore, any cell that can differentiate into all cells and tissues that make up the body is not limited thereto. Adult stem cells are extracted from umbilical cord blood, bone marrow, fat, blood, etc. and refer to primitive cells just before differentiation into cells of specific organs such as bone, liver, and blood, and have the ability to develop into tissues within the body when necessary. It refers to cells in an undifferentiated state. Germ cells are cells that transmit genetic information to the next generation through reproduction. Humans include, but are not limited to, sperm and eggs.

The stem cells may be autologous or allogeneic, may be derived from any type of animal, including humans and non-human mammals, may be stem cells derived from adults, or may be stem cells derived from embryos. For example, it may be selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem cells, but is not limited thereto.

Specifically, the type of the stem cell is not limited, but as an example that does not limit the present invention, it may be an adult stem cell, for example, an adult stem cell derived from human or animal tissue, or an adult stem cell derived from human or animal tissue. It may be selected from the group consisting of mesenchymal stem cells and mesenchymal stem cells derived from induced pluripotent stem cells derived from human or animal tissue, but is not limited thereto.

The human or animal tissue may be selected from the group consisting of umbilical cord, cord blood, blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta, but is not limited thereto.

More specifically, in the present invention, the type of adult stem cells is not limited, but as an example that does not limit the present invention, mesenchymal stem cells, for example, umbilical cord, cord blood, blood, bone marrow, fat, muscle, The mesenchymal stem cells may be derived from one or more types of tissues selected from the group consisting of nerves, skin, amniotic membrane, and placenta.

Mesenchymal stem cells can be obtained from various parts described above, but among them, adipose tissue is known to be a rich source of stem cells with various potentials. Specifically, adipose-derived stem cells (ASC) are stem cells isolated from adipose tissue that can differentiate into most mesenchymal cells such as adipocytes, osteoblasts, chondroblasts, and myofibrocytes, and are known as preadipocytes. It refers to cells that have been called stromal cells, multipotent adipose-derived cells, or adipose derived adult stem cells. In particular, the adipose-derived stem cells can show therapeutic effects on various diseases such as arthritis and myocardial infarction, and the cell proliferation rate is fast and the procedure for separating cells from adipose tissue is relatively easy, which has the advantage of reducing the burden on patients. It is known that The adipose-derived stem cells generally refer to adipose-derived mesenchymal stem cells.

Therefore, in a specific embodiment of the present invention, the mesenchymal stem cells may be adipose-derived mesenchymal stem cells (ADSCs) isolated from adipose tissue, and more specifically, human-derived adipose mesenchyme. It could be a stem cell. According to many studies, 'stem cell-derived exosomes' secreted by stem cells contain various physiologically active factors and genetic materials, and are involved in regulating cell behavior such as cell migration, proliferation and differentiation, and cell differentiation and tissue regeneration. Stem cell characteristics are reflected, and in particular, 'human fat-derived stem cell-derived exosomes' are well known as useful substances containing bioactive substances of various genetic information that have positive effects on human health.

In one embodiment of the present invention, the diameter of the exosome is 10 nm to 500 nm, 10 nm to 400 nm, 10 nm to 300 nm, 10 nm to 250 nm, 10 nm to 200 nm, and 10 nm to 150 nm. , 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 200 nm, 50 nm to 150 nm, or 80 nm to 140 nm, but is not limited thereto.

In one embodiment of the present invention, the exosomes are 1 x 10 ⁹ to 1 x 10 ¹² , 5 x 10 ⁹ to 5 x 10 ¹² , 1 x 10 per 1 mL of the cryoprotection composition of the exosomes. ⁹ to 11 1 x ¹⁰ , 9 to 5 x 10, ⁹ to 1 x ¹⁰ , ⁹ to 1 x ¹⁰ , ¹⁰ to 5 x ¹⁰ , ⁹ to 5 x 10 It may include 1 x 10 ¹¹ pieces, or 1 x 10 ¹⁰ pieces, but is not limited thereto.

As used herein, the term 'freeze protection' refers to protecting exosomes from damage that may occur during the freezing process for long-term storage of exosomes to maintain the structure, function, physical form, and/or biological activity of exosomes. do.

In one embodiment of the present invention, a composition for cryoprotection of exosomes containing a biocompatible polymer is characterized in that it is capable of maintaining the stability of exosomes by protecting them from damage that may occur during the freezing process.

