KR102315868B1 - In vivo monitoring system for neural stem cell therapy based on neuronal differentiation specific surface antigen expression - Google Patents

Abstract

The present invention can contribute to the commercialization and popularization of stem cell therapeutics by establishing the efficacy and safety evaluation technology of stem cell therapeutics, which is currently a challenge, and providing them to domestic and foreign researchers through a temperature-sensitive hydrogel-based cell delivery system. Accurate preclinical research is essential for the development of cell therapy that can be applied clinically, but the current level of domestic research is very insufficient. It can greatly contribute to the improvement of the level of practical research technology targeting Therefore, it is possible to provide a technology that can be practically used in patients by solving the problems of clinical application through in vivo monitoring of stem cells.

Description

In vivo transplantation neuronal cell tracking platform {In vivo monitoring system for neural stem cell therapy based on neuronal differentiation specific surface antigen expression}

The present invention relates to a temperature-sensitive hydrogel-based stem cell therapeutic agent and monitoring system.

Neural precursor cells (NPC) isolated from the central nervous system (CNS) of the embryo can be expanded in vitro and differentiated into various neurons and astrocytes in vitro and in vivo. (Non-Patent Documents 1 and 2). Research on neural progenitor cells is useful for exploring brain development and developing renewable sources of neurons (Non-Patent Document 3).

In addition, stem cells have been applied to various therapies. In particular, in the case of degenerative brain diseases, research results have been reported that transplanting neural precursor cells is more effective than transplanting fully differentiated nerve cells. However, treatment using stem cells has reported many problems, including low cell survival rate, difficulty in tracking differentiation of stem cells, and low cell engraftment rate after transplantation (Non-Patent Documents 4 and 5).

In the case of stem cell therapy, it is difficult to evaluate the function of the transplanted stem cells because it cannot be confirmed whether the stem cells injected into the patient function properly in vivo. In order to confirm the differentiation of the neural progenitor cells into nerve cells after transplantation of the neural progenitor cells, it is necessary to analyze the tissue samples obtained by sacrificing the transplanted subject. To date, a method for observing the differentiation of transplanted neural progenitor cells by magnetic resonance imaging (MRI) has not been developed. It is presumed that this is because there is no cell surface antigen that can specifically track differentiated neurons.

Accordingly, the present inventors intend to provide an in vitro and in vivo platform capable of verifying the efficacy and reliability of an in vivo transplanted stem cell therapeutic agent.

Brustle et al., (1997) In vitro-generated neural precursors participate in mammalian brain development. Proc Natl Acad Sci U S A 94, 14809-14814 Davis A.A. and Temple. S. (1994) A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372, 263-266 Park et al., (2006) Acquisition of in vitro and in vivo functionality of Nurr1-induced dopamine neurons. FASEB J 20, 2553-2555 Yamashita et al., (2006) Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum. J Neurosci 26, 6627-6636 Arvidsson et al., (2002) Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 8, 963-970

It is an object of the present invention to provide a temperature-sensitive hydrogel-based stem cell therapeutic agent and monitoring system.

Accordingly, the present inventors developed a micro/nanoparticle-based temperature-sensitive hydrogel system using the synthesis technology of a temperature-sensitive hydrogel with high biocompatibility and physiological activity. In addition, improvement of engraftment and differentiation rate of neural stem cells, production of drug encapsulated micro/nanoparticles for in vivo differentiation and distribution measurement, control of drug release rate and duration according to micro/nanoparticle formulation, carotid artery ligation and hypoxic environment Preclinical testing and behavioral function evaluation were performed using an animal model of hypoxia-induced ischemic brain injury through composition. As a result, it was confirmed that when micro/nano particles are produced by synthesizing natural polymers (collagen and chitosan), the release time can be controlled according to pH. At pH 7.5, it was confirmed that the loaded BSA started to be released over time, but at pH 2.0, it was not released over time. This means that the release time and pH can be adjusted according to the synthesis rate and type of natural polymer.

Accordingly, the present invention provides a temperature-sensitive hydrogel formed from a first natural polymer and a second natural polymer; And it provides a composition for tracking the transplantation and differentiation of stem cells comprising nanoparticles coated with a biocompatible polymer.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention is a temperature-sensitive hydrogel formed from a first natural polymer and a second natural polymer;

Neural stem cells transduced with a vector for tracking differentiation into neurons containing a neural cell differentiation-specific promoter, a Creb 5' UTR sequence, and a reporter gene contained in the hydrogel; and

Provided is a composition for stem cell transplantation and differentiation tracking comprising nanoparticles that are conjugated with an antibody that specifically binds to a differentiated neuronal cell surface antigen and coated with a biocompatible polymer.

First, the composition for stem cell transplantation and differentiation tracking of the present invention includes a temperature-sensitive hydrogel formed from a first natural polymer and a second natural polymer.

In the present invention, the temperature-sensitive hydrogel is a polymer showing a sudden change in solubility according to a change in temperature, and exists in a sol state at room temperature and is surgically It refers to an intelligent hydrogel that can be used as an injection formulation without surgery. The hydrogel according to the present invention may be formed from a first natural polymer and a second natural polymer.

