KR20190108918A - Nanoparticle delivery system for inducing chondrogenic differentiation of stem cells through RNA interference and method for chondrogenic differentiation using the same - Google Patents

Abstract

The present invention, as a nanoparticle transporter for inducing chondrogenic differentiation of stem cells through RNA interference, provides a nanoparticle transporter obtained by making a complex of a plasmid vector, comprising at least one microRNAs (miRNAs) targeting the RUNX2 gene and at least one short hairpin RNAs (shRNAs) targeting the RUNX2 gene, and a polyethylenimine conjugated with dexamethasone. The present invention also provides a method for differentiating stem cells into chondrocytes using the nanoparticle transporter and a pharmaceutical composition for reconstructing or regenerating cartilage tissue including stem cells transfected with the nanoparticle transporter, that is, a cell therapeutic agent.

Description

Nanoparticle delivery system for inducing chondrogenic differentiation of stem cells through RNA interference and method for chondrogenic differentiation using the same}

The present invention relates to a nanoparticle transporter for inducing cartilage differentiation of stem cells through RNA interference. More specifically, the present invention is a nanoparticle carrier for inducing cartilage differentiation of stem cells through RNA interference, one or more microRNAs (miRNAs) targeting the RUNX2 gene and one or more short hairpin RNAs targeting the RUNX2 gene A plasmid vector comprising (shRNAs) relates to a nanoparticle transporter obtained by complexing a dexamethasone conjugated polyethyleneimine. In addition, the present invention relates to a method for differentiating stem cells into chondrocytes using the nanoparticle transporter, and a pharmaceutical composition for reconstructing or regenerating cartilage tissue including stem cells transfected with the nanoparticle transporter, that is, a cell therapeutic agent.

RNA interference (RNAi) inhibits gene expression by destroying specific mRNAs and is used for downregulation of specific genes. This is a useful means of inhibiting gene expression accurately, efficiently and reliably.

Two kinds of molecules, microRNAs (miRNAs) and small interfering RNAs (siRNAs), function as RNAi. These RNAs can bind to mRNAs, thereby increasing or decreasing the expression of mRNAs to regulate various activities such as cell proliferation, growth, differentiation and death. These small RNAs have been used to induce stem cell differentiation into desired lineage. Conversely, interference of upstream signaling pathways can delay or block stem cell differentiation of specific cells.

RUNX2 plays an important role in the differentiation of stem cells into osteoblasts from early to final stages, but has been reported to prevent cartilage formation (Agarwal T, et al., Alginate Bead Based Hexagonal Close Packed 3D Implant for Bone). . Tissue Engineering ACS Appl Mater Interfaces 2016 ; 8 (47): 32132-32145; Sthanam LK, et al Biophysical regulation of mouse embryonic stem cell fate and genomic integrity by feeder derived matrices Biomaterials 2017; 119:.. 9-22; Wang C, et al Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP / PKA Pathway ACS Appl Mater Interfaces 2016; 8 (18):.. 11262-71; Toda H, et al. , Effect of hydrogel elasticity and ephrinB2- immobilized manner on Runx2 expression of human mesenchymal stem cells. Acta Biomater 2017; S1742-7061 (17): 30178-2; Boda SK, et al., Competing Roles of Substrate Composition, Microstructure, and Sustained Strontium Release in Directing Osteogenic Differentiation of hMSCs. ACS Appl Mater Interfaces 2017; 9 (23): 19389-19408; Ramaswamy J, et al., Inhibition of osteoblast mineralization by phosphorylated phage-derived apatite-specific peptide. Biomaterials 2015; 73: 120-30). Thus, silencing of RUNX2 via RNAi promotes cartilage formation in stem cells (Caron MM, et al. Hypertrophic differentiation during chondrogenic differentiation of progenitor cells is stimulated by BMP-2 but suppressed by BMP- 7.Osteoarthritis Cartilage 2013; 21 (4): 604-13; Koelling S, et al. Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis.Cell Stem Cell 2009; 4 (4): 324-35).

We have siRNA-coated poly (lactic-co-glycolic acid) nanoparticles (NPs) to reduce RUNX2 activity and also promote differentiation into cartilage. It is reported to the power bar (Jeon SY, et al, Co -delivery of Cbfa-1-targeting siRNA and SOX9 protein using PLGA nanoparticles to induce chondrogenesis of human mesenchymal stem cells Biomaterials 2014; 35 (28):.. 8236- 48). We also found that human mesenchymal stem cells (hMSCs) transfected with RUNX2-target siRNA and SOX9 plasmid DNA (pDNA) increase differentiation into cartilage and reduce differentiation into bone (Jeon SY, . et al, Co-delivery of SOX9 genes and anti-Cbfa-1 siRNA coated onto PLGA nanoparticles for chondrogenesis of human MSCs Biomaterials 2012; 33 (17):. 4413-23).

The present inventors fabricated various miRNAs and shRNAs targeting the RUNX2 gene to evaluate the cartilage forming ability, ie, the cartilage differentiation ability of stem cells. The inventors of the present invention complexed plasmid DNA (pDNA) containing miRNAs and / or shRNAs with polyethyleneimine conjugated with dexamethasone to prepare nanoparticle carriers, and then used to synthesize human mesenchymal stem cells. stem cells (hMSCs) were transfected to evaluate cartilage differentiation. Surprisingly, when we transfected hMSCs with a combination of specific miRNAs and specific shRNAs, we initially interacted with the RUNX2 gene first through time-interval silencing, and shRNAs acted later. In other words, it was found that the two-step silencing can effectively induce chondrogenesis and inhibit osteoogenesis.

Accordingly, an object of the present invention is to provide a nanoparticle transporter complexed with a plasmid vector comprising a combination of specific miRNAs and specific shRNAs as a nanoparticle transporter for inducing cartilage differentiation of stem cells through RNA interference.

In addition, an object of the present invention is to provide a method for differentiating stem cells into chondrocytes using the nanoparticle transporter.

In addition, an object of the present invention is to provide a pharmaceutical composition for cartilage tissue reconstruction or regeneration, including stem cells into which the nanoparticle transporter is introduced.

In accordance with one aspect of the present invention, a nanoparticle transporter for inducing cartilage differentiation of stem cells via RNA interference, comprising one or more microRNAs (miRNAs) targeting the RUNX2 gene and one or more short hairpin RNAs targeting the RUNX2 gene Nanoparticle carriers obtained by complexing a plasmid vector comprising (shRNAs) with polyethyleneimine conjugated with dexamethasone are provided.