As used herein, the term 'maintenance' refers to a state in which the stability of exosomes is preserved or continues to exist, or the increase or decrease thereof is delayed.

Specifically, in one embodiment of the present invention, hyaluronic acid, which is a biocompatible polymer of the composition, binds to the exosome surface due to the CD44 receptor-ligand action, thereby maintaining the structural stability of the exosome during the freezing process. .

The 'structural stability of exosomes' refers to maintaining the spherical structural form of exosomes before and after the freezing process, and is bound to the hyaluronic acid polymer residue through specific binding between the hyaluronic acid of the composition and the exosomes. This can be achieved by providing space between exosome particles to minimize structural damage to the phospholipid membrane caused by aggregation between exosome particles that occurs during the freezing process.

In another embodiment of the present invention, when the structural stability of exosomes is maintained during the freezing process using the composition, the structural stability of the exosomes can be continuously maintained even during long-term storage.

In the examples, the present inventors confirmed through transmission scanning microscopy that when stored for a long period of time after freeze-drying using the composition, the spherical vesicle shape of exosomes was maintained in a similar shape to the initial form, and the aggregation phenomenon between exosomes was also observed compared to the control group. In comparison, it was confirmed that it appeared less frequently.

As used herein, the term 'long-term storage' means storage for, for example, 2 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, or several years.

According to an embodiment of the present invention, the 'long-term storage' may be applied differently depending on the storage temperature of exosomes using the composition of the present invention, for example, storage at 4°C for 6 months to 1 year. It may be, specifically, for 6 months to 11 months, for 6 months to 9 months, for 6 months to 7 months, for 7 months to 1 year, for 7 months to 11 months, for 7 months to 9 months, 7 months to 8 months, 8 months to 1 year, 8 months to 11 months, 8 months to 10 months, 8 months to 9 months, 9 months to 1 year, 9 months to 11 months, 9 months It may be storeable for 10 months to 1 year, 10 months to 1 year, 10 months to 11 months, or 11 months to 1 year, or, for example, 15 to 30 days at 40°C. Can be stored, specifically, for 15 to 25 days, 15 to 20 days, 20 to 30 days, 20 to 25 days, 25 to 30 days, but is not limited thereto. No.

In the present invention, storage under long-term accelerated conditions means storage at 4°C for 1 year, which corresponds to the same conditions as storage at 40°C for 30 days, so in the embodiment of the present invention, storage at 40°C for 30 days The effect of maintaining the stability of exosomes under these conditions was evaluated.

In addition, in the present invention, storage under long-term real-time conditions means storage at 4°C for 30 days.

In another embodiment of the present invention, the composition is capable of maintaining the functional stability of exosomes when stored for a long period of time after freeze-drying the exosomes.

In a specific embodiment of the present invention, the functional stability is measured based on the following indicators:

(a) Size of exosomes; (b) concentration of exosomes; (c) Enzyme activity ability of exosomes; (d) total protein amount of exosomes; or (e) a combination thereof.

In one embodiment of the present invention, the above indicators can be measured to determine whether the functional stability of exosomes is maintained during long-term storage after freeze-drying.

According to an embodiment of the present invention, the functional stability of the exosomes can be determined by measuring the degree to which the values of the indicators are maintained compared to the initial values when the exosomes are stored for a long period of time.

Specifically, the evaluation of the functional stability of the exosomes can be made by observing that when the exosomes are stored for a long period of time, the indicator (a) is maintained at a level similar to the initial value or shows a low level of increase compared to the initial value, and the indicator (a) This can be achieved through b) to (e) being maintained at a level similar to the initial value or showing a low level of decrease compared to the initial value.

In one embodiment of the present invention, the composition of the present invention inhibits the increase in size of the exosomes.

In another embodiment of the present invention, the composition of the present invention inhibits a decrease in the concentration, enzyme activity ability, or total protein amount of the exosomes.

More specifically, the functional stability of the exosome may be that when the exosome is stored at 4° C. for 6 months to 1 year, the indicator (a) increases to within 15% of the initial value, for example, 15 %, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or 0%, or when exosomes are stored at 40°C for 15 to 30 days. , the indicator may be increased within 10% of the initial value, for example, within 9%, within 7%, within 5%, within 3%, within 1%, or 0%.