The first and second natural polymers are collagen, chitosan, hyaluronic acid, poly-L-lysine, poly(4-vinylpyridine/divinylbenzene), chitin, poly(butadiene/acrylonitrile) amine terminus, polyethylene imine, polyaniline, poly(ethylene glycol) bis(2-aminoethyl), poly(N-vinylpyrrolidone), poly(vinylamine) hydrochloride, poly(2-vinylpyridine), poly(2-vinylpyridineN) -oxide), poly-ε-Cbz-L-lysine, poly(2-dimethylaminoethyl methacrylate), poly(allyl amine), poly(allylamine hydrochloride), poly(N-methylvinylamine), poly (Dialyldimethylammonium chloride), poly(N-vinylpyrrolidone) and poly(4-aminostyrene) may be each selected from the group consisting of, but is not limited thereto.

In one embodiment, the first natural polymer and the second natural polymer may be collagen and chitosan, respectively.

In the present invention, the temperature-sensitive hydrogel is formed by including the first natural polymer and the second natural polymer in a concentration ratio of 0.5 to 2: 0.5 to 2, for example, 0.7 to 1.5: 0.7 to 1:5. can

The temperature-sensitive hydrogel of the present invention can be gelled by mixing the first natural polymer and the second natural polymer, and then adding a crosslinking agent to adjust the pH. In this case, as the crosslinking agent, NaOH, CaCl ₂ or FeCl ₃ may be used, but is not limited thereto.

In one embodiment, the concentration of the crosslinking agent may be 0.2 to 0.4 N.

In the following examples, it was confirmed that differentiation into neurons is possible by including neural stem cells in the temperature-sensitive hydrogel and culturing them, and proliferation of neural stem cells in the temperature-sensitive hydrogel induces differentiation into neurons It was confirmed that there was no negative effect on Through this, it was demonstrated that the temperature-sensitive hydrogel of the present invention can be utilized as a cell carrier.

In the present invention, the temperature-sensitive hydrogel may include neural stem cells therein. The neural stem cells are transduced with a vector for tracking differentiation into neurons. The vector for tracking differentiation into neurons includes a neuron differentiation-specific promoter, a Creb 5' UTR sequence, and a reporter gene.

The present inventors prepared a vector to include a neuron differentiation-specific promoter, a Creb 5' UTR sequence, and a reporter gene through previous studies. It was confirmed that differentiation tracking into

The vector refers to a plasmid, viral vector or other medium into which a promoter inducing neuronal differentiation-specific expression and a reporter gene operably linked thereto can be inserted or introduced.

The promoter for inducing the neuron-specific expression refers to a promoter of marker proteins characteristically expressed in neurons, and refers to a promoter that binds transcription factors to these promoters to express the neuron-specific marker protein. The vector of the present invention can express a large amount of the reporter gene specifically only in neurons when the reporter gene is operably linked under a promoter inducing neuron-specific expression. In the present invention, "operably linked" means that the appropriate gene sequence is linked in such a way as to enable gene expression when linked to the promoter sequence.

The promoter for inducing the neuron-specific expression is a synapsin promoter. A reporter gene can be expressed specifically in a neuron by the synapsin promoter.

Examples of the plasmid include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), Bacillus subtilus-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24 and YCp50). , Examples of the viral vector include retroviruses, adenoviruses, adeno-associated virus (AAV), animal viruses such as lentiviruses or vexinia viruses, and insect viruses such as baculoviruses. In the present invention, the vector suitable for introducing the polynucleotide according to the present invention into a host cell may be used, and preferably a vector designed to facilitate protein expression induction and isolation of the expressed protein may be used.

In one embodiment, the vector may be a viral vector, in particular a lentiviral vector.

In another embodiment, the neuron-specific promoter may be a synapsin promoter. The synapsin promoter has the characteristic of being specifically expressed only upon differentiation into neurons, and may be expressed by the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In the vector for tracking differentiation into neurons, the reporter gene may be any reporter gene in which the gene is transcribed and the translated protein can be identified in vivo or in vitro.

The reporter gene introduced into the vector may be a gene encoding a cell surface-expressed fluorescent protein. The gene encoding the fluorescent protein may be a gene encoding a red fluorescent protein. In the case of a reporter gene encoding a fluorescent protein, the presence or absence of the expressed protein can be checked without a separate process, so it is preferable for a system for tracking differentiation into neurons. The fluorescent protein may be one mutated to be expressed on the cell surface from the group consisting of mCherry, tdTomato, J-Red, DsRed, mRuby, mStrawberry, mPlum, GFP and YFP, but is not limited thereto. When the fluorescent protein gene is used as a reporter gene, the differentiation of neural stem cells into neurons can be easily traced without killing the cells (or non-invasively) using immunostaining or a method using a confocal microscope.

In one embodiment, the reporter gene may be a fluorescent protein-coding gene expressed on the surface of a differentiated cell membrane, specifically, the CherryPicker gene, which is a gene encoding mCherry. The CherryPicker gene is a fusion protein composed of the fluorescent protein mCherry and the transferrin receptor membrane anchor domain. Since the mCherry protein is expressed on the cell surface, it can be expressed on the surface of differentiated neurons, and thus can be used for tracking differentiation into neurons through magnetic resonance imaging when introduced into a living body.