In the nanoparticle transporter of the present invention, the plasmid vector may include miRNAs having a nucleotide sequence of SEQ ID NO: 1 or 2. In addition, the plasmid vector may include shRNAs having a nucleotide sequence of SEQ ID NO: 3 or 4. Preferably, the plasmid vector comprises miRNAs having the nucleotide sequence of SEQ ID NO: 1, miRNAs having the nucleotide sequence of SEQ ID NO: 2, shRNAs having the nucleotide sequence of SEQ ID NO: 3, and shRNAs having the nucleotide sequence of SEQ ID NO: 4 do.

The polyethyleneimine conjugated with dexamethasone can be obtained by reacting dexamethasone with polyethyleneimine in the presence of N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide. Preferably, the polyethyleneimine may be a branched polyethylenimine (bPEI) having a chain chain having an aminoethyl branch. In addition, the polyethylenimine of the chain structure may have a weight average molecular weight of 0.6 to 30 kDa.

The complexing may comprise (i) reacting the plasmid vector in an aqueous medium with the polyethyleneimine conjugated with dexamethasone and (ii) filtering the reaction mixture obtained in step (i) with a membrane filter. . The plasmid vector is preferably complexed at a rate of 0.75 μg or more relative to 1 μg of the dexamethasone conjugated polyethyleneimine.

According to another aspect of the present invention, there is provided a method of differentiating stem cells into chondrocytes comprising transfecting stem cells with the nanoparticle transporter. In the differentiation method of the present invention, the stem cells may be human mesenchymal stem cells. In addition, the transfection may be performed by culturing the stem cells in a serum-free medium containing the nanoparticle transporter.

According to another aspect of the invention, there is provided a pharmaceutical composition for cartilage tissue reconstruction or regeneration comprising stem cells transfected with the nanoparticle transporter. In the pharmaceutical composition of the present invention, the stem cells may be human mesenchymal stem cells.

It has been found by the present invention that when transfected with hMSCs by combining certain miRNAs and specific shRNAs, cartilage formation, ie, cartilage differentiation, can be effectively induced through time-interval silencing. That is, when hMSCs are transfected with nanoparticle carriers obtained by complexing specific miRNAs and specific shRNAs with plasmid vectors conjugated with dexamethasone conjugated polyethylenimine, miRNAs initially bind to the RUNX2 gene and act first. It has been found by the present invention that by acting at a time point, that is, by two-step silencing, cartilage formation can be effectively induced and bone formation can be suppressed. Therefore, the nanoparticle transporter may be usefully used for differentiation of stem cells into chondrocytes, and further, the stem cell into which the nanoparticle transporter is introduced may be usefully used as a pharmaceutical composition for reconstructing or regenerating cartilage tissue, that is, a cell therapeutic agent. Can be.

