KR101605528B1 - Novel hydrogel based on hyaluronic acid and use thereof - Google Patents

Abstract

The present invention relates to a hyaluronic acid-based hydrogel which comprises a cell differentiation agent or a cell activity inducing substance and is prepared using cucurbituril and polyamine, a composition for assisting cell therapy including the hydrogel, a cell differentiation agent or a cell activity inducer And a mesenchymal stem cell. Another object of the present invention is to provide a composition for cell therapy, which comprises a hyaluronic acid-based hydrogel prepared by using cucurbituril and polyamine, and a hydrogel And a mesenchymal stem cell. The hyaluronic acid-based hydrogel provided by the present invention acts as an adjuvant for supporting stem cells expressing a therapeutic substance used as a cell therapy agent, thereby promoting the secretion of the therapeutic substance and increasing the cell survival rate, The therapeutic effect can be maximized, and thus it can be widely used for the development of a cell therapeutic agent exhibiting improved efficacy.

Description

Novel hyaluronic acid-based hydrogels and their uses < RTI ID = 0.0 >

The present invention relates to a novel hyaluronic acid-based hydrogel and a use thereof, and more particularly, to a hyaluronic acid-based hydrogel prepared by using a cell differentiation agent or a cell activity inducing substance and using cucurbituril and polyamine, A cell therapy composition comprising a hydrogel, a composition for cell therapy comprising a hydrogel and a stem cell comprising the cell differentiation agent or a cell activity inducer, a hyaluronic acid-based hydrogel prepared using cucurbituril and polyamine, And a composition for cell therapy comprising hyaluronic acid-based hydrogel and stem cells prepared using cucurbiturils and polyamines.

Modern cell therapy research was initiated in 1958 by French scientists to develop a blood typing system called the HLA system and in 1973 the success of a real bone marrow transplant. In 1999, Pittinger revealed that mesenchymal stem cells present in bone marrow and adipose differentiate into various tissues, and research on cell therapy using stem cells has begun in earnest. However, when cells are used alone, it requires about 50 million to 5 billion cells to show efficacy because of its rapid clearance in the body. In case of using only cells, a support for increasing the residence time in the body Is a useful method.

Recently, various biomaterials have been developed for three-dimensional cell culture and efficient cell delivery. Among them, the most effective substances meeting this purpose have come to be regarded as hydrogels and various studies are under way. A hydrogel is a water-soluble polymer having a network structure and has a high water content without dissolving in water and has a similar structure to a tissue. Hydrogels can induce gel form by inducing covalent bonds between functional groups using highly reactive chemicals. In recent years, there has been a growing interest in injection-type hydrogels, which can fill drugs or cells without denaturation, and which can be injected into the human body with minimal scarring. In order to develop the injection type hydrogel, it is necessary to use enzymes or chemical substances, chemical reaction by light, external stimulation such as temperature or pH change, physical change by solvent change and swelling Is being attempted.

Among the representative methods using chemical reactions, Michael addition, which is a reaction between a thiol and a double bond, is used as a method of using a highly reactive chemical substance. There is PEGDA using a photopolymerization method formed by polymerizing a polymer having a reactor capable of polymerizing with commercially available Hystem-C with a light initiator. Hystem has a merit that it can form hydrogel by mixing thiol group with hyaluronic acid and simple mixing with PEGDA. It is composed of PEG derivative. When photoinitiator is added to PEGDA and ultraviolet light is injected, hydrogel There is an advantage that it can be formed. However, the photopolymerization method can cause the reaction by using the initiator. However, since initiators that have been commercialized so far have mostly cytotoxic effects and ultraviolet rays for photopolymerization are difficult to pass through the skin, It is difficult to use. In addition, PEGDA, which is commonly used in both products, has a disadvantage in that the double bond which is not involved in gel formation is potentially toxic when introduced into the body because it uses a double bond having a good reactivity.

In addition, there is CorgelTM using an enzyme which induces a chemical reaction by inducing reactive oxygen such as horseradish peroxidase, and Gelite which uses an ion reaction in which anionic polymeric substance forms a chelate by using an electrostatic attraction Is commercially available. However, the enzyme-based method is more likely to be an enzyme-mediated immune rejection reaction than the method using the native enzyme in vivo, since most of the enzymes such as horseradish peroxidase are injected before the injection. Since the method using ionic reaction is hard to maintain the state of gel in the living body for a long time because the hardness of the gel depends on the concentration of the cation, it is currently used mainly in product development research for drug delivery.

One of the ways in which hydrogels are formed by external stimuli is to utilize a temperature-sensitive system, and there is a method in which a triple block copolymer of ReGel® and PEO-PPO-PEO using a triple block copolymer of PLGA-PEG-PLGA And Matrigel (TM) using collagen derived from sarcoma cells of Engelbreth-Holm-Swarm (EHS) rats. In the temperature-sensitive system, the critical solution temperature (LCST) phenomenon is shown in detail. When the temperature is low, it is present as a liquid. When the temperature is 37oC, it becomes a solid form. It is stored at a low temperature in a liquid state and then injected into a living body when necessary to make a gel, thereby facilitating drug delivery and cell delivery. In particular, MatrigelTM has various cell growth factors, which can prolong cell survival time. However, since the temperature-sensitive injectable hydrogel forms a network structure due to the physical bonding that forms micelles, it has a disadvantage that it can not stably maintain in vivo for a long time because it has a large amount of early release and a rapid decomposition rate in vivo. In the case of Regel®, the degradation product is partially cytotoxic due to the use of a synthetic polymer. In the case of Matrigel (TM), it is a substance derived from a cancer cell of a mouse.

As a pH-sensitive system, Polyacrylic Acid-based Carbopol® product is commercialized. In this product, when the pH is 4, the viscosity is low and it is in a solution state. When the pH is high, the ionized acrylic acid group is gradually neutralized and becomes hydrophobic Physical binding due to hydrophobic reaction occurs, resulting in increased viscosity and solidification at pH 7.4, which is the physiological activity. Therefore, when injected in a liquid state, it is easy to reach the physiologically active pH of 7.4, which is advantageous in that the gel can be made simple. However, in order to inject the gel, it must be used in an acidic pH of 4, which may damage the cells during administration.

As described above, the injection-type hydrogel preparation method using a chemical reaction has a disadvantage in that it can cause serious damage to the supported cells due to the good chemically reactive substance. Therefore, the hydrogel does not introduce the functional group into the polymer, Many attempts have been made to form, and products such as ReGel (R), Matrigel (TM), and Pluronic (R) have been commercialized. However, since these products are used as a cell therapy agent in vivo, they have a weak point as described above, and are stable in the living body for a suitable period of time, and have a safe biocompatibility, biodegradability, Development of hydrogels is required.

Under these circumstances, the inventors of the present invention have found that a novel hyaluronic acid-based hydrogel prepared using a cell differentiation agent or a cell activity inducing substance and using cucurbituril and polyamine is useful as a support capable of supporting the cell therapy effect of stem cells And completed the present invention.
[Prior Art Literature Information]
1. ACS NANO, 6 (4), p.2960-2968 (March 12, 2012)
2. Korean Patent Registration No. 10-1201473 (November 15, 2012)
3. Korean Patent Publication No. 10-2007-0073225 (2007.07.10)

One object of the present invention is to provide a hyaluronic acid-based hydrogel comprising a cell differentiation agent or a cell activity inducing substance and produced using cucurbituril and polyamine.