The vector includes a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 between the synapsin promoter and a reporter gene encoding a fluorescent protein. The polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 is a partial sequence of the nucleotide sequence encoding the Creb 5'UTR of the mouse, and is interposed between the synapsin promoter and the reporter gene in the vector of the present invention. It maintains high tissue specificity of the promoter and can act as an enhancer of reporter gene expression.

In the present invention, the "neural stem cell (NSC, Neural stem cell)" has the ability to continue proliferation, that is, self-replication, and the neurons and astrocytes constituting the central nervous system. , refers to undifferentiated cells having multidifferentiation ability to differentiate into cells such as oligodendrocytes. According to an embodiment of the present invention, neural stem cells are included as cells transduced with the vector expressing the reporter gene in the present invention. The neural stem cells may be primary cultured neural stem cells or genetically modified immortalized neural stem cells. Neural stem cells may be used interchangeably with “neural progenitor cells or neural precursor cells,” and in some cases, cells derived from neural stem cells may be referred to as neural precursor cells.

In the present invention, the method for transducing the vector into neural stem cells or neural progenitor cells can be applied to a method known in the art. For example, the vector is introduced into the cell using a reagent for transduction such as lipofectamine, calcium-phosphate, positively charged polymer, liposome, nanoparticles, nucleofection (nucleofection), electric It may be performed by a method by electroporation, heat shock, magnetofection, or viral infection. In one embodiment, the introduction of the vector may be by viral infection.

After transplanting the neural stem cells or neural progenitor cells into which the vector is introduced into a patient in need of cell therapy, the transplanted neural stem cells or neural progenitor cells can be used to determine to what extent the differentiation into neurons has progressed. .

The neural stem cells or neural progenitor cells may be directly administered or transplanted into the ventricle of a patient with a neurological disease, but is not limited thereto. In addition, in order to enhance the engraftment rate of neural stem cells or neural progenitor cells, various embedding materials, such as sodium alginate, hyaluronic acid, or collagen may be used by forming a complex with the cells. . Thereafter, by taking an MRI, it is possible to check whether or not the differentiation of the transplanted cells is progressing or not.

In the present invention, neural stem cells or neural progenitor cells may be derived from mammals. The mammal may be a human, monkey, chimpanzee, dog, cat, cow, goat, pig, mouse or rat, but is not limited thereto. When the neural stem cells or neural progenitor cells are derived from a mammal, the cells may be any kind of neural stem cells or neural progenitor cells derived from any part of the brain tissue including the cerebrum or the cortex of the brain. In addition, the neural stem cells or neural progenitor cells may be derived from all developmental stages including embryos and adults, but are derived from developing embryos in the period when neurogenesis is most active in order to obtain a large amount of neural stem cells or neural progenitor cells. it may have been

The vector of the present invention can be usefully used for gene research in various nerve cells, such as in vitro cell culture experiments or clinical trials such as cell therapy.

Next, the composition for stem cell transplantation and differentiation tracking of the present invention is conjugated with an antibody that specifically binds to the surface antigen of the differentiated nerve cell and includes nanoparticles coated with a biocompatible polymer.

In the present invention, the antibody that specifically binds to the differentiated neuronal cell surface antigen may be immunoglobulin G (IgG) or immunoglobulin M (IgM), but is not limited thereto.

The biocompatible polymer of the present invention is glycol chitosan, poly-L-lysine, poly(4-vinyl pyridine/divinylbenzene), chitin, poly(butadiene/acrylonitrile) amine terminus, polyethyleneimine, polyaniline, poly(ethylene glycol) )bis(2-aminoethyl), poly(N-vinylpyrrolidone), poly(vinylamine)hydrochloride, poly(2-vinylpyridine), poly(2-vinylpyridine N-oxide), poly-ε- Cbz-L-lysine, poly(2-dimethylaminoethyl methacrylate), poly(allylamine), poly(allylamine hydrochloride), poly(N-methylvinylamine), poly(diallyldimethylammonium chloride) , poly(N-vinylpyrrolidone), chitosan, or poly(4-aminostyrene). Preferably, glycol chitosan may be used as a biocompatible polymer.

The glycol chitosan is a biocompatible and biodegradable material, and is used in various fields such as tissue engineering or drug delivery. However, in the case of high molecular weight glycol chitosan, there was a difficulty in using it as a pharmaceutical preparation due to the toxicity problem due to the positive charge it has.

As used herein, the term “nanoparticle” refers to a structure or material having a size of nanometers (nm). The nanometer size is a reduction in the size of a micron meter (10 ^-6 ) to one-thousandth, and when the size of a material is reduced to the nanometer level, various unique physical, chemical, mechanical and electronic properties are displayed.

In the present invention, the nanoparticles generally have an average size of from about 1 nm to about 500 nm, such as from about 1 nm to about 50 nm, from about 50 nm to about 100 nm, from about 100 nm to about 250 nm, about 250 nm to about 500 nm.

According to one embodiment of the present invention, the nanoparticles of the present invention may be prepared in a size of 1 to 100 nm, preferably 1 to 50 nm, more preferably 1 to 20 nm. When the size of the nanoparticles is 500 nm or more, the sedimentation rate is increased, which causes inconvenience in use.

In the present invention, the nanoparticles may be organic or inorganic nanoparticles, preferably magnetic nanoparticles.