FIG. 1 shows the results of preparation of miRUNX2 and shRUNX2 and characterization of PEI / DEX NPs complexed with pDNA containing these RNAs.
1A shows a map and sequence of a vector comprising miRUNX2 and shRUNX2.
1B shows the results of DLS and SEM analysis of PEI / DEX NPs complexed with pDNA containing miRUNX2 and shRUNX2 (scale bar, 100 nm).
1C shows the results of ξ-potential analysis of PEI / DEX NPs complexed with pDNA containing miRUNX2 and shRUNX2.
1D shows strong complexation of pDNA with PRUN / DEX NPs containing miRUNX2 and shRUNX2 in a gel retardation assay
Figure 2 shows the results of analyzing the internalization of PEI / DEX NPs by hMSCs.
2A shows the results of FACS analysis of PEI NPs and PEI / DEX NPs internalization. PEI NPs (a) and PEI / DEX NPs (b) were introduced into hMSCs.
2B shows the results of fluorescence intensity measurements of TRITC (control hMSCs (a), hMSCs (b) transfected with PEI NPs, and hMSCs (c) transfected with PEI / DEX NPs).
2C and 2D show the results of confocal laser microscopy (control hMSCs (a), hMSCs (b) transfected with PEI NPs, and hMSCs (c) transfected with PEI / DEX NPs).
Figure 3 is the result of analysis that the transfection of miRUNX2 and shRUNX2 inhibits RUNX2 expression in hMSCs.
3A shows a schematic of miRNA and shRNA sequences targeting human RUNX2 mRNA [UTR; Untranslated region].
3B shows the results of RT-PCR and Western blot analysis of hMSCs transfected with miRNA-1, miRNA-2, shRNA-1, and shRNA-2.
3C shows the results of RT-PCR and Western blot analysis of hMSCs transfected with two miRNAs, two shRNAs, and all four factors.
3D shows the results of FACS analysis of RUNX2 expression [negative control hMSCs (a), hMSCs (b) transfected with two miRNAs, hMSCs (c) transfected with two shRNAs, and hMSCs transfected with all four factors). (d)].
3E shows the results of confocal microscopy of RUNX2 [negative control hMSCs (a), hMSCs (b) transfected with two miRNAs, hMSCs (c) transfected with two shRNAs, and all four factors Magnified image of hMSCs (d), magnified image of negative control hMSCs (e) and hMSCs transfected with all four factors. The RUNX2 label is shown in green. Scale bar, 20 μm].
4 shows the results of transfection of RDtNPs complexed with pDNA containing two miRNAs, two shRNAs, and all four factors to inhibit RUNX2 expression in hMSCs.
4A shows RT- for 24, 48, and 72 hours of hMSCs transfected with RDtNPs complexed with pDNA comprising two miRNAs (a), two shRNAs (b), and all four factors (c). PCR and Western blot analysis results are shown.
4B shows 72 hour RT-PCR and Western blot analysis of hMSCs transfected with RDtNPs complexed with pDNA comprising two miRNAs, two shRNAs, and all four factors.
4C shows FACS analysis of RUNX2 expression [negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), complex with pDNA comprising two miRNAs HMSCs (c) transfected with mutated RDtNPs, hMSCs (d) transfected with RDtNPs complexed with pDNA comprising two shRNAs, and hMSCs transfected with RDtNPs complexed with pDNA comprising all four factors ( e)].
4D shows the results of confocal microscopy analysis of RUNX2 [negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), pDNA containing two miRNAs and HMSCs (c) transfected with complexed RDtNPs, hMSCs (d) transfected with RDtNPs complexed with pDNA comprising two shRNAs, and hMSCs transfected with RDtNPs complexed with pDNA containing all four factors (e). The RUNX2 label is shown in red. Scale bar, 20 μm].
5 shows that the expression of osteogenic factors is reduced in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors.
5A is a schematic diagram showing how RUNX2 deletion promotes cartilage formation.
5B shows the results of RT-PCR and Western blot analysis of RUNX2 [negative control hMSCs (a), hMSCs (b) transfected with PEI NPs complexed with empty pDNA (MOCK pDNA) and empty pDNA (MOCK pDNA) HMSCs (c) transfected with complexed PEI / DEX NPs, pDNA complexed with all four factors, hMSCs (d) transfected with RtNPs, and RDtNPs complexed with pDNA containing all four factors HMSCs (e) transfected].
Figure 6 shows the results of cartilage formation of hMSCs transfected with RDtNPs complexed with pDNA containing all four factors in a 2D system.
6A is a schematic showing how transfection of RDtNPs complexed with pDNA containing all four factors promotes cartilage formation of hMSCs in a 2D system.
6B shows the histological analysis results [ad: asian blue staining, eh: safranin-O staining. Negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), hMSCs (c) transfected with RtNPs complexed with pDNA containing all four factors, And hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors].
6C shows GAG content analysis results [complexed with negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), pDNA containing all four factors HMSCs (c) transfected with RtNPs, and hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors].
FIG. 6D shows the results of quantitative analysis of cartilage-associated gene SOX9 and osteogenic-related gene RUNX2 expression [hMSCs transfected with negative control hMSCs (a), PEI / DEX NPs complexed with empty pDNA (MOCK pDNA) ( b) hMSCs (c) transfected with RtNPs complexed with pDNA comprising all four factors, and hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors].
FIG. 6E shows the results of RT-PCR and Western blot analysis of osteogenic- and chondrogenic-related markers [hMSCs transfected with negative control hMSCs (a), PEI / DEX NPs complexed with empty pDNA (MOCK pDNA) ( b) hMSCs (c) transfected with RtNPs complexed with pDNA comprising all four factors, and hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors].
6F shows the results of confocal microscopy analysis of SOX9 and RUNX2 [negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), all four factors). HMSCs (c) transfected with RtNPs complexed with pDNA, and hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors.
Figure 7 shows the results of analysis of cartilage formation of hMSCs transfected with RDtNPs complexed with pDNA containing all four factors in the 3D system.
FIG. 7A is a schematic showing how transfection of RDtNPs complexed with pDNA containing all four factors promotes cartilage formation of hMSCs in a 3D system.
Figure 7B shows the results of RT-PCR analysis of cartilage-related markers [negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), all four factors). HMSCs (c) transfected with RtNPs complexed with pDNA comprising; and hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors].
7C shows the results of real-time-qPCR analysis of cartilage-related marker genes [nMS control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), four factors). HMSCs (c) transfected with RtNPs complexed with all-inclusive pDNA, and hMSCs (d) transfected with RDtNPs complexed with pDNA containing all four factors].
7D shows GAG content analysis results [complexed with negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), pDNA containing all four factors HMSCs (c) transfected with RtNPs, and hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors].
7E shows the histological analysis results [ad: Alcian blue staining, eh: safranin-O staining. Negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), hMSCs (c) transfected with RtNPs complexed with pDNA containing all four factors, And hMSCs (d) transfected with RDtNPs complexed with pDNA comprising all four factors. Scale bar, 100 μιη].
8 shows the results of analyzing cartilage and bone formation of hMSCs cultured in a 3D system.
8A shows Western blot analysis of SOX9, COL II, and Agrican [negative control hMSCs (a), hMSCs (b), transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), 4 HMSCs (c) transfected with RtNPs complexed with pDNA containing all four factors, and hMSCs (d) transfected with RDtNPs complexed with pDNA containing all four factors].
FIG. 8B shows the results of quantitative analysis of cartilage-related marker proteins [negative control hMSCs (a), hMSCs (b) transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), all four factors). HMSCs (c) transfected with RtNPs complexed with the containing pDNA, and hMSCs (d) transfected with RDtNPs complexed with the pDNA comprising all four factors. * P <0.05, ** P <0.05].
8C shows the results of immunohistochemical analysis of SOX9, COL II, and COL I [negative control with PEI / DEX NPs complexed with negative control hMSCs (a, e, i, and m), empty pDNA (MOCK pDNA) Infected hMSCs (b, f, j, and n), pDNAs containing all four factors and transfected with RtNPs hMSCs (c, g, k, and o), and pDNAs containing all four factors HMSCs (d, h, 1, and p) transfected with RDtNPs complexed with. Panel r shows an enlarged image of panel p. ad: SOX9, eh: COL II, il: Agrican, and mp: merged images. Scale bar, 100 μιη].
9 shows the chemical structure of a PEI / DEX polymer.
FIG. 10 shows the results of the DLS analysis [PEI alone (a), pDNA complexed with pDNA containing all four factors, PEI (b), PEI / DEX NPs (c), and pDNA containing all four factors. Complexed PEI / DEX NPs (d)].
FIG. 11 shows the results of a gel retardation assay evaluating the binding of pDNA including miRNAs and shRNAs to PEI / DEX polymers.
11A shows the results of analyzing the complexation of pDNA containing miRNAs with PEI / DEX.
11B shows the results of analyzing the complexation of pDNA containing shRNAs with PEI / DEX.
Figure 12 is the result of measuring the cytotoxic effect of PEI NPs and PEI / DEX NPs of various contents by Live / Dead assay (Live / Dead assay).
13 shows the results of analyzing GFP expression in transfected hMSCs.
FIG. 13A shows the results of FACS analysis [negative control hMSCs (a), hMSCs (b) transformed with PEI NPs complexed with GFP pDNA, and hMSCs transformed with PEI / DEX NPs complexed with GFP pDNA ( c)].
13B shows the results of confocal microscopy [negative control hMSCs (a), hMSCs (b) transformed with PEI NPs complexed with GFP pDNA, and PEI / DEX NPs complexed with GFP pDNA. hMSCs (c)].
Figure 13C shows the results of Western blot analysis [negative control hMSCs (a), hMSCs (b) transformed with PEI NPs complexed with GFP pDNA, and hMSCs transformed with PEI / DEX NPs complexed with GFP pDNA. (c)].
14 is a schematic diagram of PEI / DEX NPs conjugated with pDNA containing miRUNX2 and shRUNX2 to induce cartilage formation of hMSCs. These NPs enter hMSCs through endocytosis, exit the endosomes into cell substrates, and stimulate cartilage formation of hMSCs.

