Construction of PLGA Nanoparticles Coated with Polycistronic SOX5

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Construction of PLGA nanoparticles coated with polycistronic SOX5, SOX6, and SOX9 genes for chondrogenesis of human mesenchymal stem cells Ji Sun Park, Se Won Yi, Hye Jin Kim, Seong Min Kim, Jae-Hwan Kim, and Keun-Hong Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15354 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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ACS Applied Materials & Interfaces

Construction of PLGA Nanoparticles Coated with Polycistronic SOX5, SOX6, and SOX9 Genes for Chondrogenesis of Human Mesenchymal Stem Cells Ji Sun Park, Se Won Yi, Hye Jin Kim, Seong Min Kim, Jae-Hwan Kim,* and Keun-Hong Park* Department of Biomedical Science, College of Life Science, CHA University, 6F, CHA Biocomplex, 689 Sampyeong-dong Bundang-gu, Seongnam-si, 134-88 (Korea)

* Co-corresponding author : [email protected] & [email protected]

KEYWORDS : hMSC, nanoparticle, PEI, SOX9, polycistronic vector ABSTRACT :

Transfection of a cocktail of genes into cells has recently attracted attraction in

stem cell differentiation. However, it is not easy to control the transfection rate of each gene. To control and regulate gene delivery into human mesenchymal stem cells (hMSCs), we employed multicistronic genes coupled with a non-viral gene carrier system for stem cell differentiation. Three genes, SOX5, SOX6, and SOX9, were successfully fabricated in a single plasmid. This multicistronic plasmid was complexed with the polycationic polymer polyethylenimine, and poly (lactic-co-glycolic) acid (PLGA) nanoparticles were coated with this complex. The uptake of PLGA nanoparticles complexed with the multicistronic plasmid was tested first. Thereafter, transfection of SOX5, SOX6, and SOX9 was evaluated, which increased the potential for

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chondrogenesis of hMSCs. The expression of specific genes triggered by transfection of SOX5, SOX6, and SOX9 was tested by RT-PCR and real-time qPCR. Furthermore, specific proteins related to chondrocytes were investigated by a glycosaminoglycan/DNA assay, Western blotting, histological analyses, and immunofluorescence staining. These methods demonstrated that chondrogenesis of hMSCs treated with PLGA nanoparticles carrying this multicistronic genes was better than that of hMSCs treated with other carriers. Furthermore, the multicistronic genes complexed with PLGA nanoparticles were more simple than that of each single gene complexation with PLGA nanoparticles. Multicistronic genes showed more chondrogenic differentiation than each single gene transfection methods.


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1. INTRODUCTION

Various factors such as genes and proteins are involved in stem cell differentiation and have great potential for clinical approaches. regard.

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1-4

Several types of genes have been used in this

Plasmid DNA (pDNA) has been used for gene delivery in cancer, stem cell

differentiation, and several genomic diseases. For gene therapy, two types of gene delivery systems have been employed for safe and stable delivery.

9, 10

A carrier system employing viral

vectors has been widely used for gene delivery due to its extremely high transfection efficiency. 11-12

However, it has some drawbacks, namely, strong immunogenicity, host inflammatory

response, recombination during fabrication, and carcinogenicity, which restrict its usage in clinical trials.

13, 14

Despite its comparatively low transfection efficiency, a non-viral vector

system for gene therapy overcomes several disadvantages of the viral vector system.

15, 16

Cationic polymers are typically used and broadly tested with common vectors for gene delivery. 17-20

Cationic vectors harboring genes are easy and inexpensive to fabricate. 21, 22 Among cationic

polymers, polyethylenimine (PEI), which is a powerful gene carrier, has been widely employed, and its length (molecular weight: 1.8–25 KDa) and forms (linear or branched types) can vary. 23, 25

Chondrogenesis is regulated by complex molecular networks, including the SOX5, SOX6, and SOX9 genes. SOX9 plays a key role as a master transcriptional activator required for chondrogenesis.

26

Nevertheless, SOX9 alone is insufficient, and SOX5 and SOX6 are also

required to drive efficient chondrogenesis.

27

SOX5 and SOX6 strikingly enhance the

transcriptional activity of SOX9 by securing it to chondrocyte-specific enhancer elements in type


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II collagen (COL II), aggrecan, and other chondrocyte genes.