As used herein, the term “magnetic” refers to a magnetic property exhibited by a material. All materials interact with a magnetic field to generate an attractive or repulsive force. That is, when a magnetic field is applied to a material, it is magnetized, and according to the aspect in which the object is magnetized, it is classified into a ferromagnetic substance, a paramagnetic substance, a diamagnetic substance, a ferrimagnetc substance, and the like.

As used herein, the term “magnetic nanoparticles” refers to nanometer-sized structures or materials that are magnetic. The magnetic nanoparticles may be prepared by solution synthesis, co-precipitation, sol-gel method, high energy pulverization, hydrothermal synthesis, microemulsion synthesis, pyrolysis synthesis, or sonic chemical synthesis, but is not limited thereto.

In the present invention, the magnetic nanoparticles may be selected from the group consisting of iron (Fe), cobalt (Co), nickel (Ni), manganese (Mn), gadolinium (Gd), oxides thereof, or alloys thereof. However, the present invention is not limited thereto.

According to one embodiment of the present invention, the magnetic nanoparticles are magnetic nanoparticles made of iron or iron oxide.

According to one embodiment of the present invention, the biocompatible polymer includes a functional group capable of binding to nanoparticles, and the functional group may be an amine group, a carboxyl group, a thiol group, or a hydroxide group, preferably a hydroxyl group. It may be a side group. Therefore, a thermal decomposition technique used in manufacturing magnetic nanoparticles can be used for nanoparticle coating. Specifically, by reacting the iron oxide nanoparticles with the glycol chitosan conjugate at a high temperature, the conjugate can be wrapped on the surface of the iron oxide nanoparticles by the -OH functional group present in the polysaccharide of glycol chitosan. This method has the advantage that the conjugate is stably coated on the surface of the nanoparticles by a large amount of -OH functional groups, but when reacted at high temperature for an excessively long time, the polysaccharide itself may be decomposed.

In addition, the present invention is a temperature-sensitive hydrogel formed from a first natural polymer and a second natural polymer;

Neural stem cells transduced with a vector for tracking differentiation into neurons containing a neural cell differentiation-specific promoter, a Creb 5' UTR sequence, and a reporter gene contained in the hydrogel; and

Provided is a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising nanoparticles coated with a biocompatible polymer and conjugated with an antibody that specifically binds to a differentiated neuronal cell surface antigen as an active ingredient.

As described above, the temperature-sensitive hydrogel, the neural stem cells transduced with the vector for tracking differentiation into neurons, and the system including the nanoparticles differentiated when the neural stem cells are transplanted in vivo. When the behavioral improvement effect is observed while observing the survival and distribution of

In the present invention, the neurological disease is stroke, Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, memory loss, myasthenia gravis, progressive supranuclear palsy, multiple system atrophy, essential tremor, cortical-basal nucleus degeneration It may be selected from the group consisting of disease, diffuse Lewy body disease, and Pick's disease.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient. can be Meanwhile, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

Advantages and features of the present invention, and methods of achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

The present invention aims to establish a technology for evaluating the efficacy and safety of stem cell therapeutics, which is currently a challenge, through a temperature-sensitive hydrogel-based cell delivery system. Accurate preclinical research is essential for the development of cell therapy that can be applied clinically, but the current level of domestic research is very insufficient. It can greatly contribute to the improvement of the technical level of practical research targeting Therefore, it is possible to provide a technology that can be practically used in patients by solving the problems of clinical application through in vivo monitoring of stem cells.

1 shows that when microparticles are generated using a hydrogel synthesized with natural polymers (collagen and chitosan), the release time according to pH can be controlled.
Figures 2a to 2e show suitable NaOH concentrations of natural polymers (collagen and chitosan) and NaOH upon encapsulation (Figure 2a: 0.1 N, mixture pH: 5.5; Figure 2b: 0.2 N, mixture pH: about 6.6; Figure 2c : 0.3 N, mixture pH: 8.4 to 8.4 (optimal); Fig. 2d: 0.4 N, mixture pH: 9.6 to 10; Fig. 2e: 0.5 N, mixture pH: more than 12 to 14).
3 is a schematic diagram of a microfluidic chip for producing particles surrounded by neural stem cells in collagen/chitosan hydrogel using a hierarchical structure.
4 is a schematic diagram of the synthesis process of particles surrounded by neural stem cells in collagen/chitosan hydrogel.
Figure 5 shows the synthesized collagen / chitosan hydrogel particles, (A) is a particle prepared using mineral oil at room temperature, (B) is a particle prepared using a mineral oil heated to 37 ℃ ( magnification 200x).
Figure 6 shows the size of the particle diameter for each flow rate, (A) is a one-fold flow rate, (B) is a 1.5-fold flow rate, (C) is a two-fold flow rate (scale bar 25㎛), magnification 200x).
7 is a result of establishing the encapsulation optimization conditions by fine-controlling the concentration of NaOH.
8 shows the differentiation of neural stem cells into neurons in the temperature-sensitive hydrogel.
9 is a schematic diagram illustrating the manufacturing process of α-RFP-SPIO.
FIG. 10 is a diagram illustrating whether the binding of the α-RFP antibody to SPIO is verified by measuring the zeta-potential.
11 is a verification of whether the binding of the α-RFP antibody and SPIO through UV-visible light measurement.
12 is to verify whether the binding of the α-RFP antibody and SPIO through the iron assay.
13 is a measurement of the concentration of α-RFP antibody bound to α-RFP-SPIO.
14 shows electron micrographs of GC-SPIO and α-RFP-SPIO.
15 shows SPIO that specifically binds to a cell surface antigen.
16 is a result of confirming the distribution and engraftment in the body after transplantation of encapsulated cells.
FIG. 17 shows the results of taking MRI images of brain injury at each stage to identify the optimal transplantation time after constructing an animal model of stroke induced by hypoxia-induced ischemic brain injury.
18 is an MRI image taken 4 after transplantation into a stroke mouse by mixing α-RFP-SPIO with stem cells expressing cell surface antigen during neuronal differentiation and encapsulating with collagen/chitosan.
19 is an observation of differentiation at the transplantation site when the CherryPicker fluorescent protein system was introduced into mouse neural stem cells and then encapsulated and transplanted into a mouse stroke model.
20 is a diagram illustrating an overall technical summary of the present invention.