In addition, the stability of the exosomes may be such that when the exosomes are stored at 4°C for 6 months to 1 year, the indicators (b) to (e) are reduced to within 20% of the initial value, for example may be within 19%, within 17%, within 15%, within 13%, within 11%, within 9%, within 7%, within 5%, within 3%, within 1%, or within 0%, or exosomes When stored at 40°C for 15 to 30 days, the indicators (b) to (e) may be reduced to within 30% of the initial value, for example, within 28%, within 25%, and within 23%. It may be within, within 20%, within 18%, within 15%, within 13%, within 10%, within 8%, within 5%, within 3%, within 1%, or 0%.

Meanwhile, in the examples of the present invention, the present inventors also compared indicators for evaluating the functional stability of exosomes using a composition that did not contain a biocompatible polymer or contained a general freeze-drying protective agent, such as trehalose, as a control group. analyzed. As a result, compared to the control group, the values of the indicators were maintained at a similar level to the initial value in the experimental group using the composition of the present invention, or showed a lower increase or decrease, thereby demonstrating the functional stability of the exosome according to the composition of the present invention. It was confirmed that this was maintained effectively.

Therefore, the composition containing the biocompatible polymer of the present invention can maintain high exosome stability compared to the composition not containing the biocompatible polymer.

By using a composition for cryoprotection of exosomes containing the biocompatible polymer of the present invention, the structural stability of exosomes can be maintained during the freezing process, and the functional stability of exosomes can be maintained during long-term storage after the freezing process. there is.

Figure 1 is a schematic diagram showing that the exosome-hyaluronic acid composition of the present invention prevents membrane damage and maintains stability between exosomes during freezing through specific binding between exosomes and hyaluronic acid polymers.

Figure 2 shows the results of analyzing the specific binding force between exosomes and hyaluronic acid polymer by CD44 receptor-ligand.

Figure 3 shows the results of measuring the change in concentration of exosomes according to the hyaluronic acid content in the exosome-hyaluronic acid composition under accelerated conditions (40°C/30 days).

Figure 4 shows changes in (a) size, (b) concentration, (c) enzyme activity, and (d) protein amount of exosomes in the exosome-hyaluronic acid composition under accelerated conditions (40°C/30 days). Shows the results.

Figure 5 shows changes in (a) size, (b) concentration, (c) enzyme activity, and (d) protein amount of exosomes in the exosome-hyaluronic acid composition under real-time conditions (4°C/30 days). Shows the results.

Figure 6 shows a transmission scanning microscope image for morphological analysis of exosomes according to exosome composition.

Figure 7a shows the results of evaluating the cell migration ability of exosomes due to specific binding of exosomes and hyaluronic acid when storing exosomes.

Figure 7b shows the results of quantitative evaluation of Figure 7a.

Figure 8a shows the results of evaluating the ability to maintain the concentration of exosomes by exosome-hyaluronic acid specific binding during exosome storage.

Figure 8b shows the results of evaluating the ability to maintain the size of exosomes by exosome-hyaluronic acid specific binding during exosome storage.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example

Preparation Example 1. Extraction of exosomes effective in collagen production from human adipose stem cells

First, exosomes derived from human adipose stem cells were extracted during the process of culturing human adipose stem cells.

Preparation example 1.1. Culture of human adipose stem cells

Specifically, human adipose stem cells were cultured in general culture medium (Gibco, Cat#: 11995065), and 24 hours before exosome extraction, they were cultured in serum-free, antibiotic-free, phenol red-free medium (Gibco, Cat#: 31053028). It was replaced and cultured for 24 hours.

Preparation example 1.2. Isolation and purification of exosomes

The cell culture supernatant was recovered and centrifuged primarily at 2,000 xg for 4 to 5 minutes and secondarily centrifuged at 10,000 xg for 4 to 30 minutes to remove cell debris and waste. Then, the recovered cell culture supernatant was first centrifuged at 3,000 Afterwards, exosomes were separated and purified by filtering the filtered cell culture supernatant using a tangential flow filtration (TFF) system using a 300 kDa filter.

Example 1. Verification of specific binding force between exosomes and hyaluronic acid polymer by CD44 receptor-ligand action

Using bio-layer interferonmetry, the specific binding force between the CD44 receptor on the surface of exosomes and the CD44 ligand on the hyaluronic acid polymer chain was verified.