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28-30

Therefore, we employed a

polycistronic gene delivery system to maximize the efficiency of chondrogenic differentiation. In a previous study, we tested the ability of a multiple-gene delivery system to induce chondrogenesis of human mesenchymal stem cells (hMSCs).

31

In this study, SOX5, SOX6, and

SOX9 were delivered into hMSCs, either separately or in combination, to stimulate chondrogenesis. Chondrogenesis of hMSCs was enhanced when SOX5, SOX6, and SOX9 were delivered simultaneously compared with when only one or two genes were delivered. 32 In this study, we fabricated the three SOX genes in a single polycistronic plasmid and determined the differentiation efficiency of hMSCs treated with poly (lactic-co-glycolic) acid (PLGA) nanoparticles coated with this plasmid and loaded with dexamethasone (DEX). The polycistronic genes were transfected into hMSCs and were expressed, which caused hMSCs to differentiate into chondrocytes. Transfection of these three SOX genes and expression of chondrogenesis-related genes and proteins were confirmed by fluorescence-activated cell sorting (FACS), RT-PCR, Western blot, and confocal laser microscopy analyses. The experimental procedures are depicted in Scheme 1.

2. MATERIALS AND METHODS

2.1 Preparation of tetramethylrhodamine isothiocyanate (TRITC)-DEX-loaded PLGA nanoparticles (TRITC-DEX NPs) Nanoparticles were fabricated followed by previous study.

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Briefly, 200 mg of PLGA

(Boehringer Ingelheim, Germany), 2 mg of DEX, and 100 µg of TRITC (Sigma-Aldrich, St. Louis, MO) were emulsified with 1 mL of methylene chloride (DC) by sonication. The two


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different solutions were then mixed with 2 mL of PVA solution with isopropanol and residual DC was evaporated under a vacuum.

2.2 Characterization of TRITC-DEX NPs The sizes of nanoparticles (NPs) were measured using Zeta-sizer Nano ZS apparatus (Malvern, Southborough, MA). Briefly, the hydrodynamic diameter of NPs, a suspension state in distilled water, was then determined via cumulative analysis. The ζ-potentials were predicted based on the electrophoretic mobility of the nanospheres in aqueous medium, which were evaluated using folded capillary cells in automatic mode. DEX-loaded nanoparticles, PEI-modified DEX-loaded nanoparticles, and pDNA (polycistronic

SOX

genes)-complexed,

PEI-modified,

DEX-loaded

nanoparticles

were

completely dried in a vacuum, coated with platinum, and observed via scanning electron microscopy (SEM) to determine their mean diameters and morphologies. The ability of different delivery agents to condense pDNA was assessed by analyzing the results of a gel retardation assay using a Bio-Rad Imaging System. Briefly, samples with different nanoparticle to pDNA weight ratios were loaded onto a gel and electrophoresed at 100 V for 20 min in Tris-acetate-EDTA buffer.

2.3. Construction of the polycistronic SOX plasmid expression vector The 2A-polycistronic expression vector system was constructed using standard recombinant PCR techniques to examine the synergistic effects of human SOX5, human SOX6, and human


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SOX9 expression induced by the same CMVp. The expression vector construct was confirmed by nucleotide sequencing and appropriate restriction enzyme digestion (NruI/XhoI for CMVp plus human SOX5, XhoI/AgeI for 2A plus human SOX6, and NotI/XbaI for NLS plus human SOX9).

2.4 Cellular uptake of TRITC-DEX NPs complexed with polycistronic SOX genes 2.4.1 Cell culture hMSCs were purchased from Lonza Walkersville Inc. (Walkersville, Cat#: PT-2501). After thawing, hMSCs were cultivated in MSCGM Basal Medium (Cat#: PT-3001; Lonza Walkersville Inc.) in a humidified 5% CO2 incubator at 37°C. Experiments were performed with cells at passage #5–7 and TRITC-DEX NPs in same conditions with serum-free medium. For the detection of cell transfection ability, 293T cells and HeLa cells were cultured in same conditions.