Hereinafter, the present application will be described in detail through examples. The following examples only illustrate the present application, but the scope of the present application is not limited to the following examples.

[Example]

[Example 1] Establishment of cell encapsulation optimization conditions using collagen and chitosan

1) Selection of hydrogel material that is non-toxic to nerve cells

Collagen and chitosan were selected as substances that were not toxic to neural stem cells and differentiated neurons. After microparticles were generated using a hydrogel synthesized with collagen and chitosan, the degree of release according to pH was confirmed (FIG. 1). In FIG. 1, the release degree according to pH was confirmed in particles composed of only collagen (Collagen), particles prepared by mixing collagen and chitosan (Collagen-Chitosan), and particles coated with chitosan on collagen particles (Collagen-Chitosan Multi) did. As a result, it was confirmed that the microparticles of the present invention can control the release time according to the pH.

2) Cell encapsulation using collagen/chitosan hydrogel system

2-1) Confirmation of cytotoxicity

The gelation of collagen and chitosan is controlled by the concentration and pH of each component. That is, it is essential that the particles using a hydrogel synthesized from collagen and chitosan adjust a pH environment suitable for cell survival, unlike conventional hyaluronic acid. Collagen and chitosan exist as solutions with a low pH, and when NaOH is added, the pH increases and gelation proceeds rapidly. In addition, the concentration of NaOH to be used for encapsulation should be close to neutral pH when NaOH is finally mixed so as not to damage the cells. In addition, the gelation rate can be controlled by the concentration of NaOH, which serves to solidify into a gel. Therefore, in order to select a concentration that does not show cytotoxicity of NaOH, a substance for pH control of a mixture of collagen and chitosan, conditions optimized for gelation were established at various concentrations and pH conditions ( FIGS. 2a to 2e ).

Specifically, after differentiation by treating the neural stem cells with NaOH at various concentrations, the surviving neurons after treatment with NaOH through trypan blue staining were counted to determine a survival rate of 85% or more as non-toxic (Fig. 2a). to Figure 2e).

As a result, around 0.3N was observed as the most appropriate concentration of NaOH to use, and when NaOH was in the range of 600 to 700 μg (total 540 μl volume Scale, ^{based on the number of cells 5.4x10 6} ), the survival of neurons was It was confirmed that there was no effect, ^{and the optimal mixing ratio of collagen and chitosan was determined in the state of 5X10 7} cells based on a total volume of 540 μl [chitosan: collagen: N2 medium: NaOH = 150 μl: 150 μl: 180 μl: 54 μl ] was determined. At this time, N2 medium was DMEM/F12 (Invitrogen, Carlsbad, CA), 4.4 μM insulin, 100 mg/L transferrin, 30 nM selenite, 0.6 μM putrescine, 20 nM progesterone, 200mM L-glutamine, 8.6mM D(+) glucose, 20mM NaHCO _{3 It} refers to a medium containing (Invitrogen).

In addition, it was observed that mixing chitosan, collagen, and cell culture medium at a low temperature first, followed by NaOH, and then mixing the cells in that order, can harden slowly without damaging the cells. When mixed first, it was confirmed that a large number of cells were killed by low pH (data not shown).

2-2) Synthesis of hydrogel particles synthesized from collagen and chitosan

For the synthesis of hydrogel particles synthesized from collagen and chitosan, a previously known method was applied. Specifically, a microfluidic chip was designed for the synthesis of hydrogel particles synthesized from collagen and chitosan using a hierarchical structure, and the microfluidic chip was manufactured by injection molding ( FIGS. 3 and 4 ).

In order to prevent the gelation of the collagen/chitosan mixture mixed with collagen and chitosan before passing through the microfluidic chip, the NaOH concentration was lowered to 636 μg to slow the gelation time. Particles were collected using mineral oil heated to 37° C. (FIG. 5).