First, we wanted to confirm whether specific binding exists between exosomes and hyaluronic acid polymer. Accordingly, after hyaluronic acid polymer (MW: 1,000 kDa) was conjugated to the biosensor substrate, the protein concentration of exosomes extracted from human adipose stem cells in Preparation Example 1 (0.2, 1, and 2 mg/mL) was conjugated to the biosensor substrate. By flowing the sample onto the biosensor substrate, the binding force between hyaluronic acid polymer and exosomes was measured based on the signal displayed on the biomolecule reaction analyzer.

The results are shown in Figure 2a.

As shown in Figure 2 (a), when exosomes of various concentrations were flowed onto a biosensor substrate conjugated with a hyaluronic acid polymer, it was confirmed that as the concentration of exosomes increased, the amount of binding to the hyaluronic acid polymer also increased, thereby establishing a relationship between the two substances. It was confirmed that specific binding existed.

Next, in order to verify whether there was actually a difference in the binding ability with hyaluronic acid polymer when blocking the CD44 receptor present on the surface of exosomes, the binding ability with hyaluronic acid was compared using exosomes reacted with CD44 antibody.

Exosomes reacted with CD44 antibodies, that is, exosomes with the CD44 receptor blocked, were prepared by mixing 1 mL of exosome solution ( ^1.5x10 particles) with 10 ㎕ of anti-CD44 antibody solution and reacting at 4°C for 3 hours. The binding force between exosomes and hyaluronic acid was measured using the same method as above.

The results are shown in Figure 2b.

As shown in Figure 2b, the binding force of regular exosomes to hyaluronic acid polymer was measured to be more than two times higher than that of exosomes whose receptors were blocked by CD44 antibody. Through this, it was confirmed that the presence or absence of the CD44 receptor on the surface of the exosome actually had a significant effect on specific binding to the hyaluronic acid polymer.

Example 2. Optimization of hyaluronic acid content in composition for maintaining exosome concentration

In order to determine the optimal content of hyaluronic acid in the composition for maintaining exosome concentration, freeze-dried formulation samples of compositions for each hyaluronic acid content were analyzed using a nanoparticle tracking analysis system (NanoSight LM10; Malvern Instruments, Malvern) for 9 days under accelerated conditions at 40°C. , UK) was used to quantitatively analyze the change in exosome concentration according to the hyaluronic acid content in the composition.

First, hyaluronic acid (MW: 1,000 kDa) polymer was dissolved step by step in 0.25 mL of 10 mM phosphate buffer solution by content using a centrifugal mixer for co-viscosity (step 1: 0 wt) %(w/v), Step 2: 0.005 wt%(w/v), Step 3: 0.01 wt%(w/v), Step 4: 0.05 wt%(w/v), Step 5: 0.1 wt%( w/v), step 6: 0.5 wt% (w/v)), mixed with 0.25 mL of exosome solution (1 x 10 ⁹ particles) and lyophilized for 24 hours. As a control, a sample dissolved in the same amount of 10 mM phosphate buffer solution containing 0.5 wt% (w/v) of trehalose and then freeze-dried was used.

Then, for nanoparticle tracking analysis, the freeze-dried sample was dissolved in 0.5 mL of 10 mM phosphate buffer solution, then 0.25 mL of 400 U/mL hyaluronidase solution was added, and then incubated for 10 minutes at room temperature. It was reacted for several minutes and used as a sample solution for nanoparticle tracking analysis.

The results are shown in Figure 3.

As shown in Figure 3, after 9 days of storage of the composition, the exosome concentration of the stage 4, 5, and 6 samples remained similar to the initial concentration on day 0, whereas the samples containing the trehalose lyophilization protectant and 2 In samples below this stage, it was confirmed that the exosome concentration was reduced by more than 30%.

Experimental Example 1. Evaluation of maintaining exosome stability in the exosome-hyaluronic acid composition

Experimental Example 1.1. Evaluation of maintenance of exosome stability in exosome-hyaluronic acid composition under long-term accelerated conditions

In this example, the hyaluronic acid content (step 4, 0.05 wt%) optimized in Example 2 was used under 'accelerated conditions for 30 days at 40℃', which corresponds to 'real-time conditions for 1 year at 4℃'. An attempt was made to evaluate the effect of maintaining exosome stability according to the present invention using an exosome-hyaluronic acid composition containing it.