It is another object of the present invention to provide a cell therapy aid composition comprising the hydrogel.

It is still another object of the present invention to provide a composition for cell therapy comprising a hydrogel and a stem cell comprising the cell differentiation agent or a cell activity inducing substance.

Another object of the present invention is to provide a composition for assisting cell therapy comprising hyaluronic acid-based hydrogel prepared using cucurbituril and polyamine.

It is still another object of the present invention to provide a composition for cell therapy comprising the hydrogel and stem cells.

In an embodiment of the present invention, there is provided a hyaluronic acid-based hydrogel comprising a cell differentiation agent or a cell activity inducing substance and produced using cucurbituril and polyamine.

The term "cell differentiation agent" of the present invention means a substance capable of promoting cell differentiation of stem cells. Generally, a retinoid (ATRA (all-trans-retinoic acid) , Vitamin A and its derivatives including PtA (Pantothenic Acid), vitamins and their derivatives such as vitamin D and its derivatives, EGF (Epidermal Growth Factor), and dexamethasone (Dexa) And various growth factors such as bone morphogenetic protein (BMP), and preferably VEGF (Vascular Endothelial Growth Factor), HGF (Hepatocyte Growth Factor) or PDGF (Platelet Growth Factor) derived growth factor, Brain Derived Neurotropic Factor (BDNF) or ATRA to promote neural differentiation, Dexa, Cyclic-RGD or BMP to promote bone differentiation, ATRA, EGF, Panthenol, PtA, etc., and more preferably ATRA, Dexa, etc., but is not particularly limited thereto.

The term "cell activity inducing substance" of the present invention means a substance that expresses a molecule that induces a specific activity or enhances a specific gene by inducing gene expression of a cell. The term " Retinoids (ATRA) Glucocorticoids (generic name for steroid hormones), Cholesterol, Vitamin D and its derivatives, Tyroid hormone, Trichostatin A (TSA) and its induction, including Dexamethasone (Dexa) A hormone or a ligand capable of binding to a Nucleic Receptor or a promoter such as a substance, Fatty Acids, Prostaglandin, or Sterol, and is preferably ATRA, glucocorticoid, tricostatin (Trichostatin (A)), sterol, and the like, but are not particularly limited thereto.

The term "Cucurbituril, CB [n]" of the present invention means an amber hollow molecule in which n glycourols are gathered, wherein n is not particularly limited, but is preferably 1 to 10,000 More preferably 5, 6, 7, 8 or 10, most preferably 6. CB [n] can interact with various guest molecules to form a complex by utilizing the internal hydrophobic space. The guest molecules include, but are not limited to, ionic or polar materials And preferably a compound having a functional group such as an amino group or a carboxylic acid, and more preferably a compound having various organic substances such as a gaseous compound, an aliphatic compound and an aromatic compound, a pesticide, a herbicide, an amino acid, Metal ions, organometallic ions and the like, and most preferably polyamine compounds.

The term "polyamine" of the present invention means a biogenic amine widely present in the biological system and refers to an aliphatic hydrocarbon having two or more primary amino groups. The polyamines are present in many tissues in which protein and nucleic acid synthesis are active. It is especially useful as a tumor marker because it is present in many cancer tissues and also in blood or urine. The physiological actions of polyamines include stabilization of nucleic acids, promotion of nucleic acid and protein synthesis, post-translational modification (acetylation, phosphorylation) of proteins, activation of various enzymes, and stabilization of cell membranes. The polyamine is not particularly limited, but the polyamine is preferably a C1-C20 alkyl group having at least one amine group; A C2-C20 alkenyl group; A C2-C20 alkynyl group; A C1-C20 alkoxy group; A C1-C20 aminoalkyl group; A C4-C20 cycloalkyl group; A C4-C7 heteroarylcyclo group; A C6-C20 aryl group; A C5-C20 heteroaryl group; A C1-C20 alkylsilyl group; A substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or an adamantane having a C5-C20 heteroaryl group; A substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group, or a ferrocene or metallocene having a C5-C20 hetero aryl group; A carbene having a substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; A substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; A cycloalkyl or crown ether having a substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; An amino-protected amino acid having a substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; A substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; An alkaloid having a substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; A substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; An oligonucleotide having a substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; A substituted or unsubstituted C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group; And a nanoparticle having a C1-C20 alkyl group, a C6-C20 aryl group or a C5-C20 heteroaryl group, and more preferably a C1-C20 aminoalkyl group having at least one amine group, a C5-C20 A C1-C20 aminoalkyl group containing a metallocene, a C5-C20 heteroaryl group containing a metallocene, and the like, and most preferably, a cyano group such as ptresin, catavulin, spermidine, Perinum, diaminohexane (DAH), and the like, but are not particularly limited thereto.

The term "hyaluronic acid" of the present invention is a kind of mucopolysaccharide composed of amino acid and uronic acid, and is a polymer in which N-acetylglucosamine and glucuronic acid are alternately linked in a chain form and have a molecular weight of 200,000 to 400,000 Compound, similar to the pectin of a plant, hydrolyzed by hyaluronidase. In vivo, it exists in the vitreous body of the eye and the umbilical cord. It is highly viscous and plays a role in preventing invasion of bacteria and penetration of poison. For the purpose of the present invention, the hyaluronic acid is used as a basic skeleton for forming a hydrogel.

The term "hyaluronic acid-based hydrogel" of the present invention refers to a hyaluronic acid derivative to which a cucurbituril-coupled hyaluronic acid derivative and a polyamine are bonded, non-covalently linked by the specific hydrophobic / hydrophilic action of the cucurbituril and polyamine Means a hydrogel formed. Since the hydrogel does not use a chemically reactive reagent or an initiator having the possibility of causing toxicity, there is little possibility of inducing cytotoxicity, and the possibility of immune rejection is low because no external enzyme is used. It is possible to form a hydrogel irrespective of the influence of ions and to maintain the cells stably for a long time as a cell therapy agent because the host-guest molecular action is very strong. However, if necessary, the replacement ratio of hyaluronic acid can be controlled, Can be freely adapted to the application.

The term "hydrogel " of the present invention means a gel comprising water as a dispersion medium, wherein the hydrosol loses fluidity due to cooling, or the hydrophilic polymer having a three-dimensional network structure and a microcrystalline structure contains water, Or the like. The hydrogel of the electrolytic polymer is highly absorbent and is widely used as an absorbent polymer in various fields. In the hydrogel, the expansion ratio may change discontinuously due to the phase transition by temperature, pH, and the like.

According to one embodiment of the present invention, a hyaluronic acid derivative to which DAH is coupled, a hyaluronic acid derivative to which dexamethasone and MonoCB [6] are bound, and a hyaluronic acid derivative to which retinoic acid and MonoCB [6] (Fig. 3, Fig. 5, and Example 2-4). As a result of evaluating their toxicity in vivo, it was confirmed that they did not show any toxicity in vivo (Fig. 4), and the hyaluronic acid derivatives (Example 3-1).

In another embodiment, the present invention provides a cell therapy adjuvant composition comprising the hydrogel.