The synthesis of the particles was prepared by the following method:

□ Stem cell encapsulation protocol in collagen/chitosan hydrogel

1. Reagent Preparation

- Collagen: 8.91 mg/mL (in 0.02 N acetic acid) [Corning, 354249]

- Chitosan: 30 mg/mL (in 0.35 N acetic acid) [TCI, C0831]

- NaOH: 1 N (in N ₂ medium)

-N ₂ medium + neural stem cells (fixed)

- Mineral Oil / 2% Span80

2. Beads Manufacturing

(1) Mixing

- Volume & Ratio

Total mixture: 600 μl (collagen:chitosan=5 mg/ml:5 mg/ml)

NaOH
(1N) 8.91mg/ml Collagen
(0.02N AA) 30mg/ml Chitosan
(0.35N AA) DMEM
_(10X) DMEM
_(1X) PBS
(1X) D.W. pH
(strip) 35 μl 337 μl 100 μl 30 μl 30 μl 30 μl 38 μl 7.4

① Collagen - Chitosan Stirring / 30~50sec ② DMEM - PBS - NaOH - DW

(2) Inlet1

30mL/Hr

Mineral Oil / 50uM Span80

(3) Inlet2

- 2mL/Hr

*Use a 25㎛ pore size stencil

3. Gelling

- The temperature of the container receiving the collagen/chitosan capsules was adjusted to 40°C using a hot plate.

- The collected beads were incubated for 30 minutes in an incubator at 37°C.

4. Oil removal

① Centrifuge

a. The resulting beads were transferred to an E-tube and centrifuged (100G, 10 seconds).

b. After centrifugation, the filtrate was removed and the remaining oil remaining in the pellet was removed using an oil pad.

c. The pellet was redispersed in the medium.

As a result, as shown in FIG. 5, when mineral oil at room temperature was used (A in FIG. 5), the average diameter size tended to gradually increase due to interparticle bonding, but when mineral oil maintained at 37°C was used. As a result (B in FIG. 5), the gelation proceeded rapidly, and collagen-chitosan particles having a more stable diameter were obtained. That is, the present invention can provide particles using a temperature-sensitive hydrogel synthesized from collagen and chitosan, which forms a gel near the temperature of the human body.

In addition, when the cell particles encapsulated with collagen/chitosan were prepared using the microfluidic chip, when the production time approached 5 minutes, the collagen/chitosan gelled and the microfluidic chip was blocked. In order to solve this problem, as a result of checking the particle diameter size for each flow rate, it was confirmed that as the flow rate increases, the phenomenon that the collagen/chitosan hydrogel particles block the microfluidic chip decreases. It was confirmed that the clogging phenomenon could be minimized by increasing the injection rate (FIG. 6).

In addition, as a result of observing cells encapsulated with collagen-chitosan hydrogel through a fluorescence microscope (FIG. 7), it was observed that the cells were encapsulated inside the hydrogel. In addition, through Fig. 7, it shows the appropriate conditions for encapsulating close to the single cell level through the fine control of NaOH (NaOH 0.257 ~ 0.263N) established in the present invention.

3) Encapsulation of cell surface antigen-expressing neural stem cells using collagen and chitosan

Collagen and chitosan, which are temperature-sensitive hydrogel components that have been confirmed to have no intracellular toxicity by replacing hyaluronic acid, were introduced into single cell encapsulation technology. Since the cell encapsulation has to surround the cells as a single cell unit and harden to a higher viscosity than the temperature-sensitive hydrogel that surrounds the entire cell, the concentration of collagen and chitosan used in the past was increased. Since collagen and chitosan react very sensitively to pH, experiments were performed under various conditions to establish optimal conditions.

As described in Korean Patent 10-2019-0003253, after introducing a lentivirus expressing CherryPicker fluorescent protein by the Synapsin1 promoter into neural stem cells, the neural stem cells are mixed with collagen and chitosan to perform cell encapsulation It was observed with a fluorescence microscope (FIG. 8).

4) Confirmation of effect of temperature-sensitive hydrogel when neural stem cells differentiate into neurons

In order to check whether the temperature-sensitive hydrogel interferes with neuronal differentiation, neural stem cells expressing CherryPicker fluorescent protein on the cell surface were hardened into a temperature-sensitive hydrogel, and 200 μM ascorbic acid (AA, Sigma, St Louis) was added for 5 days. , MO) was induced to differentiate in serum-free N2 medium. The culture was maintained at 37° C. in a 5% CO ₂ incubator, and the medium was changed once every two days.

As a result (FIG. 8), the neural stem cells differentiated into neurons in the hydrogel and the shape of the cytoplasm stretched out like a tree branch was observed, and neuron-specific Class III β-tubulin (TUJ1), a neuron marker, was added to the hydrogel. It was confirmed that the expression was distributed throughout the Therefore, it can be seen that even when the temperature-sensitive hydrogel is used for cell transplantation, it does not interfere at all in the differentiation of the transplanted cells into mature neurons.

[Example 2] Development of cell surface antigen-binding α-RFP-SPIO fusion based differentiated cell tracking technology

1) Preparation of α-RFP-SPIO fusion

Superparamagnetic iron oxide (SPIO) is a nanocontrast agent capable of MRI tracking, and a system that can be followed by MRI after transplantation was established by combining cells to be transplanted into the animal brain and SPIO that binds to cell surface-specific antigens.

1-1) Preparation of α-RFP-Glycol Chitosan-SPIO (α-RFP-SPIO)

As a pre-step for binding the α-RFP antibody to SPIO, GC-SPIO was prepared by binding glycol chitosan to SPIO. In order to conjugate the antibody to GC-SPIO coated with glycol chitosan, glutaraldehyde was used as a linker. This is done using the EDC (1-ethyl-3-(4-dimethylaminopropyl)/NHS (N-hydroxysuccinimide) reaction between the aldehyde group of the terminal part of glutaraldehyde and the amine group present in lysine of the antibody. α-RFP-SPIO was prepared by conjugation (FIG. 9).