As a negative control, a freeze-dried sample of the DPBS composition was used, a composition containing trehalose (0.05 wt%, Tre), a composition containing hyaluronic acid (0.05 wt%, HA), and a composition containing hyaluronic acid (0.05 wt%) and trehalose (0.05 wt%). wt%), samples of the freeze-dried formulation of the composition (HA+Tre) were used as experimental groups, respectively.

Experimental Example 1.1.1. Evaluation of maintenance of exosome concentration in exosome-hyaluronic acid composition

The exosome concentration in the exosome-hyaluronic acid composition was measured using a nanoparticle tracking analysis system (NanoSight LM10; Malvern Instruments, Malvern, UK), and the efficiency of maintaining the concentration of exosomes in the composition was determined by the decrease in exosome concentration compared to the initial concentration. It was evaluated through change in concentration.

The results are shown in Figure 4a.

As shown in Figure 4 a, after 30 days of storage, the 4th stage hyaluronic acid-containing sample (HA) of the present invention showed a 35% decrease compared to the initial exosome concentration, which was compared to the sample containing nothing other than exosomes ( The concentration maintenance efficiency was 2.3 times higher than that of DPBS and 1.8 times higher than that of the trehalose-containing sample (Tre).

On the other hand, in the sample containing both hyaluronic acid and trehalose (HA+Tre), the exosome concentration showed no significant difference from the sample containing hyaluronic acid (HA).

From the above results, it was found that additionally adding trehalose to hyaluronic acid did not have a significant effect on maintaining the concentration of exosomes.

Experimental Example 1.1.2. Evaluation of exosome size maintenance in exosome-hyaluronic acid composition

The size of exosomes in the exosome-hyaluronic acid composition was measured using a nanoparticle tracking analysis system (NanoSight LM10; Malvern Instruments, Malvern, UK), and the efficiency of maintaining the size of exosomes in the composition was determined by the size of the exosomes decreasing compared to the initial level. Evaluated through changes.

The results are shown in Figure 4c.

As shown in Figure 4c, after 30 days of storage, compared to the size of exosomes on day 0, the sample containing nothing except exosomes (DPBS) decreased by 23%, and the sample containing trehalose (Tre) decreased by 23%. It was confirmed that the size increased by about 15%. On the other hand, it was confirmed that the hyaluronic acid-containing sample (HA) and the sample containing both hyaluronic acid and trehalose (HA+Tre) showed the best maintenance ability in terms of size change by showing an increase of less than 5%.

Experimental Example 1.1.3. Evaluation of maintenance of exosome enzyme activity ability in exosome-hyaluronic acid composition

The concentration of exosomal acetylcholinesterase (AchE) was measured using the Amplite Colorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, CA, US). Enzyme activity was evaluated. By measuring the change in the enzyme activity ability of the exosomes, which decreases compared to the initial level of the exosomes in the exosome-hyaluronic acid composition, it is possible to evaluate whether the composition can maintain the enzyme activity ability of the exosomes.

The results are shown in Figure 4c.

As shown in Figure 4c, after 30 days of storage, it was confirmed that the exosome enzyme activity ability decreased by 14% in the hyaluronic acid-containing sample (HA, HA+Tre) of the present invention compared to the initial day 0, which is The maintenance efficiency of enzyme activity was 2.1 times higher than that of the sample without moths (DPBS) and 1.6 times higher than that of the sample containing trehalose (Tre). From the above results, it was found that the hyaluronic acid-containing composition of the present invention is very useful not only for maintaining the concentration of exosomes, but also for maintaining the enzymatic activity ability of exosomes.

Experimental Example 1.1.4. Evaluation of maintenance of total exosome protein amount in exosome-hyaluronic acid composition

By measuring the change in the total amount of protein of exosomes in the exosome-hyaluronic acid composition through protein quantification (BCA assay), the efficiency of maintaining the total amount of protein of exosomes in the composition was evaluated.

The results are shown in Figure 4d.

As shown in Figure 4 d, after 30 days of storage, it was confirmed that the amount of protein in exosomes decreased by 15% in the hyaluronic acid-containing samples (HA, HA+Tre) of the present invention compared to the initial day 0, which is The efficiency of maintaining the total protein amount in exosomes is 1.7 times higher than that of the sample without trehalose (DPBS) and 1.6 times higher than that of the sample containing trehalose (Tre).

From the above results, it was found that the hyaluronic acid-containing composition of the present invention was very effective in maintaining the total protein amount of exosomes even under long-term accelerated conditions.