In the present specification, "chondrogenesis" and "chondrogenic differentiation to cartilage" are used in the same sense unless otherwise indicated. Also, "osteogenesis" and "osteogenic differentiation" are used in the same sense unless otherwise indicated.

The term "microRNAs (miRNAs)" also refers to double-stranded RNAs (dsRNAs) that are present in cells and induce RNAi phenomena. The term “miRNAs targeting the RUNX2 gene” refers to miRNAs that induce RNAi phenomena for the RUNX2 gene. All of the RUNX2 genes are known in Gene Bank and the like, but play an important role in the differentiation of stem cells into bone cells, but are known to prevent cartilage formation.

In addition, the term “short hairpin RNAs” (shRNAs) are dsRNAs that are artificially introduced into cells to induce RNAi, which are introduced into cells into plasmids or vector-based systems to provide siRNAs intracellularly. Say. The siRNA refers to oligomeric dsRNAs that induce RNAi phenomena.

The present invention provides a nanoparticle transporter for inducing cartilage differentiation of stem cells through RNA interference, including one or more microRNAs (miRNAs) targeting the RUNX2 gene and one or more short hairpin RNAs (shRNAs) targeting the RUNX2 gene. Provided is a nanoparticle carrier obtained by complexing a plasmid vector to a polyethyleneimine conjugated with dexamethasone.

Nanoparticle transporters of the present invention are transporters in the form of nanoparticles complexed with plasmid vectors comprising a combination of specific miRNAs and specific shRNAs as differentiation induction vectors. The form of the nanoparticle carrier is usually spherical, that is, nanospheres, but is not particularly limited. In addition, the nanoparticle carriers may, for example, have a diameter of 10-500 nm, preferably 80-200 nm.

In the nanoparticle transporter of the present invention, the plasmid vector may include miRNAs having a nucleotide sequence of SEQ ID NO: 1 or 2. In addition, the plasmid vector may include shRNAs having a nucleotide sequence of SEQ ID NO: 3 or 4. Preferably, the plasmid vector comprises both miRNAs of SEQ ID NOs: 1 and 2 and shRNAs of SEQ ID NOs: 3 and 4. That is, in one embodiment, the plasmid vector has miRNAs having the nucleotide sequence of SEQ ID NO: 1, miRNAs having the nucleotide sequence of SEQ ID NO: 2, shRNAs having the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence of SEQ ID NO: 4 Contains shRNAs. As a vector that can be used as the plasmid vector, a plasmid commonly used in the field of genetic engineering can be used, and the miRNAs and shRNAs can be inserted by conventional methods using an appropriate restriction enzyme.

Polyethyleneimine conjugated with the dexamethasone may be prepared through a carbodiimide reaction. For example, the polyethyleneimine conjugated with dexamethasone can be obtained by reacting dexamethasone with polyethyleneimine in the presence of N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide. The reaction can be carried out at room temperature for about 24 hours in an organic solvent such as dimethyl sulfoxide. After the reaction is completed, unreacted substances and the like can be removed by dialysis through an appropriate dialysis membrane (eg, MWCO 1,000 Da) as necessary. The obtained dexamethasone conjugated polyethyleneimine can be dried according to a conventional method such as lyophilization. Preferably, the polyethyleneimine may be a branched polyethylenimine (bPEI) having a chain chain having an aminoethyl branch. In addition, the polyethylenimine of the chain structure may have a weight average molecular weight of 0.6 to 30 kDa, for example about 25 kDa.

The complexation is achieved by reacting a plasmid vector in an aqueous medium (e.g., distilled water, etc.) with dexamethasone conjugated polyethylenimine, thereby self-assembling nanoparticles by electrostatic attraction between the anionic pDNA and the cationic polymer. assembly nanoparticles). In addition, by filtering with an appropriate pore size membrane filter (eg, 0.2 μm membrane filter), the size of the nanoparticles can be adjusted to the desired size. Thus, in one embodiment, the complexing comprises (i) reacting the plasmid vector in an aqueous medium with the polyethyleneimine conjugated with dexamethasone and (ii) filtering the reaction mixture obtained in step (i) with a membrane filter. It may include the step. In the complexing, the plasmid vector is preferably reacted at a rate of 0.75 μg or more with respect to 1 μg of the polyethyleneimine conjugated with dexamethasone.

Nanoparticle transporter according to the present invention is effectively introduced into the stem cells by the endocytosis, exit the endosomes, can be internalized into the cell substrate (cytosol) to induce differentiation of stem cells. This is shown in FIG. 14 as a schematic diagram. Therefore, the nanoparticle transporter according to the present invention can be usefully used as a nanoparticle transporter for inducing cartilage differentiation of stem cells through RNA interference.

In addition, the nanoparticle transporter according to the present invention can be usefully used to selectively differentiate stem cells into chondrocytes. Accordingly, the present invention provides a method for differentiating stem cells into chondrocytes, comprising transfecting stem cells with the nanoparticle transporter. In the differentiation method of the present invention, the stem cells may be human mesenchymal stem cells. In addition, the transfection may be performed by culturing the stem cells in a serum-free medium containing the nanoparticle transporter. For example, stem cells may be cultured in a serum-free medium containing the nanoparticle transporter for about 4 hours to induce the inflow of the nanoparticle transporter into the cells. In addition, cells obtained from the culture, that is, cells transfected with the nanoparticle transporter may be cultured through two-dimensional or three-dimensional culture according to a conventional method.

Nanoparticle transporters according to the invention can also be used with stem cells as cell therapeutics for cartilage tissue reconstruction or regeneration. Accordingly, the present invention provides a pharmaceutical composition for reconstructing or regenerating cartilage tissue, that is, a cell therapeutic agent, comprising stem cells transfected with the nanoparticle transporter. In the pharmaceutical composition of the present invention, the stem cells may be human mesenchymal stem cells.