The present inventors have already developed a hyaluronic acid-based hydrogel and a method for producing the hyaluronic acid-based hydrogel using the interaction between the cucurbituril and the polyamine (Korean Patent Registration No. 1201473), and through the present invention, the hyaluronic acid- Was developed. Particularly, when the hyaluronic acid-based hydrogel is bound to a cell differentiation agent or a cell activity inducing substance, the activity of the mesenchymal stem cell, which is an active ingredient of the cell treatment agent, is enhanced by the bound cell differentiation agent or cell activity inducing substance Respectively.

According to one embodiment of the present invention, recombinant mesenchymal stem cells (MSCs) expressing the therapeutic gene Interleukin 12 (IL-12) mutant or BDNF (Brain Derived Neurotrophic Factor) 1). These were mixed with the hydrogel to evaluate their efficacy. As a result, they remained in the body after 50 days of using C / D-RD-HA hydrogel (Example 3-3) -DH-hydrogel showed higher IL-12 expression than C / D-HA hydrogel (FIG. 10), indicating that C / D-HA hydrogel was more effective than Matrixen or C / D- IL-12 hydrogel was used as a support, the expression level of IL-12 was high and the expression of IL-12 was detected up to 15 days (Fig. 11 (A)) and the mesenchymal stem (FIG. 12 (A)), and C / D-DR-H, a mesenchymal stem cell into which the therapeutic gene IL-12M was introduced A hydrogel MSC (Fig. 12 (B)), and it was confirmed that the anticancer activity remained the same even in repeated administration (Fig. 13).

In another embodiment, the present invention provides a composition for cell therapy comprising a hydrogel and a stem cell comprising the cell differentiation agent or a cell activity inducing substance.

It was confirmed that the hyaluronic acid-based hydrogel of the present invention maintains the cell-holding durability and promotes therapeutic gene expression and anti-cancer efficacy of the cell therapy agent. Therefore, not only the anti-cancer therapeutic agent but also the therapeutic gene for treatment of various intractable diseases Hyaluronic acid hydrogel was expected to be applied to the development of stem cell therapy. In addition, the hydrogel containing the cell differentiation agent / cell activity inducer not only sustained the expression time of the gene but also exhibited the enhanced therapeutic effect and confirmed its applicability as a cell therapy agent.

The term "stem cell" of the present invention means multipotent undifferentiated cells capable of differentiating into various types of cells including osteoblasts, chondroblasts, adipocytes and the like. For the purpose of the present invention, the stem cells may be mesenchymal stem cells, hematopoietic stem cells, neural crest stem cells, or stem cells derived from mouse, rat, dog, ), Endothelial stem cells and the like, or may be a natural stem cell such as a cell stem cell transplantation gene or an intrinsic cell therapy gene by overexpressing it, The stem cells may be transformed into genetically modified stem cells. The cytokine, the growth factor, the nutritional factor, and the like can be used as the component useful for the cell therapy to be expressed in the genetically modified stem cell, and preferably the interleukin-1, interleukin-6, interleukin- Growth factors such as EGF, BMP family, VEGF, FGF, HGF, TRAIL, HSV-TK, and other nutrients such as interferon gamma, cytokine such as tumor necrosis factor (TNF), and Brain Derived Neurotrophic Factor Etc. However, the present invention is not particularly limited thereto. In addition, the vector used for introducing the gene into the stem cell is not particularly limited, but adenovirus, naked DNA, poxvirus, lentivirus and the like can be used.

The composition for cell therapy provided in the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent depending on the administration mode. Specifically, it is possible to use a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and any of the above components, And other additives conventionally used, such as buffers. In addition, depending on the purpose of administration, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, A target organ or tissue specific antibody or other ligand can be used in combination with the carrier. The carrier, excipient, or additive may be any conventional formulation, and the carrier, excipient, or additive is not limited by the above examples.

The composition or the mixture may be appropriately administered to a subject according to the conventional method, route of administration, and dose used in the art depending on the purpose or necessity. Examples of the administration route include oral, non-oral, subcutaneous, Intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intramuscularly, intraperitoneally, Non-oral injections include intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, and intradermal administration. The amount and the frequency of administration of the composition for cell therapy of the present invention to be actually administered can be appropriately selected depending on the type of symptoms to be prevented or treated, , Health condition, diet, age and weight of the individual, and the severity of the disease.

In another embodiment, the present invention provides a cell therapy adjuvant composition comprising a hyaluronic acid-based hydrogel prepared using topical cucurbiturils and polyamines.

The present inventors confirmed that the hyaluronic acid-based hydrogel itself can enhance the activity of the mesenchymal stem cell, which is an active ingredient of the cell therapy agent, as compared with the conventional cell therapy agent.

According to one embodiment of the present invention, recombinant mesenchymal stem cells (MSCs) expressing the therapeutic gene Interleukin 12 (IL-12) mutant or BDNF (Brain Derived Neurotrophic Factor) 1). When they were mixed with the above-mentioned hydrogels, the efficacy was evaluated. As a result, Matrixen used as a conventional cell therapy agent was degraded within 10 days and Matrigel was used only in 3 weeks. However, C / D- (Example 3-3), and when C / D-HA hydrogel was used in vitro, it showed higher IL-12 expression compared to Matrigel (Fig. 10) In the case of using Matrixen as a support, the expression level of IL-12 was high when C / D-HA hydrogel was used as a support, and IL-12 expression was detected up to 15 days (FIG. 11 (A) Intermediate lobe into which the therapeutic gene IL-12M is introduced (FIG. 12 (A)), and the C / D-HA hydrogel MSC, which is a mesenchymal stem cell into which the therapeutic gene IL-12M was introduced, )), And it was confirmed that the anticancer activity remained the same at the time of repeated administration (Fig. 13).

In another embodiment, the present invention provides a composition for cell therapy comprising hyaluronic acid-based hydrogel and stem cells prepared using topical cucurbituril and polyamine.

The hyaluronic acid-based hydrogel provided by the present invention acts as an adjuvant for supporting stem cells expressing a therapeutic substance used as a cell therapy agent, thereby promoting the secretion of the therapeutic substance and increasing the cell survival rate, The therapeutic effect can be maximized, and thus it can be widely used for the development of a cell therapeutic agent exhibiting improved efficacy.