Experimental Example 1.2. Evaluation of maintenance of exosome stability in exosome-hyaluronic acid composition under long-term real-time conditions

In this example, under 'real-time conditions at 4°C for 30 days', the exosome-hyaluronic acid composition containing the optimized hyaluronic acid content (step 4, 0.05 wt%) in Example 2 was used in the present invention. We sought to evaluate the effect of maintaining exosome stability.

As a negative control, a freeze-dried sample of the DPBS composition was used, and a trehalose-containing (0.05 wt%) composition (Tre), a hyaluronic acid-containing (0.05 wt%) composition, hyaluronic acid (0.05 wt%), and trehalose (0.05 wt%) were used. ) Freeze-dried formulation samples of the composition (HA+Tre) containing both were used as experimental groups.

Experimental Example 1.2.1. Evaluation of maintenance of exosome concentration in exosome-hyaluronic acid composition

The concentration of exosomes in the exosome-hyaluronic acid composition was measured using a nanoparticle tracking analysis system (NanoSight LM10; Mlavern Instruments, Malvern, UK).

The results are shown in Figure 5a.

As shown in Figure 5 a, after 30 days of storage, the hyaluronic acid-containing sample (HA) of the present invention showed a 20% decrease compared to the initial exosome concentration, which was compared to the sample containing nothing except exosomes (DPBS). The concentration maintenance efficiency was 2.1 times higher than that of the trehalose-containing sample (Tre) and 1.7 times higher than that of the trehalose-containing sample (Tre).

From the above results, it was found that the hyaluronic acid-containing composition of the present invention is very useful in stably maintaining exosomes under long-term real-time conditions.

Experimental Example 1.2.2. Evaluation of exosome size maintenance in exosome-hyaluronic acid composition

The size of exosomes in the exosome-hyaluronic acid composition was measured using a nanoparticle tracking analysis system (NanoSight LM10; Malvern Instruments, Malvern, UK).

The results are shown in Figure 5c.

As shown in Figure 5c, compared to the initial day 0, the hyaluronic acid-containing sample (HA) showed a size increase of 6% after 30 days of storage, while the sample without exosomes (DPBS) and trehalose In the case of the containing sample (Tre), increases were confirmed by 15% and 13%, respectively.

From the above results, it was found that the hyaluronic acid-containing composition of the present invention is useful in stably maintaining the size of exosomes under long-term real-time conditions.

Experimental Example 1.2.3. Evaluation of maintenance of exosome enzyme activity ability in exosome-hyaluronic acid composition

The enzymatic activity of exosomes in the composition was measured using an acetylcholine degrading enzyme assay.

The results are shown in Figure 5c.

As shown in Figure 5c, after 30 days of storage, the enzymatic activity ability of exosomes of the hyaluronic acid-containing sample (HA, HA+Tre) and trehalose-containing sample (Tre) of the present invention is almost compared to the initial day 0. It was confirmed that no difference was maintained. On the other hand, the sample containing no exosomes (DPBS) showed the enzyme activity of exosomes reduced by 8% compared to the initial day 0.

From the above results, it can be seen that the hyaluronic acid-containing composition of the present invention is very useful not only for maintaining the concentration of exosomes under long-term real-time conditions, but also for maintaining the enzymatic activity ability of exosomes. there was.

Experimental Example 1.2.4. Evaluation of maintenance of total exosome protein amount in exosome-hyaluronic acid composition

The total amount of protein of exosomes in the composition was measured using protein quantification (BCA assay). The results are shown in Figure 5d.

As shown in Figure 5 d, under real-time conditions, there was almost no change in the total protein amount of exosomes in the hyaluronic acid-containing samples (HA, HA+Tre), trehalose-containing samples (Tre), and DPBS samples on the 30th day of storage. confirmed.

From the above results, it was found that the hyaluronic acid-containing composition of the present invention is very effective in maintaining the total protein amount of exosomes even under long-term real-time conditions.

Experimental Example 2. Morphological analysis of exosomes according to exosome-hyaluronic acid composition under long-term accelerated conditions

In order to perform morphological analysis of exosomes according to the exosome-hyaluronic acid composition under long-term acceleration conditions, the same freeze-dried formulation of each composition used in Experimental Example 1 was used to analyze exosomes according to each composition through a transmission scanning microscope. Changes in the shape of the silverfish were compared and analyzed. Specifically, the freeze-dried formulation of each composition was stored for 30 days under accelerated conditions at 40°C, and then the change in the shape of exosomes according to each composition was compared with the spherical exosome shape on day 0.