The pharmaceutical composition of the present invention may include stem cells transfected with a nanoparticle transporter as described above, and may include a pharmaceutically acceptable carrier, and the parenteral of liquids, suspensions, emulsions, lyophilizers, etc. according to conventional methods. It may be formulated into a dosage form (eg, injection). The pharmaceutically acceptable carrier may include an aqueous diluent or solvent such as phosphate buffered saline, purified water, and sterile water. If desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives may be included. In addition, the pharmaceutical composition according to the present invention may be prepared in the form of a construct for cartilage tissue reconstruction or regeneration using a conventional support such as BD Matrigel ™ Matrix (BD biosciences).

In the pharmaceutical composition of the present invention, the dosage of stem cells transfected with the nanoparticle transporter varies depending on the condition and body weight of the patient, the extent of the disease, the dosage form, the route of administration, and the duration, for example, the nanoparticles. Stem cells transfected with a carrier may be administered at a dose of about 5 × 10 ⁶ cells, eg in the form of an injection, in a single dose. The administration may be administered once or several times a day as needed. In addition, the unit formulation of the pharmaceutical composition of the present invention may contain stem cells transfected with a nanoparticle transporter of about 5 X 10 ⁶ cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention and do not limit the scope of the invention.

1. Materials and test methods

(1) material

Tetramethlyrhodamine (TRITC), dimethyl sulfoxide (DMSO), and dexamethasone (DEX) were purchased from Sigma-Aldrich (St Louis, MO, USA). Cationic polymer branched polyethylenimine (bPEI, 25 kDa) was purchased from Polysciences (Warrington, PA, USA). Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS), alpha minimum essential medium, antibiotics, and trypsin-EDTA are thermo Fisher Scientific Purchased from Thermo Fisher Scientific Incorporation.

(2) miRUNX2  And shRUNX2  Produce

PCR fragments containing KpnI / NotI restriction sites obtained using human genomic DNA were ligated into pTracer ™ -EF / V5-His A (Invitrogen, Carlsbad, CA, USA) to miR-30a Overexpression vectors were constructed for (miRBase accession number: M10000088-SEQ ID NO. provided by Origene SC400322) and miR-30d (miRBase accession number: M10000255-SEQ ID NO. provided by Origene SC400326). For miR-30a, primer sets of F: 5'-CGC GGTACC CACTTGCCTATTTACTTCTTGTGG-3 'and R: 5'-CAG GCGGCCGC GGGAAATATTGCCCTACTACG-3' were used, and for miR-30d, F: 5'-CAT GGTACC ATCCTCATGTAGGATATCAG-3 A primer set of 'and R: 5'-CAC GCGGCCGC ACTAAAATTTCATTGACAGG-3' was used. Underlined sequences indicate restriction sites.

Two target sequences for silencing of RUNX2 were obtained using the Gene Link shRNA Design Guidelines website (http://www.genelink.com/sirna/shrnai.asp). I.e., 5'-GATCC GCACGCTATTAAATCCAAA GAAGCTTG TTTGGATTTAATAGCGTGC TTTTTTGGAAGC -3 ' and 5'- GGCCGCTTCCAAAAAA GCACGCTATTAAATCCAAA CAAGCTTC TTTGGATTTAATAGCGTGC G-3 ' joined to (annealing) the to produce a RUNX2 shRNA 1, 5'- GATCC GGTTCAACGATCTGAGATT GAAGCTTG AATCTCAGATCGTTGAACC TTTTTTGGAAGC-3 ' and Two double-stranded oligonucleotides were obtained by annealing the 5′-GGCCGCTTCCAAAAAA GGTTCAACGATCTGAGATT CAAGCTTC AATCTCAGATCGTTGAACC G-3 ′ to generate RUNX2 shRNA 2. RUNX2 sense and antisense sequences are underlined and hairpin loop structures with HindIII restriction sites are italicized. The resulting double-stranded oligonucleotide was inserted into a pGSH1-GFP shRNA vector (Genlantis, San Diego, CA, USA) digested with BamHI and NotI. For differentiation induction, GFP sequences were removed and UNX2 shRNA 1/2 was inserted into the pGSH1 vector. All constructs were confirmed by sequencing.

(3) RNA-based NPs  Produce

(3-1) TRITC -Labeled bPEI  Preparation of containing polymer

bPEI (500 mg) and TRITC (1 mg) were dissolved in 5 mL of DMSO, and the resulting solution was dialyzed in the dark for 2 days (MWCO 1,000 Da). The sample was then lyophilized for 3 days.

(3-2) DEX - Conjugated TRITC -Labeled bPEI  Preparation of containing polymer

DEX-conjugated TRITC-labeled PEI (TRITC-DEX-PEI) was synthesized via conventional carbodiimide reactions. PEI (250 mg, 10 μmol) was added to 10 mL of DMSO and stirred until complete dissolution. Then, DEX (25 mg, 50.76 μmol), N, N-dicyclohexylcarbodiimide (DCC) (16 mg, 77.55 μmol), and N-hydroxysuccin prepared in 5 mL of DMSO Imide (N-Hydroxysuccinimide, NHS) (16 mg, 139.02 μmol) was added to the PEI solution and reacted at room temperature for 24 hours. Thereafter, TRITC (1 mg) dissolved in DMSO (3 mL) was added dropwise to the solution at room temperature for 12 hours. The final solution was dialyzed against distilled water using a dialysis membrane (MWCO 1,000 Da) for 3 days. The final product was obtained by lyophilization.

(3-3) RNA-based NPs  Produce

The polymer containing TRITC-labeled bPEI prepared in (3-1) was dissolved in distilled water and reacted for 15 minutes by adding the miRUNX2 and shRUNX2 vectors prepared in (2), respectively. By this reaction, self-assembly nanoparticles were formed by electrostatic attraction between anionic pDNA and cationic polymer. The reaction mixture was filtered with a 0.2 μm membrane filter to prepare RNA-based NPs (RtNPs) containing TRITC-labeled bPEI.

Also, in the same manner as above, using a polymer containing DEX-conjugated TRITC-labeled bPEI prepared in (3-2) instead of a polymer containing TRITC-labeled bPEI prepared in (3-1). RNA-based NPs (RDtNPs) were prepared, each dissolved in distilled water and containing DEX-conjugated TRITC-labeled bPEI.