FIG. 1 (A) shows that host-guest non-covalent binding between CB [6] and DAH plays a role of cross-linking agent when CB [6] and DAH, which are the main molecule in mesenchymal stem cells, (B) shows the process of formation of a hydrogel (C / D-HA hydrogel). (B) shows a process for producing a therapeutic agent or cell differentiation element combined with hyaluronic acid and mesenchymal stem cells (MSC) It is a schematic diagram of stem cell (Engineered MSC) and its action. Tag1, Tag2 are therapeutic substances or cell differentiation factors. For example, All-trans-Retinoic Acid, Dexamethasone, Epidermal Growth Factor, and Pantothenic Acid. CB [6] -HA and DAH-HA are the skeleton constituents of hydrogel manufacturing. Genetically modified mesenchymal stem cells (Engineered MSCs) are cells into which the gene of interest has been introduced to secrete therapeutic or differentiating factors. The present invention is applicable to all stem cell therapeutic agents including Engineered MSC.
2 (A) shows a manufacturing process of the engineered MSC. By using an IL-12 mutant (IL-12M ), Brain Derived Neurotrophic Factor (BDNF), or Enhanced Green Fluorescent Protein (EGFP) adenovirus, and iron ions (Fe ^{3 +)} for gene transfer to the MSC, the transgenic MSC The production method is shown to be produced. (B) is a schematic diagram of recombinant adenovirus production.
(A) is a schematic diagram of synthesis of C / D-HA hydrogel, (B) shows the synthesis of monoallyloxyCB [6] (C) shows the change in peak of NMR after addition of spermine, which is a guest material strongly binding to CB [6] 10% of CB [6] was introduced.
4 is a result of a single subcutaneous toxicity test using C57BL / 6 mouse of monoCB [6] -HA and DAH-HA, which are components of hyaluronic acid hydrogel, (A) is a scheme of the test group used for the toxicity test (B) showed that the dead mass was above 2 mg / head because there was no dead animal until the 15th day after administration, (C) indicates normal until 15 days as a result of general symptoms, (D) There is no significant difference compared to the control group (G1), and (E) is the result of necropsy. No toxicologic abnormalities associated with the drug substance were observed in the autopsy results.
Figure 5 is a schematic diagram of Dexamethasone-CB [6] and its 1H NMR spectrum, (A) is a schematic diagram of synthesized Dexamethasone-CB [6] 6]. (C) is a 2-dimensional 1H NMR result showing that Dexamethasone and CB [6] are bonded to each other.
6 shows the synthesis of HA-Dexamethasone conjugate (HA-Dexa) and 1H NMR analysis (A) is a schematic diagram of HA-Dexamethasone synthesis (B) Peaks of the < / RTI >
7 is a synthesis and 1H NMR analysis of HA-All-trans-Retinoic Acid conjugate (HA-ATRA). (A) is a schematic diagram of HA-ATRA synthesis. BOP refers to benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, HOBt refers to anhydrous 1-hydroxybenzotriazole, and EDC refers to 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide. (B) shows that all-trans-retinoic acid and HA peak coexist in the peak of HA-ATRA.
8 is using the adenovirus expressing EGFP Fe ^{3 +} transduction in Rat bone marrow-derived MSC, and wherein created MSC / EGFP a C / D-HA hydrogel, C / DD-HA hydrogel, C / DR-HA hydrogel, C / D-DR-HA hydrogel, or Matrigel, Matrixen as a control cell support, as compared to the fluorescence imaging analysis. Imaging analysis was measured at the beginning of day 5 and every 10 days from day 10. (C / DD-HA hydrogel, C / DR-HA hydrogel, and C / D-RD-HA) in the case of introducing stem cell differentiation and activation inducer into the hydrogel hydrogel) cell retention, which clearly shows the usefulness of the introduction of the active factor.
FIG. 9 shows the results of the measurement of the residual volume of the Matrigel, Matrixen, C / D-HA hydrogel and C / D-RD-HA hydrogels.
FIG. 10 shows the results of comparing the persistence of IL-12 expressed by various cell supports with MSC (MSC / IL-12M) in which IL-12M gene was introduced in vitro up to 21 days. (C / D-DR-HA) loaded with Matrixen, Matrigel and C / D-HA hydrogel alone or Dexamethasone (100uM) and All-trans-Retinoic Acid (100uM) in MSC / IL- IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12 The two-tailed t-test was expressed as * in the expression of IL-12 when C / D-DR-HA was used when using the existing Matrigel and C / D-HA hydrogels of the present invention.
Figure 11 shows that IL-12 and C / D-HA hydrogel and C / D-RD-HA hydrogel filled with engineered MSCs with IL-12M gene were injected intratumorally into C57BL / 6 mice with B16F10 tumors, IFN-gamma expression. Matrixen was used as a control for the cell support. The concentration of IL-12 (A) and the concentration of IFN-gamma (B) present in the serum after the administration of each supporter was analyzed using an ELISA kit of R & D systems. MSCs without transfected genes and engineered MSCs without MSCs (MSC / IL-12M) were used as controls. The two-tailed t-test was expressed as * in the case of using Matrixen and C / D-HA hydrogels, which were used previously, and when using C / D-DR-HA hydrogel.
Figure 12 shows the tumor volume after administration of C / D-HA hydrogel and C / D-RD-HA hydrogel filled with Engineered MSC with IL-12M gene to C57BL / 6 mice with B16F10 tumors. (A) and the survival rate (B) of mice with tumors were compared. Matrigel and Matrixen were used as control supports, and MSC without gene insertion and engineered MSC (MSC / IL-12M) without support were used as controls.
Figure 13 shows antitumor efficacy results after repeated administration of engineered MSC-loaded hydrogel in a B16F10 tumor-bearing C57BL / 6 mouse model. MSC / IL-12M alone, MSC / IL-12M using C / D-RD-HA hydrogel as cell scaffold, or cell scaffold control at 6 days and 20 days after administration of B10F10 tumor cells to 57BL / Matrixen was injected into MSC / IL-12M as a drug, and (A) tumor size and (B) survival rate were observed in mice over time.
14 shows the results of MSC / BDNF expression by introducing adenovirus expressing BDNF gene into GFP MSCs, which are derived from adipose tissue of GFP transgeneic mouse, using Fe ^{3 +} (C / D-HA, C / D-HA and C / D-RD-HA) and Matrigel and Matrixen as control cells were used as cell supports. , and the expression times of therapeutic agents were compared in engineered MSCs. The expression level of BDNF inducing the proliferation and differentiation of neural stem cells was analyzed by ELISA. When C / D-RD-HA was used as a support, the highest concentration of BDNF expression was observed at 21 days, and the two-tailed t-test was denoted by *.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Manufacture of cell therapy

(MSCs) expressing IL-12 mutant or Brain Derived Neurotrophic Factor (BDNF) were prepared and used as a cell therapy agent. The recombinant mesenchymal stem cells (MSCs)

Example  1-1: MSC / IL Production of -12M

Interleukin 12 (IL-12) is a p70 dendritic protein having two p35 and p40 units. In the present invention, the p40 homodimer is reduced by modifying a glycosylation site important for the formation of a heterodimer of the p40 unit, IL-12 (IL-12M, Nature Biotechnology, 2004) was used as a mutagenic form capable of maximizing the activity of IL-12.

IL-12M gene was cloned into a shuttle vector and introduced into the adenovirus vector (pAd / EASY) and BJ5183 through homologous recombination to construct the adenovirus expressing the IL-12M. The final adenovirus DNA (pAd / IL-12M). The recombinant adenovirus expressing IL-12M was prepared by introducing the pAd / IL-12M into an adenovirus producing cell line 293 cell line.

Then, 50 MOI and Fe ^{3 +} (50 uM) of recombinant adenovirus expressing IL-12M were added to bone marrow-derived mesenchymal stem cells (MSCs) present in the bones of the rat's femur (tibia) And MSCs (MSC / IL-12M) producing IL-12M were cultured in Low Glucose DMEM medium supplemented with 1% antibiotics antimycotics.