The results are shown in Figure 6.

As shown in Figure 6, the exosome morphology of the hyaluronic acid-free sample (DPBS) and the trehalose-containing sample (Tre) shows a distorted, irregular shape in the form of a spherical endoplasmic reticulum, while the hyaluronic acid-containing sample of the present invention (HA, It was confirmed that the exosomes (HA+Tre) maintained a spherical shape similar to the exosomes on day 0. In addition, in terms of aggregation between exosomes, it was confirmed that less aggregation between exosomes occurred in the hyaluronic acid-containing sample (HA) compared to the control samples.

From the above results, it was found that the hyaluronic acid-containing composition of the present invention was very effective in maintaining not only the function but also the shape of exosomes even under long-term accelerated conditions.

Experimental Example 3. Evaluation of maintenance of exosome stability according to the presence or absence of CD44 receptor in exosomes during long-term storage of exosomes

In this experimental example, the purpose was to confirm whether maintaining the stability of exosomes during long-term storage of exosomes according to the exosome-hyaluronic acid composition confirmed in Experimental Examples 1 and 2 is an effect caused by CD44-specific binding between hyaluronic acid and exosomes. did.

A lyophilized formulation sample of the DPBS composition was used as a negative control group, and a lyophilized formulation sample of a trehalose-containing (0.05 wt%, Tre) composition and a hyaluronic acid-containing (0.05 wt%, HA) composition was used as an experimental group, respectively. Additionally, , the CD44 receptor-blocked exosome prepared in Example 1 was applied and used as a negative control for each experimental group.

Experimental Example 3.1. Evaluation of cell migration ability of exosomes by CD44-specific binding between hyaluronic acid and exosomes

First, cell migration ability was evaluated to confirm the maintenance of exosome stability by specific binding to hyaluronic acid polymer during long-term storage of exosomes, that is, the ability to maintain the functional properties of exosomes.

To evaluate the cell migration ability of exosomes, a wound healing assay using cells was performed. After scraping the central area of the dish where fibroblasts were cultured, cells were treated under accelerated conditions (40°C on day 0 and day 10 of normal exosome composition and CD44 receptor-blocked exosome composition, respectively, after 40 hours) The difference in width of the occupied space was confirmed through a microscope.

The results are shown in Figure 7.

As shown in Figure 7a, when fibroblasts were treated with a general exosome composition on day 0 of storage under accelerated conditions at 40°C, there was significant cell migration ability between compositions and cell migration ability by the presence or absence of CD44 receptor blocking of exosomes. It was confirmed that there was no difference.

However, when the exosome composition was processed on the 10th day of storage under accelerated conditions at 40°C, in the case of a general exosome composition with intact CD44 receptors, the hyaluronic acid-containing sample (HA/Exo) was changed to the hyaluronic acid-free sample (DPBS/Exo). ), the cell migration efficiency was 1.5 times higher than that of the trehalose sample (Tre/Exo), and 1.4 times higher than that of the hyaluronic acid-containing CD44 receptor-blocked exosome sample (HA/b-Exo). It showed the efficiency of movement ability (Figure 7b).

As a result, it was confirmed that the specific binding between the CD44 receptor expressed in exosomes and hyaluronic acid has an effective effect on the cell migration ability of exosomes.

Experimental Example 3.2. Evaluation of changes in concentration and size of exosomes due to CD44-specific binding between hyaluronic acid and exosomes

Next, in order to confirm the maintenance of exosome stability by specific binding to hyaluronic acid polymer during long-term storage of exosomes, that is, the ability to maintain the physical properties of exosomes, changes in concentration and size of exosomes were evaluated.

Specifically, to evaluate changes in the concentration and size of exosomes, an antigen-antibody reaction using an anti-CD44 antibody was performed under accelerated conditions (hyaluronic acid-free treatment group (DPBS) for 10 days at 40°C, trehalose treatment group ( Tre), and changes in the concentration and size of exosomes in the hyaluronic acid treatment group (HA) were measured, wherein the experimental and comparison groups were regular exosomes and exosomes that blocked the CD44 receptor that specifically binds to hyaluronic acid, respectively. was used.

The results are shown in Figure 8.