2.4.2 Cellular internalization For flow cytometry, hMSCs were seeded in a 6-well plate (3 × 105 cells per well) in same conditions. hMSCs were incubated with 0.2 mg/mL pDNA (empty vector)-complexed TRITCDEX NPs for 0.5–6 h. Alternatively, hMSCs were incubated with various concentrations (0.01– 0.2 mg/mL) of pDNA (empty vector)-complexed TRITC-DEX NPs for 6 h. The cells were suspended in PBS and analyzed using the Guava EasyCyte System (Guava Technologies, Hayward, CA) equipped with a 583/26-nm laser. Data show the mean fluorescence signals of 10,000 cells.


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For confocal imaging, hMSCs were seeded in 35-mm glass bottom dishes (2 × 105 cells/well). Fluorescence was monitored in three channels, namely, FITC; excitation, 488 nm; emission, 518 nm), TRITC; excitation, 558 nm; emission, 583 nm), and 4,6-diamidino-2phenylindole (DAPI) (for DAPI labeling; excitation, 358 nm, emission, 461 nm).

2.5 Transfection hMSCs were plated in a 6-well plate (2 × 105 cells/well). The pDNA (polycistronic SOX genes)complexed PEI-modified DEX-loaded nanoparticles was added into them. Thereafter, they were cultured for 6 h. The transfection efficiency was calculated by Western blotting and fluorescence confocal microscopy (LSM 880, Zeiss).

2.6 Biochemical assays of glycosaminoglycan (GAG) production The samples and negative controls were extracted, rinsed with 2.5 mL of PBS, homogenized with a pellet grinder (Fisher Scientific, MA), and digested in 500 µL of papain digestion buffer (0.05 M sodium acetate, 5 mM L-cysteine HCl, and 10 mM EDTA, pH 5.5) at 65°C for 3 h. Thereafter, the specimens underwent three freeze/thaw/sonication cycles (30 min at -80°C, 30 min at room temperature, and 3 min of sonication) to completely extract DNA from the cytoplasm. Similarly, the GAG content was determined by a dimethylmethylene blue dye assay.

2.7 Pellet culture


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To prepare each pellet, aliquots of cells were centrifuged for cell mass. The pre-cultures cell masses were divided into four and cultured in complete medium (containing 10% FBS and 1% antibiotics) at 37°C in 5% CO2 for 21 days. The detailed methods were described in previous studies. 31

2.8 The evaluation of gene and protein expressions in cells After 3 weeks of pellet culture in vitro system, all kinds of mRNAs and proteins were extracted from them. Matured chondrocyte-related gene and protein productions from cultured pellets were evaluated using real-time PCR and Western blot analyses, respectively. The detailed methods were described in previous studies. 31

2.9 Histology and immunohistochemistry Briefly, samples were harvested at 21 days after treatment of cells with different types of nanoparticles. hMSCs transfected with pDNA (empty vector), pDNA (empty vector)-complexed TRITC-DEX NPs, pDNA (polycistronic SOX genes)-complexed PEI, and pDNA (polycistronic SOX genes)-complexed, PEI-modified, DEX-loaded nanoparticles were fixed in 4% paraformaldehyde. Briefly, cryosections (10 µm) were stained with Alcian blue, Safranin-O, and Masson’s trichrome for histological evaluation. Immunohistochemical analyses were conducted to identify COL II (Millipore, Temecula, CA) and SOX9 (Millipore) by applying specific antibodies (1:200) in a humid environment. Samples were then examined with a Zeiss LSM 880 confocal microscope (Carl Zeiss, Gottingen, Germany).


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In other experiments, sections were stained with a Histostain-SP Kit (AEC, Broad Spectrum, Bulk; Invitrogen). The sections were incubated with primary antibodies (anti-SOX-9, Millipore), incubated for 1 h with a broad spectrum secondary antibody, and washed three times with PBS. Signals were developed with AEC (3-amino-9-ethylcarbazole) solution following the manufacturer’s instructions. The detailed methods were described in previous studies. 31

2.11 Statistical analyses Data are presented as the mean ± standard deviation. An analysis of variance was performed to statistically compare the experimental groups. A paired t-test was performed to compare the results.