1-2) Characterization of α-RFP-SPIO

[Measurement of zeta potential value]

As a result of measuring the zeta-potential value in the SPIO coated with glycol chitosan (GC) (FIG. 10), the result was obtained that the zeta potential value of GC-SPIO was about 40 to 60 mv, and the antibody was conjugated In the case of the gated antibody-GC-SPIO, a change in the zeta potential value was observed with a negative charge. In the case of zeta size, when a ligand or antibody is conjugated to nanoparticles, the hydrodynamic size is reduced. It was found that it was anchored to SPIO.

[UV-visible light spectrophotometer]

In the case of UV-visible light experiment, there is no reference to the exact absorbance peak for SPIO, and as a result of measuring each material with and without antibody, peak shifting (peak) when the antibody is conjugated shifting) occurred, and it was inferred that the α-RFP antibody bound to GC-SPIO through this change ( FIG. 11 ).

[Iron assay]

When a standard curve was created using an iron assay kit, the R-squared value was 0.9944, indicating a high correlation. 10 μl of GC-SPIO and α-RFP-GC-SPIO were added to each well, and absorbance values were measured. When the results were derived, the concentrations were 0.59 mM and 0.17 mM, respectively. mol) to obtain a concentration of α-RFP-GC-SPIO in the unit of 9.5 μg/ml ( FIG. 12 ).

When the RFP antibody was used to form a standard curve at a concentration of 0 to 2 (mg/ml), a high correlation was obtained with an R-squared value of 0.9938. The α-RFP-GS-SPIO sample (2 mg/ml) was put into N=5 and measured through soft max pro 5X at 562 nm wavelength. BCA (Bicinchorinic acid) analysis method measures the amount of protein through color change when buffer is added. Since SPIO itself is dark purple, it is regarded as a constant (blank) value, and the value obtained by measuring SPIO at 562 nm is a constant. and subtracted as a constant from the measured values of each sample. As a result (FIG. 13), it was found that about 630 μg/ml of α-RFP antibody was attached to 2000 μg/ml of α-RFP-GC-SPIO.

[Electron Microscope Observation]

As a result of observing the nanoparticle morphology of GC-SPIO and α-RFP-SPIO through an electron microscope (FIG. 14), it was verified that both of them formed nanoparticles of an appropriate size.

2) Analysis of cell surface antigen binding specificity of α-RFP-SPIO

In order to check whether the synthesized α-RFP-SPIO specifically binds well to the RFP antigen, neural stem cells expressing CherryPicker fluorescent protein on the cell surface and synthesized α-RFP-SPIO are mixed with Prussian blue staining method was confirmed as As a result (FIG. 15), it was observed that α-RFP-SPIO was specifically bound to neural stem cells expressing Cherrypicker fluorescent protein and exhibited a blue color. In addition, it was confirmed that fluorescence was well expressed when reacting with a secondary antibody (Alexa 488) that binds to the RFP antibody of SPIO by immunostaining method, which is consistent with Cherrypicker expression (FIG. 15).

3) Confirmation of stability and function by implanting encapsulating cells into animals

When the encapsulated neural stem cells were transplanted into the animal brain, it was confirmed whether the neural stem cells could survive and differentiate in vivo. After combining the neural stem cells expressing CherryPicker fluorescent protein with α-RFP-SPIO, single-cell encapsulation was performed. The cells were transplanted into the rat brain the next day, and the transplanted cells were observed by tissue staining 3 days after surgery. As a result (FIG. 16), it was confirmed that cells expressing CherryPicker fluorescent protein were gathered at the transplanted site, and TUJ1 protein, a neuronal marker, was also expressed, indicating that the transplanted cells were differentiated into neurons.

[Example 3] Confirmation of effect in stroke model

A stroke animal model was established through hypoxic-ischemic brain injury, and brain injury MRI images were taken for each period to identify the optimal transplantation time (FIG. 17).

Mouse neural stem cells encapsulated with α-RFP-SPIO and hydrogel were observed through MRI 4 weeks after transplantation in the acute phase of the stroke animal model ( FIG. 18 ). As a result, only the SPIO-RFP tagging mNPCs group was able to follow up SPIO binding to transplanted cells and cell surface specific antigen (RFP) by MRI.

In addition, 4 weeks after transplantation of non-capsuling mNPCs (RFP tagging mNPCs) and SPIO and hydrogel-encapsulated mNPCs in the brain, the results of observing the survival and differentiation of cells through histological examination (Fig. 19), it was observed that the transplanted cells were stained with βIII-tubulin, a neuronal marker, and GFAP, an astrocyte marker.