Example  1-2: MSC / BDNF Production

To prepare recombinant adenovirus expressing human BDNF, a codon optimized BDNF (BDNFco) gene was synthesized and cloned into pShuttle vector to produce pShuttle / BDNFco. (PAd / BDNF) was prepared by introducing pShuttle / BDNFco into COS7 cell line to confirm expression and introducing pShuttle / BDNFco through homologous recombination in adenovirus vector (pAd / EASY) and BJ5183 strain Respectively. The recombinant adenovirus expressing BDNF was prepared by introducing pAd / BDNF into 293 cell lines, an adenovirus producing cell line. The rAd / BDNF-TK vector was developed to produce autologous stem cells capable of producing both proteins simultaneously in the cells. The HSV-TK sequence used in this study is a codon-optimized sequence, and the BDNF gene and the HSV-TK gene can be simultaneously expressed in a single vector through IRES (internal ribosome entry site).

Then, recombinant adenovirus 50 MOI and Fe ^{3 +} (50 uM) expressing BDNF were added to 105 fat-derived stem cells extracted from GFP transgenic mice, and the cells were treated with 10% FBS and 1% antibiotics antimycotics Glucose MSCs (MSC / BDNF) producing BDNF were prepared by culturing in DMEM medium.

Example  1-3: MSC / EGFP Production

Engineered MSCs were constructed to express the desired gene by infecting mesenchymal stem cells with a recombinant adenovirus (rAd) expressing EGFP. As a strategy for increasing the infection efficiency of adenovirus in MSC, a 50 μM concentration of Fe ^{3 +} was used as a cation. Adenovirus expressing EGFP and Fe ³⁺ were added and the medium left at room temperature for 30 minutes was used for MSC infection. In order to induce transfection into MSCs, conditions with high expression rates were selected by variously applying the range of MOI (multiplicity of infection) of recombinant adenovirus. At this time, for the high transfer efficiency of the gene expressing the mesenchymal stem cells, the expression was confirmed according to the presence or absence of Fe ^{3 +} , and at the same time, 50MOI, which is high in expression rate and not giving cytotoxicity to the stem cell itself, Respectively. MSC / EGFP, an MSC expressing EGFP, was prepared by infecting bone marrow-derived rat-derived mesenchymal stem cells (rBM-MSC) with recombinant adenovirus vector and iron ion for 30 minutes.

Example  2: Synthesis of hyaluronic acid derivative

Example  2-1: DAH - HA  Derivative synthesis

A hyaluronic acid solution having a concentration of hyaluronic acid (molecular weight: 235,000) of 5 mg / ml for distilled water was prepared. After activating the carboxyl group of hyaluronic acid by using 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (EDC) and sulfo-N-Hydroxysuccinimide (Sulfo-NHS), diaminohexane, DAH ), And the mixture was reacted at room temperature for about 2 hours while maintaining the pH at about 4.8 with 1.0 N hydrochloric acid. At this time, the addition amounts of EDC, Sulfo-NHS and DAH were 4 times, 1 time, and 20 times, respectively, as to the carboxyl groups of hyaluronic acid.

The reaction product was dialyzed with 100 mM sodium chloride solution, 25% ethanol solution and distilled water, and lyophilized to obtain HA-DAH having a hydrazide group introduced therein. The introduction rate of hyaluronic acid was determined by 1H-NMR (DPX300, Bruker, Germany) and it was confirmed that about 50 mol% was introduced.

Example  2-2: MonoCB [6] - HA  Derivative synthesis

A hyaluronic acid solution having a concentration of hyaluronic acid (molecular weight: 100,000) of 5 mg / ml was prepared. The carboxyl group of hyaluronic acid was activated by EDC (1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide) and HOBt (Hydroxybenzotriazole), cystamine was added, Was maintained at about 4.8 for about 8 hours at room temperature. At this time, the addition amounts of EDC, HOBt, and cystamine were 4 times, 4 times, and 0.5 times, respectively, with respect to the carboxyl group of hyaluronic acid. The reaction product was dialyzed with 100 mM sodium chloride solution and distilled water. The purified material was disrupted by disulfide groups present in cystamine using 4 times the amount of cystamine substituted with tris (2-carboxyethyl) phosphine, and thiol-substituted hyaluronic acid (HA-SH) was obtained through recrystallization using ethanol. The introduction rate of the thiol group was determined by 1H-NMR (DPX300, Bruker, Germany), and it was confirmed that about 50 mol% was introduced.

To increase the substitution rate of CB [6] to be introduced into hyaluronic acid, CB [6] substituted with a monohydroxy group was dissolved in anhydrous DMSO and reacted with 10 equivalents of sodium hydride at 60 ° C for 12 hours. After that, 10 equivalents of allylbromide was slowly added and the reaction was continued for another 24 hours. The reaction product was washed with 3 liters, 2 liters, and 1 liter of diethyl ether, and the resulting precipitate was dissolved in acetonitrile and dried. Aluminum oxide columns were used to obtain CB [6] substituted with monoallyl groups using 20% distilled water and acetonitrile mixture to 40% mixture at 5% intervals as mobile phase.

HA-SH was dissolved in distilled water at 3 mg / ml, CB [6] and TCEP containing allyl groups were dissolved in a quartz tube and reacted in a photoreactor for 3 hours. At this time, the addition amounts of CB [6] and TCEP into which the allyl group was introduced were 2 times and 2 times as much as the carboxyl groups of hyaluronic acid, respectively. This reaction product was purified by dialysis in distilled water and lyophilized to obtain hyaluronic acid (monoCB [6] -HA hydrogel) into which CB [6] was introduced. As shown in FIG. 3, the introduction rate of CB [6] was confirmed to be about 10% of the total hyaluronic acid monomer through introduction of CB [6] through 1H-NMR measurement.

Example  2-3: DAH - HA Wow MonoCB [6] - HA Toxicity evaluation

4 (A) is a scheme chart of the test group used in the toxicity test. The excipient (G1) and the single agent group (G5, G6) were subcutaneously administered subcutaneously to the mouse of the C57BL / 6 strain as the test substance I (DAH-HA) and the test substance II (MonoCB [6] -HA) G2 and G3 are the mixtures of the respective test substances, and the compound-administered group G4 is the test substances I and II sequentially administered. After collecting blood, they were lavaged by ligation of the posterior vena cava and abdominal aorta. All organs and sites of administration were examined visually. Figure 4 (B) shows mortality. Fig. 4 (C) shows the general symptoms associated with the administration of the test substance, Fig. 4 (D) shows the weight change, and Fig. 4 (E) shows the necropsy result. When the test substance monoCB [6] -HA and DAH-HA were administered to the C57BL / 6 strain mice in a single subcutaneous route, no deaths were observed in the combination and single drug administration groups, and the general symptoms, Autopsy findings showed no gross abnormality related to the administration of the test substance. In addition, no difference was observed between the combination administration group and each single administration group. Therefore, under the test conditions, approximate lethal dose (ALD) by combination administration of monoCB [6] -HA and DAH-HA is considered to exceed 2 (1 + 1) mg / Therefore, the single dose available in mice will be 2 (1 + 1) mg / head.

Example  2-4: Cells Particulate agent  Or a hyaluronic acid derivative into which a cell activity inducing substance is introduced

Example  2-4-1: Synthesis of dexamethasone-incorporated hyaluronic acid derivative

Example  2-4-1-1: CBs with dexamethasone incorporated [6] ( Dexamethasone -CB [6]) Synthesis

In the case of a cell differentiation agent such as Dexa or a cell activator inducing a hydroxyl group, it was dissolved in acetone to introduce a carboxyl group. Specifically, Dexa prepared a solution of 10 mg / ml for anhydrous acetone. Succinic anhydride and DMAP (4-dimethylaminopyridine) were added to the reaction mixture and reacted at room temperature for 8 hours. Dexa into which the carboxyl group was introduced was precipitated in acetone, which was filtered to prepare purified Dexa-COOH.