3. RESULTS AND DISCUSSION The size and morphology of nanoparticles were monitored by dynamic light scattering and SEM analyses (Fig. 1A). The size of the nanoparticles was increased by coating with PEI and PEI/pDNA comparing to control (Fig. 1A, a-c). DEX-loaded PLGA nanoparticles, PEI-coated DEX-loaded nanoparticles, and PEI/pDNA-coated DEX-loaded nanoparticles had a diameter of 130, 139.5, and 150 nm, respectively. This confirmed that DEX-loaded PLGA nanoparticles were easily coated with PEI and PEI/pDNA. In SEM, DEX-loaded nanoparticles had a more blurry appearance when coated with PEI or PEI/pDNA (Fig. 1A, d-f). It is possible that DEXloaded nanoparticles became less compact when complexed with PEI or PEI/pDNA. This also shows that DEX-loaded PLGA nanoparticles were coated with PEI or PEI/pDNA for gene delivery.


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To further characterize DEX-loaded nanoparticles coated with PEI or PEI/pDNA, their ζpotentials were determined (Fig. 1B). pDNA (polycistronic SOX genes) and DEX-loaded PLGA nanoparticles had negative ζ-potentials (Fig. 1B, a, c). The negative ζ-potential of DEX-loaded PLGA nanoparticles became positive after PEI coating (Fig. 1B, b, d, & e). These results were due to the highly positive charged backbone of PEI. A gel retardation assay was also performed to determine the complexation of DEX-loaded PLGA nanoparticles and PEI/pDNA (Fig. 1C). PEI and PEI/pDNA-coated DEX-loaded PLGA nanoparticles showed tight complexation. pDNA complexed with PEI alone showed the same results. When DEX-loaded PLGA nanoparticles not coated with PEI were assayed, their migration was not retarded by pDNA. This result shows that PEI not only complexed with pDNA but also coated PLGA nanoparticles. However, although PEI has the potential to retard pDNA migration and to transfect genes, cytotoxicity increases when it is used alone. 31 In a previous study, SOX5, SOX6, and SOX9 were individually delivered into hMSCs to promote chondrogenesis.

31, 32

Although the combination of SOX5, SOX6, and SOX9 effectively

promoted chondrogenesis in that study, it was difficult to regulate and control the delivery of a single gene. For effective gene delivery, we constructed a polycistronic plasmid harboring these three SOX genes. The map of this plasmid is shown in Figure 2A. The appropriate positioning of the three genes was confirmed by restriction enzyme digestion (Fig. 2B). The expression levels of the three genes in hMSCs were confirmed by RT-PCR and real-time qPCR (quantitative polymerase chain reaction) (Fig. 2C & D). mRNA expression of SOX5, SOX6, and SOX9 was confirmed using single plasmids and the polycistronic plasmid (Fig. 2C). The expression levels were also confirmed using polycistronic SOX trio genes (Fig. 2D). The expression levels were dramatically increased when the polycistronic SOX genes were


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transfected into hMSCs. The SOX6 and SOX9 mRNA levels were highly increased compared with control hMSCs not transfected with the polycistronic SOX genes. Although the level of SOX5 mRNA was relatively low in transfected hMSCs, the expression of SOX5 is very weak in control hMSCs not transfected with the polycistronic SOX genes. To verify the uptake of DEX-loaded PLGA nanoparticles, hMSCs treated with TRITCconjugated DEX-loaded PLGA nanoparticles were analyzed by FACS (Fig. 3A). As the amount of TRITC-conjugated DEX-loaded PLGA nanoparticles increased, red fluorescence shifted to the right (Fig. 3A, a). Uptake was also examined after various amounts of time (Fig. 3A, b). Uptake increased over time, and reached around 98% after 6 h. Therefore, hMSCs retained TRITC-conjugated DEX-loaded PLGA nanoparticles in the cytosol. During their uptake, TRITC-conjugated DEX-loaded PLGA nanoparticles localized near the nucleus (Fig. 3B, b). They were internalized into cells, encapsulated within endosomes and escaped from endosome (Fig. 3B, b-1, b-2, & b–3). This showed that TRITC-conjugated DEXloaded PLGA nanoparticles entered hMSCs via endocytosis and were positioned in the cytosol to deliver specific materials. To further characterize the internalization of TRITC-conjugated DEXloaded PLGA nanoparticles, the membranes of hMSCs were stained (Fig. 3C). Initially, red particles were not observed in hMSCs treated with TRITC-conjugated DEX-loaded PLGA nanoparticles (Fig. 3C, a). At later time points, red particles were observed near the cell membrane (Fig. 3C, b & c) and moved toward the nucleus (Fig. 3C, d). This result indicates that DEX-loaded PLGA nanoparticles can deliver specific drugs into hMSCs near to the nucleus. To confirm the delivery of the polycistronic SOX genes and single SOX trio genes into hMSCs, 293T cells, and HeLa cells, protein expression was assayed by Western blotting (Fig.