(4) NPs  Characterization

The size and shape of the NPs were analyzed using a scanning electron microscope (SEM) and a Zeta Sizer Nano ZS instrument (Malvern, Southborough, MA, USA). Cumulative analysis was performed on various types of NPs. Size analysis of NPs was performed 20 times each. Gel retardation assay was performed to determine the optimal ratio of NPs and RNA.

(5) cell culture

Bone marrow-derived hMSCs (PT-2501; Lonza, Walkersville, MD, USA) were obtained from proximal femur samples of 22 year old men obtained during hip replacement surgery. The patient provided informed consent, and the Ethical Committee approved the use of the sample for the study. hMSCs were cultured in alpha minimal essential medium containing 10% FBS and 1% antibiotics (antibiotics-antimycotics, AA).

(6) NPs  Cellular uptake

Cell monolayers (1.5 × 10 ⁵ cells per well) in 6-well plates were preincubated in serum-free medium, treated with 2.5 μg of RtNPs and RDtNPs for 4 hours, and Dulbecco's phosphate buffered saline (Dulbecco washed with phosphate buffered saline (DPBS) and analyzed by fluorescence-activated cell sorting (FACS; Becton Dickinson, San Jose, Calif., USA). Cell membranes and endosomes were stained using standard protocols.

(7) transient transfection transfection )

Serum-containing medium was exchanged with serum free medium. hMSCs were transfected with RDtNPs for 4 hours and analyzed by FACS (Guava Technologies, Hayward, CA, USA), Western blotting, and fluorescence confocal microscopy (LSM 880 META, Zeiss).

(8) 3D cell culture

Transfected hMSCs were centrifuged for 3 minutes at 1,300 rpm in a 15 mL conical tube. Cell pellets were incubated for two weeks in medium containing 10% FBS and 1% AA, followed by an additional week in serum-free medium. Culture medium was changed every 2-3 days.

(9) mRNA  And protein analysis

After incubation for 3 weeks in a three-dimensional (3D) system, mRNA was extracted using TRIzol (Invitrogen), and the protein was extracted using a radioimmunoprecipitation buffer (radioimmunoprecipitation buffer). mRNA and protein were analyzed by RT-PCR / quantitative PCR (qPCR) and Western blotting, respectively.

10 Glycosaminoglycans ( Glycosaminoglycan , GAG) analysis

Total glycosaminoglycan (GAG) was extracted according to standard protocols. Lysates were prepared in papain buffer (300 μL) and GAG was detected using the GAG Assay Kit (Blyscan, UK).

(11) histological analysis and Immunofluorescence  analysis

Cells cultured for 3 weeks in a two-dimensional (2D) or three-dimensional (3D) system are washed with DPBS, 15% at 4 ° C. for 1 hour with 1% paraformaldehyde (PFA) in DPBS or with 4% PFA in DPBS. Fixed at 4 ° C. each for minutes. The samples incubated in the 3D system were then embedded in an optimal cutting temperature material (TISSUE-TEK 4583; Sakura Finetek USA), frozen at -20 ° C for 1 hour, and then 8-10 Cut into μm-thick pieces.

Samples were stained with hematoxylin and eosin, Alcian blue, and safranin-O according to standard protocols. The immobilized sample was washed twice with water, blocked for 5 minutes in 0.1% Triton X-100, and washed twice with PBS containing 0.1% bovine serum albumin (BSA). Samples were then incubated with the primary antibody for 2 hours at room temperature, washed with PBS containing 0.1% BSA and incubated with fluorescent secondary antibody for 45 minutes. Finally the samples were washed, stained with DAPI for 5 minutes and placed on confocal microscope.

(12) statistical analysis

Statistical analysis was performed using Student's t-test. * P <0.05 and ** P <0.01 were considered statistically significant.

2. Results and Discussion

pDNA comprising miRUNX2 and shRUNX2 were complexed with PEI / DEX polymer (FIG. 1). Two miRNAs containing 30 bases [miRNA30a (SEQ ID NO: 1) and miRNA30d (SEQ ID NO: 2) and two shRNAs (SEQ ID NO: 3 and SEQ ID NO: 4) were constructed to express the expression of RUNX2 that promotes bone differentiation of hMSCs. Inhibition (FIG. 1A).

The chemical structures of PEI-conjugated DEX labeled TRITC and PEI-conjugated DEX not labeled TRITC are shown in FIG. 9. PEI has a molecular weight of 25 KDa. PEI / DEX polymers are conjugated with TRITC to enable their visualization in cell tracking and bioimaging experiments.

The manufactured pDNA and PEI / DEX polymers formed nanosphere-shaped NPs. These NPs were characterized by dynamic light scattering (DLS) (FIG. 10). The size of NPs increased with complexation with pDNA. PEI is much larger than PEI / DEX polymers because PEI / DEX polymers are more tightly aggregated than PEI.

pDNAs containing miRUNX2 and shRUNX2 were complexed with PEI / DEX polymers (FIG. 1B). DLS analysis shows that these NPs have a diameter of 115.9 nm (FIGS. 1B, a), and SEM shows that they have a morphology similar to nanospheres and have a diameter of about 110 nm (FIGS. 1B, b). ). These results indicate that the size and shape of NPs can easily transfer genes into cells.

Surface charges of PEI / DEX polymers with or without complexes with pDNA including miRUNX2 and shRUNX2 were analyzed (FIG. 1C). pDNAs comprising miRUNX2 and shRUNX2 were negatively charged (FIG. 1C, a), while PEI / DEX polymers complexed with these pDNAs were positively charged (FIG. 1C, b-e). This indicates that negatively charged RNAs are complexed and their charges are shielded by complexing with PEI / DEX polymers.

Strong complexation was observed between pDNA and PEI / DEX polymers (FIG. 1D). The level of complexation increased with increasing concentrations of NPs. This indicates that cationic PEI / DEX polymers can be complexed through ionic interactions with pDNAs including anionic miRUNX2 and shRUNX2.

NPs were complexed with increasing concentrations of pDNA including miRUNX2 and shRUNX2 (FIG. 11). Complexation was first observed when 0.75 μg of pDNA was used.

Cell influx of NPs consisting of PEI and PEI / DEX was measured (FIG. 2A). NPs entered hMSCs after 30 minutes (Figures 2A, a and b). The percentage of cells in which these NPs were internalized differed after 1 hour, but did not differ after 2 hours. This indicates that NPs can be easily internalized. After 4 hours, NPs were internalized in more than 90% of hMSCs.