A solution of Dexa-COOH at a concentration of 10 mg / ml for anhydrous DMSO was prepared. The addition of DCC and NHS corresponding to 3 molar equivalents of the carboxyl group of Dexa-COOH results in urea-type precipitation. After removal of urea, the amine-introduced cucurbituril [6] (CB [6]) is reacted with Dexa-COOH 2 molar equivalents of TEA and 3 molar equivalents of TEA were added for 3 days to allow Dexa and CB [6] to bond. This reaction product was dialyzed against DMSO and ultrapure water using a dialysis tube of MWCO 500 and lyophilized to obtain Dexa-CB [6]. The reaction of Dexa-CB [6] was confirmed by 1H NMR as shown in FIG.

Example  2-4-1-2: CB [6] with dexamethasone incorporated ( Dexamethasone -CB < / RTI > [6]) HA - Dexa ) synthesis

HA-DAH was prepared in the same manner as in Example 2-1 except that the reaction time was changed to 10 minutes. The DAH was 30%. This HA-DAH solution was prepared in distilled water at a concentration of 5 mg / ml. On the other hand, ion exchange resin (Dowex 50WX8, Sigma-aldrich, US) was mixed with hydroxy tetrabutylammonium to allow tetrabutylammonium to bind to the resin. The resin was again mixed with the HA-DAH solution, stirred for 8 hours, and then filtered and lyophilized to prepare HA-DAH / TBA. The HA-DAH / TBA was dissolved again in anhydrous DMSO at a concentration of 5 mg / ml. Then, trauture reagent (Thermo scientific, US) and dexamethasone mesylate (steraloids, US) HA-Dexamethasone was prepared by linking the amine of HA with dexamethasone using an opening reaction. At this time, the amounts of Traut's reagnet and dexamethasone mesylate were 2 times and 2 times as much as the DAH introduced into hyaluronic acid, respectively. The reaction product was dialyzed with 100 mM sodium chloride solution and distilled water, and lyophilized to obtain HA-Dexa. In FIG. 6, introduction rate of Dexamethasone was determined by 1H-NMR (DPX400, Bruker, Germany), and it was confirmed that about 9 mol% was introduced.

Example  2-4-2: Retinoic acid  Synthesis of introduced hyaluronic acid derivative

Example  2-4-2-1: Retinoic acid  The introduced CB [6] ( All - trans - Retinoic Acid -CB [6]) Synthesis

In the case of ATRA, a cell-dividing cellulolytic agent or cell activator was dissolved in anhydrous DMSO to prepare a solution having a concentration of ATRA of 10 mg / ml. Addition of DCC and NHS corresponding to 3 molar equivalents of the carboxyl group of ATRA results in urea-type precipitation. After removal of urea, the amine-introduced cucurbituril [6] (CB [6] TEA was added in an amount of 3 molar equivalents and reacted for 3 days to bind ATRA and CB [6]. This reaction product was dialyzed against DMSO and ultrapure water using a dialysis tube of MWCO 500 and lyophilized to obtain ATRA-CB [6]. The reaction of ATRA-CB [6] was confirmed by 1H NMR.

Example  2-4-2-2: Retinoic acid  The introduced CB [6] ( All - trans - Retinoic Acid -CB < / RTI > [6]) HA - ATRA ) synthesis

1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP, TCI, Japan) was prepared by dissolving All-trans-Retinoic Acid (Sigma-aldrich, US) in anhydrous DMSO at 10 mg / ATRA-DAH was synthesized by reacting with DAH. At this time, DAH, BOP, and HOBt were 10 times, 4 times, and 4 times, respectively, by molar ratio with respect to ATRA. The reaction product was dialyzed with 100 mM sodium chloride solution and distilled water, and lyophilized to obtain ATRA-DAH. Confirmation of the introduction of ATRA was confirmed by ESI-MS to have a molecular weight of 398 and a proper synthesis.

On the other hand, ion exchange resin (Dowex 50WX8, Sigma-aldrich, US) was mixed with hydroxy tetrabutylammonium to allow tetrabutylammonium to bind to the resin. The resin was again mixed with HA, stirred for 8 hours, filtered and lyophilized to prepare HA-TBA.

HA-TBA was dissolved in anhydrous DMSO at 2 mg / ml, and HA-ATRA was synthesized by adding the ATRA-DAH synthesized in the above. At this time, the addition amounts of ATRA-DAH, BOP and HOBt were 10%, 4-fold, and 4-fold relative to the carboxyl groups of HA, respectively. The reaction product was dialyzed with 100 mM sodium chloride solution and distilled water, and lyophilized to obtain HA-ATRA. In FIG. 7, the introduction rate of All-trans-Retinoic Acid was determined by 1H-NMR (DPX400, Bruker, Germany) and it was confirmed that about 9 mol% was introduced.

Example  3: Hyaluronic acid In hydrogel Supported  Assessment of the persistence of expressed substances in cells

We wanted to use hyaluronic acid hydrogel as an effective cell delivery system to complement existing cell therapy agents that are rapidly clearance in the body. SKH-1 nude mice were observed via imaging after administration to confirm that the administered stem cells were maintained for a long time by the use of C / D-HA hydrogel and its derivatives. Specifically, when the engineered rat BM-MSC (MSC / EGFP) prepared using the adenovirus expressing EGFP was filled with the cell supporter, the analysis was performed using IVIS imaging equipment to confirm the persistence of the genetic material expressed in the MSC Respectively.

Example  3-1: Hyaluronic acid Hydrogel  Produce

The C / D-HA hydrogel was prepared by mixing 20 mg / ml of DAH-HA prepared in Example 2-1 and 20 mg / ml of monoCB [6] -HA prepared in Example 2-2 at room temperature. In the case of C / DD-HA, C / DR-HA and C / D-RD-HA hydrogels bound to cell differentiation agent / cell activity inducer (Dexa, Retinoic acid) , MonoCB [6] -HA prepared in Example 2-2, Dexa-CB [6] prepared in Example 2-4-1-1 and HA-ATRA prepared in Example 2-4-2-2 Respectively. The main molecules: guest molecules = 1: 1 were commonly used for DAH-HA, DD-HA, DR-HA and D-RD-HA containing MonoCB [6] -HA and guest molecules. When the hyaluronic acid hydrogel was allowed to stand at room temperature, the gel was changed into gel form within several tens of minutes depending on the concentration and conditions, and thus the experiment was carried out rapidly.