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4A, S2A, S3A). The exogenous SOX5, SOX6, and SOX9 proteins migrated at 150 KDa (red arrows). The endogenous SOX5, SOX6, and SOX9 proteins migrated at 110, 130, and 90 KDa, respectively. Therefore, the polycistronic SOX genes and single SOX trio genes were transfected into hMSCs and translated into proteins. To determine the transfection of the polycistronic SOX genes and single SOX trio genes into hMSCs, 293T cells, and HeLa cells, immunohistological analysis was performed using specific monoclonal antibodies tagged with Cy5.5 (SOX5), TRITC (SOX6), and FITC (SOX9) after 3 days of culture (Fig. 4B, S2B, S3B). Although control hMSCs slightly expressed SOX9 protein, hMSCs transfected with the individual genes were highly labeled by each of the corresponding anti-SOX antibodies (Fig. 4B, a, f, & k). Therefore, SOX9 protein and mRNA were present in the cytosol and nuclei of hMSCs. SOX9 protein was labeled by a monoclonal antibody and is shown in green (Fig. 4B, c, g, k, o, & s). In contrast to the delivery of single genes, hMSCs treated with the polycistronic plasmid and single SOX trio genes exhibited three colors representing SOX5 (Cy5.5), SOX6 (TRITC), and SOX9 (FITC) (Fig. 4B, m, n, o, q, r, & s). Therefore, transfection of the polycistronic SOX genes and single SOX trio genes induced expression of SOX5, SOX6, and SOX9 proteins. Thus, emerald, red, and green, which represented SOX5, SOX6, and SOX9, respectively, were vivid in the cytosol of hMSCs. The same findings were made in 293T and HeLa cells (Fig. S2B & S3B). To confirm the chondrogenesis of hMSCs using the polycistronic SOX9 genes, several delivery vehicles were used in the in vitro culture system. Before assaying chondrogenesis, the cytotoxicity of the delivery vehicles complexed with pDNA was determined (Fig. S1). We did not observe cytotoxicity in any samples when a low dose was used. However, cytotoxicity increased as the PEI concentration increased. Therefore, use of a high PEI concentration with gene delivery vehicles should be avoided if possible.


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Early, middle, and late chondrogenesis marker genes were first detected by RT-PCR (Fig. 5A). In the early stage, the fibronectin marker gene was expressed, after which its expression declined. In the middle stage, specific marker genes that were not expressed in the early stage were expressed at days 5 and 7. Finally, the most important marker gene of chondrocytes, COL II, was expressed. Therefore, the viability and stability of hMSCs in the early and middle stages were not affected by addition of the polycistronic SOX genes. hMSCs differentiated into chondrocytes upon delivery of the polycistronic SOX genes. The levels of unique marker genes involved in the maturation of chondrocytes were analyzed in vitro by RT-PCR (Fig. 5B). Genes associated with chondrogenesis were expressed upon delivery of polycistronic SOX trio pDNA-coated DEX-loaded PLGA nanoparticles and single SOX trio genes coated PLGA nanoparticles. COL II was highly expressed upon transfection of these nanoparticles. hMSCs transfected with the polycistronic SOX plasmid complexed with PEI alone showed increased expression of genes such as COL II. However, COMP and aggrecan gene expression was lower in these cells than in hMSCs transfected with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles. As a very important component of matured chondrocytes, GAG was tested in a twodimensional culture system (Fig. 5C). The GAG content normalized by the DNA content was highest in hMSCs treated with polycistronic SOX pDNA-coated DEX-loaded nanoparticles. Therefore, not only the polycistronic SOX genes but also DEX is an important factor for the chondrogenesis of hMSCs. To detect extracellular matrix (ECM), including cartilage tissue formation, we evaluated histological images of specific ECM components such as proteoglycan and polysaccharides (Fig.