To measure the cytotoxic effect, live and dead cells were labeled using Cell-Counting Kit-8 and subjected to Live / Dead assay (FIG. 12). . These assays indicate that no type of NP caused cell death.

Fluorescence luminescence was measured after treatment of hMSCs with NPs composed of PEI or PEI / DEX (FIG. 2B). Fluorescence was not observed in control untreated hMSCs (FIG. 2B, a). However, hMSCs treated with PEI NPs or PEI / DEX NPs showed fluorescence, and fluorescence intensity was higher in PEI / DEX NPs compared to PEI NPs (FIGS. 2B, b and c).

HMSCs treated with NPs composed of PEI or PEI / DEX were imaged by fluorescence microscopy. All of these NPs were easily delivered to the cytosol by endocytosis (FIGS. 2C and D). Membrane staining confirmed their internalization (FIGS. 2C, b and c). All of these NPs entered early endosomes or were released from early endosomes (FIG. 2D, b and c).

Transfection of GFP pDNA using these NPs was investigated (FIG. 13). FACS analysis showed that PEI NPs and PEI / DEX complexed with GFP pDNA were introduced into hMSCs (FIG. 13A), and then 34% and 36% of the cells fluoresced (FIG. 13A, b and c), respectively. . Confocal laser microscopy and Western blot analysis also showed that hMSCs expressed GFP following transfection of GFP pDNA with these NPs (FIGS. 13B and C).

We designed two miRNAs (SEQ ID NOs: 1 and 2) and two shRNAs (SEQ ID NOs: 3 and 4) targeting RUNX2 (Figure 3A). After transfecting hMSCs with pDNA containing these RNAs, mRNA and protein expression of RUNX2 were measured by RT-PCR and Western blotting, respectively (FIGS. 3B and C). MRNA and protein levels of RUNX2 were reduced 24 hours after transfection of each miRNA and shRNA (FIG. 3B). This indicates that the prepared RNAs inhibited RUNX2 expression in hMSCs. In addition, mRNA and protein expression of RUNX2 was further reduced when treated with two miRNAs or two shRNAs, and was most decreased when all four factors were treated simultaneously (FIG. 3C).

We performed FACS and confocal laser microscopy analysis (FIGS. 3D and E). In FACS analysis, 73.47% of control hMSCs expressed RUNX2, and RUNX2 expression was reduced to 54.30%, 60.32%, and 42.7% for two miRNAs, two siRNAs, and hMSCs transfected with all four factors, respectively. (Figure 3D, bd). These results indicate that these RNAs reduce the expression of RUNX2 in hMSCs. In addition, RUNX2 expression was most reduced by simultaneous delivery of all four factors. In confocal microscopy analysis, the RUNX2 label (shown in green) gradually disappeared upon treatment of hMSCs with two miRNAs, two siRNAs, and all four factors (Figure 3E, b-d). RUNX2 expression was most decreased in hMSCs treated with all four factors; RUNX2 was not detected in the nuclei of these cells (Fig. 3E, f). However, RUNX2 was detected in the nucleus and cell substrate of control hMSCs (Fig. 3E, e). These results indicate that shRUNX2 and miRUNX2 decreased the nuclear and cellular substrate levels of RUNX2 in hMSCs, respectively.

hMSCs were analyzed via RT-PCR, Western blotting, FACS, and confocal laser microscopy for up to 72 hours after transfection of RDtNPs complexed with pRUNA and miRUNX2 and / or shRUNX2 (FIG. 4). RUNX2 expression was reduced by transfection of RDtNPs complexed with pDNA containing two miRNAs, two siRNAs, and all four factors, but transfection of PEI / DEX NPs complexed with empty pDNA (MOCK pDNA) Not by. Reduction of gene and protein expression of RUNX2 was maintained for the longest after transfection of RDtNPs complexed with pDNA containing all four factors (Figure 4A, c). Gene and protein expression of RUNX2 was reduced for up to 48 hours in hMSCs transfected with RDtNPs complexed with pDNA containing two miRNAs (Figure 4A, a). In contrast, hMSCs transfected with RDtNPs complexed with pDNA containing two shRNAs reduced RUNX2 expression only later (Figure 4A, b). These results indicate that miRUNX2 reduces RUNX2 expression immediately after transfection and shRUNX2 maintains this effect at later time points. Thus, transfection of all four factors is suitable for rapid and sustained inhibition of RUNX2 expression. FACS and confocal microscopic analysis also showed that transfection of RDtNPs complexed with pDNA containing all four factors reduced expression of RUNX2 in hMSCs (FIGS. 4C and D).

In the course of bone formation, RUNX2 induces the expression of asterix (OSX), β-catenin, and Dlx-5 (FIG. 5A, a). In contrast, increased SOX9 expression leads to the differentiation of stem cells into chondrocytes (FIGS. 5A, b). Osteogenesis-related genes and proteins were analyzed by RT-PCR and Western blot analysis (FIG. 5B). Inhibition of RUNX2 expression disrupts bone formation and promotes different forms of differentiation, such as cartilage formation. Expression of the bone formation-related genes OSX, β-catenin, and Dlx-5 was inhibited in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors, but with control hMSCs and empty pDNA (MOCK pDNA). Not so in hMSCs transfected with complexed PEI / DEX NPs.

Cartilage formation was examined by histological analysis, GAG content analysis, RT-PCR, and western blotting (FIG. 6). hMSCs were seeded in 6-well dishes, treated with various NPs and incubated for 3 weeks (FIG. 6A). In samples stained with Alcian blue and safranin-O, blue and orange labels representing proteoglycans and polysaccharides were clearly detected in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors ( 6B, d and h). However, staining was less intense in hMSCs transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA), indicating low levels of differentiation into chondrocytes. These results indicate that transfection of RDtNPs complexed with pDNA containing all four factors promoted the differentiation of hMSCs into chondrocytes and reduced the differentiation of hMSCs into osteocytes. In addition, in 2D culture systems, the GAG content was high in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors (Figure 6C, d).

Levels of cartilage-related genes and proteins were increased in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors (FIGS. 6D and E). In these hMSCs, the expression of the cartilage-associated gene SOX9 was increased while the expression of RUNX2 was decreased. These results confirm that transfection of RDtNPs complexed with pDNA containing all four factors increases the differentiation of hMSCs into cartilage and also reduces the differentiation of hMSCs into bone.