Example  3-2: Hyaluronic acid In hydrogel Supported MSC Evaluation of the persistence of the expressed substance

MSC (EGFP) expressing 5 X 10 ⁵ EGFP was injected subcutaneously into C57BL / 6 mouse together with injectable hyaluronic acid hydrogel made by the host-guest molecule to produce mesenchymal stem cells The survival and survival rates of GFP were confirmed by GFP imaging equipment. MSC / EGFP was injected subcutaneously with C / D-HA hydrogel or Matrigel and Matrixen as a control supporter. By measuring the size of the hydrogel over time, it was possible to confirm the residence time in the body. And cell persistence was confirmed. As shown in FIG. 8, it was observed at the 50th day of observation using C / D-HA hydrogel and an inducing agent, and it can be confirmed that the persistence of the cells is maintained for a long period of time as compared with the G1 to G3 groups. In the case of C / DD-HA, C / DR-HA and C / D-RD-HA hydrogels bound with cell differentiation agent / cell activator (Dexa, Retinoic acid) hydrogel, the higher intensity of EGFP expression was observed, indicating an increase in persistence.

Example  3-3: Hyaluronic acid Hydrogel  Assessment of residual duration in the body

To assess how long the hyaluronic acid hydrogel remained in the body, C / D-HA and C / D-RD-HA hydrogels were subcutaneously injected into the mice and confirmed with IVIS imaging equipment.

Specifically, photographs taken with IVIS imaging equipment were analyzed for size of hydrogel using ImageJ program, and Matrigel and Matrixen were loaded onto C57BL / C-D-HA hydrogel and C / D-RD- Six mice were injected subcutaneously and the size of the hydrogel was measured over time to evaluate the duration of the hydrogel in the body.

As shown in FIG. 9, it was confirmed that Matrixen was decomposed within 10 days and Matrigel was decomposed in 3 weeks. When C / D-HA hydrogel and C / D-RD-HA hydrogel were used, It was confirmed that it remained in the body.

In other words, it can be seen from the results that the hyaluronic acid hydrogel of the present invention can maintain the durability for a long time in the body.

Example  4: hyaluronic acid In hydrogel  Introduced MSC / IL Evaluation of efficacy of -12

Example  4-1: Expression evaluation

Example  4-1-1: in vitro  Expression evaluation

The expression of IL-12 was confirmed by ELISA after incubation with MSC / IL-12M in which IL-12M adenovirus was introduced into injectable hyaluronic acid hydrogel prepared by the action of the host-guest molecule.

MSC / IL-12 2X104 cells in which MSC / IL-12M with hydrogel was introduced and Matrixen and Matrigel, which are control cell supporters, were cultured in a 6-well cell culture plate in the presence of 10% fetal bovine serum 1% antibiotics The expression of IL-12 was measured in culture medium after 3, 5, 7, 10, 14, 17, and 21 days after glucose DMEM (Gibco, US) As shown in FIG. 10, the expression levels of IL-12 in Matrixen and MSC / IL-12 without cell support were lower than those of Matrigel and C / D-HA hydrogels. In particular, when C / D-HA hydrogel was used, the concentration of IL-12 expressed on day 21 was higher than that of Matrigel.

Did you introduce Retinoic acid (RA) and Dexamethasone (Dexa) into C / D-HA hydrogel? IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, As shown in FIG. 10, when C / D-RD-HA hydrogel was used as a cell support of MSC / IL-12M, IL-12 expression was higher than C / D-HA hydrogel.

Example  4-1-2: in vivo  Expression evaluation

When hyaluronic acid hydrogel was used as a cell scaffold for engineered cell therapy, MSC / IL-12, the concentration of the expression product in the blood was measured after administration of the mouse tumor in order to confirm the increase in gene expression.

In order to evaluate the persistence of IL-12 expression in vivo of MSC / IL-12M with C / D-HA hydrogel, 1 × 10 ⁶ of B16F10 was injected subcutaneously into C57BL / 6 mice at 6 weeks of age to make a melanoma model . MSC / IL-12M alone group and C / D-HA or C / D-RD-HA hydrogel filled MSC / IL-12M group at the time when the diameter of the tumor reached 5 mm (after 5 days of tumor formation) The expression of IL-12 and IFN-gamma in serum was determined by ELISA at 0, 1, 3, 6, 10, and 15 days after injecting Matrixen-loaded MSC / Respectively.

As shown in FIG. 11 (A), at 1 day after administration, expression was over 1000 pg / ml in all groups regardless of whether or not the suppository was used. On the other hand, after 3 days, / D-HA hydrogel was used as a support, the expression level of IL-12 was high when C / D-RD-HA hydrogel was used as a support rather than C / D-HA hydrogel and expression of IL- Can be detected.

In the case of IFN-gamma concentration induced by IL-12 activity, C / D-RD-HA hydrogel was the most abundant when used as a scaffold than Matrixen and C / D-RD-HA hydrogels at 3 days after administration The IFN-gamma concentration was measured in blood using Matrixen, C / D-RD-HA hydrogel, and C / D-RD-HA hydrogel as cell supports at the end of 6 days. In the case of using C / D-RD-HA hydrogel as a support, high IFN-gamma concentration was shown up to 15 days after administration compared to other test groups.

Example  4-2: Evaluation of anticancer efficacy

To investigate the effect of C / D-HA hydrogel on the antitumor effect of MSC / IL-12M, a melanoma model was prepared by injecting 1 × 10 ⁶ of B16F10 subcutaneously into 6-week-old C57BL / 6 mice. MSC / IL-12M without cell sup- port and MSC / IL-12M filled with C / D-HA hydrogel or C / D-HA-DR- HA hydrogel at the time when the diameter of the tumor reached 5 mm IL-12M group and MSC / IL-12M filled with Matrigel or Matrixen as a control support were injected into the tumor to confirm the tumor size and survival rate. At this time, the number of MSC / IL-12M used was 1 × 10 ⁵ .

Example  4-2-1: Evaluation of tumor growth inhibition efficacy

B16F10 mouse melanoma cell line was used to compare anticancer efficacy. 1 × 10 ⁶ cells were subcutaneously injected 5 days later, when the tumor diameter became 5 mm or more, 1 × 10 ⁵ cells of MSC / IL-12M were administered in combination with each cell scaffold (MSC alone, MSC / IL MSC / IL-12M / Matrigel, MSC / IL-12M / Matrixen, MSC / IL-12M / C / D-HA hydrogel, MSC / IL-12M / C / D-DR-HA hydrogel). As shown in FIG. 12 (A), tumor size changes were observed at intervals of two days until the 25th day after tumor injection (21 days after engineered MSC administration).

As shown in FIG. 12 (A), tumor size was increased in all groups from day 10 (10 days after tumor injection) to about 5 days after therapy, while treatment groups using MSC / IL- Lt; RTI ID = 0.0 > 6-10 days. ≪ / RTI > On the other hand, after 19 days, Matrigel or Matrixen group showed a tendency to increase in tumor size, but C / D-HA hydrogel group still showed delayed tumor growth. On day 25, tumors grew slowly in the C / D-HA hydrogel group, but the tumor growth was still suppressed in the MSC / IL-12M / C / D-DR-HA hydrogel group. Therefore, it was concluded that the anticancer effect of the stem cell therapeutic agent sup- ported with the therapeutic gene IL-12M was superior in the order of C / D-DR-HA hydrogel, C / D-HA hydrogel, Matrigel and Matrixen.