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5D). Orange and blue labeling following staining with Alcian blue and Safranin-O staining, respectively, was vivid upon treatment with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles (Fig. 5D, d & h). This staining was also vivid upon treatment with the polycistronic SOX plasmid complexed with PEI. However, in the case of control treatment and treatment with mock-coated DEX-loaded PLGA nanoparticles, specific ECM components were not detected by Alcian blue and Safranin-O staining (Fig. 5D, a, b, f, & g). Therefore, hMSCs not transfected with SOX genes did not undergo chondrogenesis, while transfection of the polycistronic SOX plasmid and single SOX trio genes, either in complex with PEI or on the surface of DEX-loaded PLGA nanoparticles, stimulated differentiation into cartilage cells. By magnifying the images of Masson's trichrome staining, the typical cellular formation of lacunae representing matured chondrocytes was observed in hMSCs treated with the polycistronic SOX plasmid complexed with PEI or polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles (red arrows) (Fig. 5E, c, d, & e). In these images, collagen fibers surrounded the cells upon treatment with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles (yellow arrows) (Fig. 5E, d). Therefore, polycistronic SOX pDNA-coated DEXloaded PLGA nanoparticles caused hMSCs to differentiate. This result is coincident with the magnified images of Safranin-O and Alcian blue staining (Fig. S5A & B). hMSCs transfected with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles exhibited vivid staining and clear lacunae formation in these images (Fig. S5A & B, d). In a three-dimensional (3D) culture system, chondrogenesis of hMSCs treated with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles was evaluated by RT-PCR (Fig. 6A). The typical marker genes of SOX9, COL II, aggrecan, and COMP were highly expressed in these hMSCs, and COL II expression was clearly observed. However, type I


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collagen (COL I), which is a unique marker gene of bone formation, was not expressed in these hMSCs. Therefore, transfection of polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles caused hMSCs to undergo chondrogenesis. Real-time qPCR was performed to quantify the expression levels. Upon transfection of hMSCs with the polycistronic SOX genes, the COL II gene was highly expressed, while the other delivery vehicles and the mock gene did not affect gene expression (Fig. 6B). The expression levels of marker genes including COMP and aggrecan were much higher in hMSCs transfected with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles than in any other groups. Therefore, polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles are a potent candidate with which to induce chondrogenesis of hMSCs. For the proof of more powerful chondrogenesis in hMSCs, the polycistronic SOX pDNAcoated DEX-loaded PLGA nanoparticles and each single SOX trio genes were tested for their potentials. When the single genes coated onto the PLGA nanoparticles were used in transfection, the exact amount of internalized genes into cells were not predictable. It means that the most important genes of SOX9 gene was not transfected into cells. The auxiliary factors of SOX5 and SOX6 genes have to support the function of SOX9 genes for chondrogenesis. Thus, the exact SOX trio genes evenly internalized into hMSCs were key role to regulate the chondrogenesis. The polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles GAG, the major component of tissues, was assayed to investigate cartilage tissue formation due to chondrogenesis of transfected hMSCs (Fig. 7A). The exact GAG contents of various hMSCs were measured, and the released GAG content was calculated and normalized by the total DNA content. In this way, the GAG/DNA contents (normalized GAG contents) of hMSCs treated in