We also investigated the differentiation of hMSCs into cartilage cultured in a 3D system. hMSCs were seeded in 6-well dishes, treated in various ways, detached, pelleted and incubated for 3 weeks (FIG. 7A). The cartilage formation of the aggregated cells was then analyzed. HMSCs transfected with RDtNPs complexed with pDNA containing all four factors expressed cartilage-associated genes SOX9, COL II, and aggrecan (FIG. 7B). However, hMSCs transfected with PEI / DEX NPs complexed with empty pDNA (MOCK pDNA) or hMSCs transfected with RDtNPs complexed with pDNA containing all four factors did not express these genes. In addition, the results of quantitative analysis showed that the expression of SOX9, COL II, and Agrican were 6, 1.5, and 5.5 fold in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors compared to control hMSCs, respectively. High (FIG. 7C). These results indicate that DEX and four factors promote differentiation of hMSCs into cartilage.

The GAG content was 18-fold higher in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors compared to hMSCs transfected with PEI / DEX NPs complexed with control hMSCs and empty pDNA (MOCK pDNA) (FIG. 7D ). In addition, the GAG content was 1.5-fold higher in hMSCs transfected with RDtNPs complexed with all four factors compared to hMSCs transfected with RDNPs complexed with all four factors (Figure 7D, c and d).

Cell aggregates were stained with alcian blue and safranin-O (FIG. 7E). No blue or orange label was observed in hMSCs transfected with control hMSCs and PEI / DEX NPs complexed with empty pDNA (MOCK pDNA). In contrast, clear labeling was observed in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors.

We performed Western blot analysis of SOX9, Agrican, and COL II (FIG. 8A). Levels of SOX9 were high in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors. All expressed protein-associated chondrogenesis of hMSCs showed high scores and strong bands in hMSCs treated with four factors complexed with PEI / DEX polymers. Expression of Agrican and COL II, respectively, involved in the early and final stages of cartilage differentiation were also high in these hMSCs. Protein expression was quantified (FIG. 8B). Similar to Western blot analysis, levels of COL II and Agrican were increased in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors.

In immunohistologic analysis, SOX9 and COL II (represented in green and red, respectively) were detected near pellets of hMSCs cultured for 3 weeks (FIG. 8C). This label was covered in hMSCs transfected with RDtNPs complexed with pDNA containing all four factors. SOX9 labeling was also detected in pellets of hMSCs transfected with RDtNPs complexed with pDNA containing all four factors, but the intensity was greater than that of hMSCs transfected with RDtNPs complexed with pDNA containing all four factors. Low. COL II did not stain strongly on these pellets. These results indicate that transfection of RDtNPs complexed with pDNA containing all four factors induces the production of specific extracellular matrix proteins and also induces the differentiation of hMSCs into mature chondrocytes.

3. Conclusion

It has been demonstrated by the present invention that PEI / DEX NPs readily enter hMSCs. In addition, transfection of miRUNX2 and shRUNX2 depletes endogenous RUNX2 for up to 72 hours. Analysis of cartilage markers indicated that internalization of RDtNPs complexed with pDNA, including miRUNX2 and shRUNX2, promoted cartilage production of hMSCs. These results indicate that inhibition of bone formation through knockdown of RUNX2 promotes cartilage formation in hMSCs.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Nanoparticle delivery system for inducing chondrogenic          differentiation of stem cells through RNA interference and method          for chondrogenic differentiation using the same <130> PN0829 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA <400> 1 tgtaaacatc ctcgactgga ag 22 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA <400> 2 tgtaaacatc cccgactgga ag 22 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> shRNA <400> 3 gcacgctatt aaatccaaa 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> shRNA <400> 4 ggttcaacga tctgagatt 19

Claims (14)

Nanoparticle carriers for inducing cartilage differentiation of stem cells via RNA interference, comprising: one or more microRNAs (miRNAs) targeting the RUNX2 gene and one or more short hairpin RNAs (shRNAs) targeting the RUNX2 gene Nanoparticle carrier obtained by complexing with dexamethasone conjugated polyethyleneimine. The nanoparticle transporter of claim 1, wherein the plasmid vector comprises miRNAs having a nucleotide sequence of SEQ ID NO: 1 or 2. 3. The nanoparticle transporter of claim 1, wherein the plasmid vector comprises shRNAs having a nucleotide sequence of SEQ ID NO: 3 or 4. 4. According to claim 1, wherein the plasmid vector miRNAs having a nucleotide sequence of SEQ ID NO: 1, miRNAs having a nucleotide sequence of SEQ ID NO: 2, shRNAs having a nucleotide sequence of SEQ ID NO: 3, and shRNAs having a nucleotide sequence of SEQ ID NO: 4 Nanoparticle transport comprising a. The nanoimide of claim 1, wherein the polyethyleneimine conjugated with dexamethasone is obtained by reacting dexamethasone with polyethyleneimine in the presence of N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide. Particle carrier. The nanoparticle carrier according to claim 5, wherein the polyethyleneimine is a branched polyethylenimine having a aminoethyl branch. 7. The nanoparticle carrier according to claim 6, wherein the polyethylenimine of the chain structure has a weight average molecular weight of 0.6 to 30 kDa. The process of claim 1 wherein said complexing comprises (i) reacting said plasmid vector in an aqueous medium with said dexamethasone conjugated polyethyleneimine and (ii) filtering the reaction mixture obtained in step (i) with a membrane filter. Nanoparticle transport comprising a. The nanoparticle carrier according to claim 1, wherein the plasmid vector is complexed at a ratio of 0.75 μg or more with respect to 1 μg of the polyethyleneimine conjugated with dexamethasone. 10. A method of differentiating stem cells into chondrocytes, comprising transfecting the stem cells with the nanoparticle transporter according to claim 1. 11. The method of claim 10, wherein said stem cells are human mesenchymal stem cells. The method of claim 10, wherein said transfection is performed by culturing said stem cells in a serum-free medium comprising said nanoparticle transporter. A pharmaceutical composition for cartilage reconstruction or regeneration comprising stem cells transfected with a nanoparticle transporter according to any one of claims 1 to 9. The pharmaceutical composition of claim 13, wherein the stem cells are human mesenchymal stem cells.

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