As shown in Figure 8a, in the case of existing exosomes (EXO) expressing CD44 on the 10th day of storage, the concentration of exosomes decreased by 6% in the hyaluronic acid treatment group (HA) compared to the initial day 0, but hyaluronic acid It was confirmed that the concentration group (DPBS) and the trehalose-treated group (Tre) decreased by 52% and 24%, respectively.

However, in the case of exosomes (b-EXO) in which the CD44 receptor was blocked, it was confirmed that the exosome concentration in the DPBS group and the trehalose treatment group showed a similar decrease in concentration as that of exosomes expressing CD44, but hyaluronic acid treatment In group (HA), it was confirmed that the concentration decreased by 35%.

Meanwhile, as shown in Figure 8b, on the 10th day of storage, the size of exosomes in the DPBS group and the trehalose treated group was confirmed to increase by 16% and 8%, respectively, which is similar to the results in Figure 8a. The size of exosomes in the group and the trehalose-treated group was maintained at a similar level regardless of the presence or absence of CD44 receptor blocking.

However, in the case of the hyaluronic acid treatment group (HA) for normal exosomes (EXO) with intact CD44 receptors, the size of exosomes increased by 4%, whereas for exosomes with CD44 receptors blocked (b-EXO), the size of exosomes increased by 4%. It showed a size increase of 12%.

As a result, it was confirmed that the specific binding between the CD44 receptor expressed in exosomes and hyaluronic acid has an effective effect on maintaining the concentration and size of exosomes.

In summary, from the above results, the maintenance of functional/structural stability of exosomes according to the exosome-hyaluronic acid composition during long-term storage of exosomes of the present invention is an effect caused by CD44 receptor-ligand binding between exosomes and hyaluronic acid. I was able to confirm.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention.

Claims (13)

1. A composition for cryoprotection of exosomes, comprising a biocompatible polymer having a network structure.
2. The method of claim 1, wherein the biocompatible polymer is hyaluronic acid, collagen, alginate, starch, chitosan, gelatin, dextran, and cellulose. A composition for cryoprotection of exosomes, selected from the group consisting of cellulose, alginic acid, chondroitin sulfate, and heparin.
3. The composition for cryoprotection of exosomes according to claim 2, wherein the biocompatible polymer is hyaluronic acid.
4. The composition for cryoprotection of exosomes according to claim 3, wherein the hyaluronic acid binds to the CD44 receptor of exosomes.
5. The composition for cryoprotection of exosomes according to claim 1, wherein the exosomes are stem cell-derived exosomes.
6. The method of claim 5, wherein the stem cells are mesenchymal stem cells derived from one or more types of tissues selected from the group consisting of umbilical cord, cord blood, blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. cell), a composition for cryoprotection of exosomes.
7. The composition for cryoprotection of exosomes according to claim 6, wherein the stem cells are adipose-derived stem cells (ASCs).
8. The composition for cryoprotection of exosomes according to claim 1, wherein the composition contains 0.01 to 1.0 wt% (w/v) of hyaluronic acid polymer.
9. The composition for cryoprotection of exosomes according to claim 1, wherein the composition includes 1 x 10 ⁹ to 1 x 10 ¹² exosomes per 1 mL of the total composition.
10. The composition for cryoprotection of exosomes according to claim 1, wherein the composition inhibits the decrease in stability of exosomes during the freezing process compared to a composition not containing a biocompatible polymer.
11. The composition for cryoprotection of exosomes according to claim 10, wherein the stability of the exosomes is measured based on the following indicators:

    (a) Size of exosomes;

    (b) concentration of exosomes;

    (c) Enzyme activity ability of exosomes;

    (d) total protein amount of exosomes; or

    (e) Combination of these.
12. The method of claim 11, wherein the composition:

    (i) When stored at 4°C for 6 months to 1 year, the indicator in (a) is inhibited to increase within 15% of the initial value, or

    (ii) A composition for cryoprotection of exosomes, which inhibits the indicator of (a) from increasing to within 10% of the initial value when stored at 40°C for 15 to 30 days.
13. The method of claim 11, wherein the composition:

    (i) When stored at 4°C for 6 months to 1 year, the indicators (b) to (e) are inhibited to decrease within 20% of the initial value, or

    (ii) A composition for cryoprotection of exosomes, which inhibits the indicators of (b) to (e) from being reduced to within 30% of the initial value when stored at 40°C for 15 to 30 days.

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