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different ways were determined. hMSCs treated with polycistronic SOX pDNA-coated DEXloaded PLGA nanoparticles had higher contents, while control and mock-transfected hMSCs had lower contents. Therefore, polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles caused hMSCs to undergo chondrogenesis and become chondrocytes. Morphological and functional changes of hMSCs treated with polycistronic SOX pDNAcoated DEX-loaded PLGA nanoparticle were histologically analyzed (Fig. 7B & C). hMSCs treated with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles produced the ECM components of cartilage tissue, proteoglycan and polysaccharide, as visualized by blue and orange labeling upon Safranin-O and Alcian blue staining, respectively. Additionally, treatment with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles increased the number of cells stained with H&E (Fig. S6). However, control and mock-treated hMSCs were not highly compact and did not exhibit widely stained surrounding matrices (Fig. 7). Furthermore, lacunae formation was observed not only in sites surrounding the cells but also at the core site (Fig. 7B & C, d). The generation of specific proteins such as SOX9, COL II, aggrecan, and COMP is also important in cartilage tissue formation. The specific proteins released from hMSCs treated with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles were evaluated by Western blotting (Fig. 8A). SOX9, COL II, aggrecan, and COMP were highly expressed in hMSCs treated with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles. Therefore, transfection of polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles induced hMSCs to undergo chondrogenesis. By contrast, control and mock-treated hMSCs did not exhibit high levels of these proteins comparing to single SOX trio gene transfected cells. This result


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indicated that the SOX9 gene in polycistronic Trio genes expressions were lasted for 96 h (Fig. S4). Thus, this produced SOX9 genes caused to encourage the chondrogenesis of hMSCs Production of the cartilage tissue-specific proteins COL II and SOX9 by hMSCs treated with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles was evaluated to judge chondrogenesis of these hMSCs. In 3D cultures of these hMSCs, cell pellets were markedly stained by antibodies against COL II and SOX9 (Fig. 8B, d & i). COL II was widely distributed and SOX9 was located near lacunae (white arrows) (Fig. 8B, m, n). Magnified images of cells treated with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles showed the clear lacunae formations (Fig. S7). Cell pellets were not stained for COL II and SOX9 when hMSCs were not treated with polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles or the polycistronic SOX plasmid complexed with PEI alone. COL II was clearly detected with DAB staining (Fig. S8). Therefore, hMSCs not transfected with the polycistronic SOX genes did not differentiate into chondrocytes and remained undifferentiated.

4. Conclusion In this study, a polycistronic plasmid harboring three SOX genes was fabricated and its ability to induce chondrogenesis of hMSCs was tested. Polycistronic SOX pDNA-coated DEXloaded PLGA nanoparticles are a suitable candidate for a gene delivery system that allows safe and stable cellular uptake and enhances gene expression in hMSCs. Exogenous polycistronic SOX5, SOX6, and SOX9 genes triggered chondrogenesis of hMSCs not only in an in vitro culture system but also in a 3D pellet culture system.


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ACKNOWLEDGMENT This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF-2014R1A2A1A09002838) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Develo pment Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI14C0322 ).


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Figure captions Scheme 1. Schematic diagram of polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles. Upon transfection, polyplexes of the nanoparticles entered hMSCs by endocytosis, escaped from endosomes, and localized in the cytosol, causing the differentiation of hMSCs.

Figure 1. Size distributions, SEM images, ζ-potentials, and gel retardation assays of polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles. A: Dynamic light scattering and SEM images. a & d: DEX-loaded PLGA nanoparticles, b & e: PEI-modified DEX-loaded PLGA nanoparticles, c & f: polycistronic SOX pDNA-coated DEXloaded PLGA nanoparticles. a-c: Dynamic light scattering analysis & d-f: SEM images. Scale bar, 500 nm. B: ζ-potential assay. a: pDNA (polycistronic SOX genes only), b: PEI, c: DEX-loaded PLGA nanoparticles, d: PEI-modified DEX-loaded PLGA nanoparticles, e: polycistronic SOX pDNAcoated DEX-loaded PLGA nanoparticles. C: Gel retardation assay of pDNA, DEX DEX-loaded PLGA nanoparticles complexed with pDNA, PEI-complexed pDNA., and polycistronic SOX pDNA-coated DEX-loaded PLGA nanoparticles

Figure 2. Fabrication of the polycistronic plasmid harboring three SOX genes and confirmation by restriction enzyme digestion.


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A: Gene map of the fabricated polycistronic SOX plasmid used in this study. The SOX5, SOX6, and SOX9 genes were fabricated as a polysictronic formation. B: Confirmation of the expressions of the polycistronic SOX genes. The each SOX5, SOX6, and SOX9 were expressed C: Determination of the mRNA levels of SOX5 (a), SOX6 (b), and SOX9 (c) when they were transfected into cells. *P

Construction of PLGA Nanoparticles Coated with Polycistronic SOX5

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