<110> INDUSTRY-UNIVERSITY COOPERATION FOUNDATION HANYANG UNIVERSITY <120> In vivo monitoring system for neural stem cell therapy based on neuronal differentiation specific surface antigen expression <130> P20U10C0814 <150> KR 10-2019-0021691 <151> 2019-02-25 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 480 <212> DNA <213> Artificial Sequence <220> <223> Synapsin promoter_pSYN <400> 1 ctagactgca gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg 60 ggtgggggtg cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt 120 ccccaaattg cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg 180 tgcgcactgc cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc 240 caccgccgcc tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc 300 cttcccggcc accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg 360 aggcgcgaga taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg 420 ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcggga 480 480 <210> 2 <211> 147 <212> DNA <213> Artificial Sequence <220> <223> Creb 5' UTR <400> 2 cggctgggag aagccgagtg ttggtgagtg acgcggcgga ggtgtagttt gacgcggtgt 60 gttacgtggg ggagagaata aaactccagc gagatccggg ccgcgaacga aagcagtgac 120 ggaggagctt gtaccaccgg tgactag 147

Claims (12)

A temperature-sensitive hydrogel formed from a first natural polymer and a second natural polymer;
Neural stem cells transduced with a vector for tracking differentiation into neurons containing a neural cell differentiation-specific promoter, a Creb 5' UTR sequence, and a reporter gene contained in the hydrogel; and
A neural line comprising nanoparticles coated with biocompatible polymer and conjugated with an antibody that specifically binds to the surface antigen of neurons differentiated from transplanted neural stem cells, expressed by the vector for tracking differentiation into neurons A composition for tracing stem cell transplantation and differentiation. According to claim 1,
The first natural polymer and the second natural polymer are collagen, chitosan, hyaluronic acid, poly-L-lysine, poly(4-vinylpyridine/divinylbenzene), chitin, poly(butadiene/acrylonitrile) amine Terminal, polyethyleneimine, polyaniline, poly(ethylene glycol)bis(2-aminoethyl), poly(N-vinylpyrrolidone), poly(vinylamine)hydrochloride, poly(2-vinylpyridine), poly(2- VinylpyridineN-oxide), poly-ε-Cbz-L-lysine, poly(2-dimethylaminoethyl methacrylate), poly(allyl amine), poly(allylamine hydrochloride), poly(N_methylvinylamine) ), poly(diallyldimethylammonium chloride), poly(N-vinylpyrrolidone) and poly(4-aminostyrene), which are each selected from the group consisting of transplantation and differentiation tracking composition of neural stem cells. According to claim 1,
Biocompatible polymers include collagen, chitosan, poly-L-lysine, poly(4-vinylpyridine/divinylbenzene), chitin, poly(butadiene/acrylonitrile) amine terminus, polyethyleneimine, polyaniline, poly(ethylene glycol)bis (2-aminoethyl), poly(N-vinylpyrrolidone), poly(vinylamine) hydrochloride, poly(2-vinylpyridine), poly(2-vinylpyridineN-oxide), poly-ε-Cbz- L-Lysine, poly(2-dimethylaminoethyl methacrylate), poly(allyl amine), poly(allylamine hydrochloride), poly(N_methylvinylamine), poly(diallyldimethylammonium chloride), poly( N-vinylpyrrolidone) and poly(4-aminostyrene) composition for transplantation and differentiation tracking of neural stem cells, each selected from the group consisting of. According to claim 1,
The temperature-sensitive hydrogel is in a sol state at room temperature, and gels at 30° C. or higher. A composition for implantation and differentiation tracking of neural stem cells. According to claim 1,
When the temperature-sensitive hydrogel is formed, the first natural polymer and the second natural polymer are contained in a concentration ratio of 0.5 to 2: 0.5 to 2, the composition for transplantation and differentiation of neural stem cells. According to claim 1,
The composition for tracking the transplantation and differentiation of neural stem cells, wherein the formation of a temperature-sensitive hydrogel is performed by gelation with a crosslinking agent at a concentration of 0.2 to 0.4 N. According to claim 1,
The nanoparticles are inorganic nanoparticles, a composition for tracking transplantation and differentiation of neural stem cells. 8. The method of claim 7,
The inorganic nanoparticles are magnetic nanoparticles selected from the group consisting of iron (Fe), cobalt (Co), nickel (Ni), manganese (Mn), gadolinium (Gd), oxides thereof or alloys thereof magnetic nanoparticles), a composition for tracking transplantation and differentiation of neural stem cells. 9. The method according to any one of claims 1 to 8,
The biocompatible polymer comprises a functional group capable of binding to nanoparticles, a composition for tracking transplantation and differentiation of neural stem cells. 10. The method of claim 9,
The functional group is an amine group, a carboxyl group, a thiol group, or a hydroxide group, a composition for tracking transplantation and differentiation of neural stem cells. A temperature-sensitive hydrogel formed from a first natural polymer and a second natural polymer;
Neural stem cells transduced with a vector for tracking differentiation into neurons containing a neural cell differentiation-specific promoter, a Creb 5' UTR sequence, and a reporter gene contained in the hydrogel; and
An antibody that specifically binds to the surface antigen of neurons differentiated from transplanted neural stem cells, expressed by the vector for tracking differentiation into neurons, is conjugated, and nanoparticles coated with a biocompatible polymer are used as an active ingredient. A pharmaceutical composition for the prevention or treatment of neurological diseases, including. 12. The method of claim 11,
The nervous system disease is stroke, Parkinson's disease, dementia, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, memory loss, myasthenia gravis, progressive supranuclear palsy, multiple system atrophy, essential tremor, cortical-basal nucleus degeneration, diffuse Lewy body A pharmaceutical composition for preventing or treating neurological diseases that is selected from the group consisting of diseases and Pick's disease.

Images