Example  4-2-2: Evaluation of survival rate increasing efficacy

Survival rates of the respective treatment groups used in Example 4-2-1 were examined. Rats with a diameter of 15 mm or more and a volume of 1700 ㎣ or more were regarded as dead and the data were summarized. 12 (B). All mice were dead for up to 30 days in the MSC / IL-12M group without the cell supporter, and 40 days in the Matrixen and Matrigel control cells. On the other hand, increased survival was observed up to 45 days in the group with C / D-HA hydrogel. In the MSC / IL-12M / C / D-DR-HA hydrogel group, mice survived at a higher rate than the MSC / IL-12M / C / D-HA hydrogel group at 31-37 days after tumor administration. All mice died as in the MSC / IL-12M / C / D-HA hydrogel group.

It was confirmed that MSC supporter's survival increases with C / D-DR-HA hydrogel, C / D-HA hydrogel, Matrigel and Matrixen. The difference in survival rates between C / D-HA hydrogel and Matrigel was statistically significant in both log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test.

Example  4-3: Repeated administration of anti-cancer efficacy

In order to confirm the increase of anti-cancer effect by repeated administration of MSC / IL-12M filled with hyaluronic acid hydrogel, 1 × 10 ⁶ of B16F10 was injected subcutaneously at 6 weeks of C57BL / 6 mouse to make a disease model . MSC / IL-12M filled with C / D-RD-HA hydrogel, or MSC / IL-12M filled with Matrixen as a control was administered to the tumor on days 6 and 20 after tumor formation, And the growth of the tumor was confirmed with time. At this time, the number of MSC / IL-12M used was 1 × 10 ⁵ .

Example  4-3-1: Evaluation of tumor growth inhibition efficacy

Similar to the single-dose study, B16F10 mouse melanoma cell line was used to evaluate the anticancer efficacy of MSC / IL-12M. 1 X 10 ⁶ cell after the subcutaneous injection, the MSC / IL-12M was repeated administration into the tumor cells in a variety of substrate and the combination (MSC alone control, MSC / IL-12M, MSC / IL-12M / Matrixen, MSC / IL- 12M / C / D-RD-HA hydrogel). As shown in FIG. 13, the tumor size change was measured at intervals of two days after the tumor injection, and when the diameter of the tumor was 17 mm or more, the subject was considered to have died at the sacrifice. The mice that did not transfer IL-12M after the first administration showed no anticancer effect. In the MSC / IL-12M group expressing IL-12, the therapeutic substance, the tumor growth was delayed until day 18 and the second administration was performed on day 20. When MSC / IL-12M was loaded onto C / D-RD-HA hydrogel, tumor growth was delayed until day 32. In the group using Matrixen as a support for MSC / IL-12M, ~ 5 days, and then increased sharply.

Example  4-3-2: Evaluation of survival rate increase efficacy

MSC / IL-12M carried on a C / D-RD-HA hydrogel was repeatedly administered to a tumor-producing mouse using the same method as in Example 4-3-1 to observe the survival rate of the mice. In the case of a single dose, mice surviving up to about 80 days or more after the repeated administration were identified, while the maximum survival time after tumor administration was 50 days. In addition, in the case of single administration, deaths were observed from 33 days after the administration of the tumor cells, whereas in the repeated administration, deaths occurred after 45 days. Therefore, it was found that the therapeutic effect of the cell therapy agent containing the hyaluronic acid hydrogel was enhanced by the repeated administration of the therapeutic agent to increase tumor growth inhibition and antitumor survival rate.

Based on this example, it can be confirmed that the hyaluronic acid hydrogel of the present invention improves cell maintenance persistence and therapeutic gene expression and anti-cancer efficacy of a cell therapy agent. Hyaluronic acid hydrogel could be applied to the development of stem cell therapy for treatment of various intractable diseases according to the therapeutic gene to be introduced as well as the chemotherapeutic agent. In addition, the hydrogel containing the cell differentiation agent / cell activity inducer not only sustained the expression time of the gene but also exhibited the enhanced therapeutic effect and confirmed its applicability as a cell therapy agent.

Example  5: MSC / BDNF from BDNF On the expression of hyaluronic acid Hydrogel  Efficacy evaluation

C / D-HA hydrogel, or a cell scaffold to which a cell differentiation agent or a cell activity inducer has been applied has the same effect in MSCs expressing not only IL-12 used for chemotherapy but also other genes, The same effect was also observed in mesenchymal stem cells derived from E. coli.

In this example, BDNF was used as a therapeutic gene and MSC derived from fat cells of GFP transgenic mice was used as MSC. MSC / BDNF was prepared by introducing BDNF-TK-expressing adenovirus into MSC (Experimental Example 1-3). The mice were plunged at 1, 5, 9, 15, and 21 days after the injection of 100 ul of cell supernatant filled with MSC / BDNF of 5 × 10 5 subcutaneously in C57BL / 6 mice. The concentration was confirmed by ELISA. Matrigel, Matrixen, C / D-HA hydrogel and C / D-DR-HA hydrogel were used as cell supports. As shown in FIG. 14, the expression of BDNF was significantly higher in the groups using C / D-HA hydrogel and its derivatives than in the other groups from 9th day after administration. In addition, when Dexamethasone and All-trans-Retinoic Acid were used (C / D-RD-HA), high BDNF expression was observed at 21 days after administration of C / D-HA hydrogel alone . In the control group Matrigel or Matrixen, the expression of BDNF was not significantly different from that of the hydrogel at 5 days after injection,

As a result, the use of C / D-HA hydrogel and the addition of hydrogel, which is a cell differentiation agent or cell activator, increased the duration of expression of BDNF as well as IL-12, Suggesting the high utility of cellular therapeutic agents composed of derivatives.

Claims (21)

A hyaluronic acid-based hydrogel prepared using cucurbituril and diaminohexane, and further comprising dexamethasone, retinoic acid or a combination thereof,
A cell scaffold for a stem cell therapeutic agent for increasing the persistence of a stem cell therapeutic agent in the body and improving therapeutic efficacy.
delete delete delete delete delete delete delete The method according to claim 1,
Wherein the cucurbituril comprises 1 to 10,000 glycourols.
delete The method according to claim 1,
Wherein the hydrogel is formed by covalently bonding a cucurbituril-coupled hyaluronic acid derivative and a diaminohexane-linked hyaluronic acid derivative by the specific hydrophobic / hydrophilic action of the cucurbituril and diaminohexane, Cell scaffold for stem cell therapy.
delete 11. A stem cell treatment agent comprising the cell support and the stem cell according to any one of claims 1, 9 and 11.
14. The method of claim 13,
The stem cell may be a mesenchymal stem cell, a hematopoietic stem cell, a neural crest stem cell, an endothelial stem cell, ), And a combination thereof.
15. The method of claim 14,
Wherein the cell therapy gene is capable of expressing a gene selected from the group consisting of cytokines, growth factors, nutritional factors, or combinations thereof.
16. The method of claim 15,
Wherein the cytokine is selected from the group consisting of interleukin-1, interleukin-6, interleukin-12, interferon gamma, tumor necrosis factor (TNF), and combinations thereof.
16. The method of claim 15,
Wherein the nutrient factor is BDNF (Brain Derived Neurotrophic Factor).
16. The method of claim 15,
The growth factors include EGF (Enhanced Green Fluorescent Protein), Bone Morphonic Protein (BMP), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), Hepatocyte Growth Factor (HGF), TRAIL HSV-TK (Herpes simplex virus thymidine kinase), or a combination thereof.
14. The method of claim 13,
Wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
delete delete

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