Regulation of Cell Signaling Factors Using PLGA Nanoparticles

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Regulation of Cell Signaling Factors Using PLGA Nanoparticles Coated/Loaded with Genes and Proteins for Osteogenesis of Human Mesenchymal Stem Cells Ji Sun Park, Se Won Yi, Hye Jin Kim, Seong Min Kim, and Keun-Hong Park* Department of Biomedical Science, College of Life Science, CHA University, 6F CHA Bio-complex, 689 Sampyeong-dong Bundang-gu, Seongnam-si, 463-400, Korea S Supporting Information *

ABSTRACT: Transfection of specific genes and transportation of proteins into cells have been a focus of stem cell differentiation research. However, it is not easy to regulate codelivery of a gene and a protein into cells. For codelivery into undifferentiated cells (human mesenchymal stem cells (hMSCs)), we used biodegradable carriers loaded with Runt-related transcription factor 2 (RUNX2) protein and coated with bone morphogenetic protein 2 (BMP2) plasmid DNA (pDNA) to induce osteogenesis. The released gene and protein were first localized in the cytosol of transfected hMSCs, and the gene then moved into the nucleus. The levels of internalized PLGA nanoparticles were tested using different doses and incubation durations. Then, transfection of BMP2 pDNA was confirmed by determining mRNA and protein levels and acquiring cell images. The same techniques were used to assess osteogenesis of hMSCs both in vitro and in vivo upon internalization of PLGA NPs carrying the BMP2 gene and RUNX2 protein. Detection of specific genes and proteins demonstrated that cells transfected with PLGA NPs carrying both the BMP2 gene and RUNX2 protein were highly differentiated compared with other samples. Histological and immunofluorescence analyses demonstrated that transfection of PLGA nanoparticles carrying both the BMP2 gene and RUNX2 protein dramatically enhanced osteogenesis of hMSCs. KEYWORDS: cocktail, BMP2, RUNX2, PLGA nanoparticles, hMSCs

1. INTRODUCTION In general, surgical procedures are used in conventional medicine to treat tissue loss or damage.1−4 However, there remain several hurdles to overcome, such as inflammation induced by transplanted materials and the high cost of largescale tissue culture systems.5,6 From this point of view, one of the best choices in regenerative medicine is artificial tissue formation that is harmonious with natural tissues. Stem cells, which are located in whole tissues, have been used as cell therapeutic agents for organizing tissue formation and for inducing paracrine effects on the proliferation and differentiation of surrounding cells.7−12 To induce stem cell differentiation into the desired cell lineage, it is necessary to provide specific signals, and exogenous genes, drugs, and growth factors have been used in this regard.13−16 Stem cell differentiation into osteoblasts is partially regulated by changes to the surrounding microenvironment, including the addition of growth factors. To regulate osteogenesis, bone morphogenetic proteins (BMPs) act in various signaling pathways associated with cellular functions.17−21 BMPs can regulate bone formation and repair as ectopic inducers.22−24 BMPs, which bind to different cell membrane receptors, can regulate two downstream signaling pathways. One is Smad-dependent, and the other is © XXXX American Chemical Society

Smad-independent. When bound to BMP receptors (BMPRs), they activate signals to induce transcription of the Runt-related transcription factor 2 (RUNX2) and osterix target genes. The Smad-dependent and -independent pathways are activated to modulate expression of the RUNX2 and osterix target genes.25−31 Thus, these two pathways play important roles in osteogenesis induced by BMP2 signaling. In this study, we investigated polyethylenimine (PEI)/ plasmid DNA (pDNA)-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with RUNX2 protein. These biodegradable PLGA NPs were used to codeliver the RUNX2 protein and BMP2 gene into human mesenchymal stem cells (hMSCs) to induce osteogenesis via two steps. The first step is the delivery of RUNX2 protein, which is a potent signaling factor involved in the early, middle, and last stages of osteogenesis, to the cytosol of hMSCs. The released RUNX2 protein triggered osteogenesis of hMSCs in the early stage. The second step is movement of the transfected BMP2 gene into the nucleus to generate BMP2 protein, which was released from hMSCs by exocytosis. Released BMP2 bound to its receptors Received: July 8, 2016 Accepted: October 19, 2016

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DOI: 10.1021/acsami.6b08343 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Views of RUNX2 Protein-Loaded BMP2 pDNA-Coated PLGA NPs for Induction of Osteogenesis in hMSCs

coated RUNX2 protein-loaded carriers were prepared for transfection of hMSCs. Recombinant human RUNX2 protein-loaded PLGA NPs coated with PEI were prepared based on a complex ion reaction. Briefly, PLGA (100 mg) and RUNX2 protein (50 μg) were dissolved in dichloroethane (DM) (10 mL). An emulsified solution of PLGA was placed on ice, and DM solution was then added to poly(vinylacohol) solution with stirring. The emulsification procedure was performed again with ultrasonication using the mixed solutions, and DM was evaporated. The size distribution of the carriers was evaluated by dynamic light scattering. The charges of the carriers were checked by determining their ζ-potentials. The shapes of the nanocarriers were determined by scanning electron microscopy (S-3000N, Hitachi Co. Ltd., Japan). To certify the drug-loading efficiencies, fluorescence-loaded NPs were imaged. BMP2 pDNA was analyzed for tight complexation with protein-loaded NPs by the gel retardation method. 2.4. Cell Culture. Bone-marrow-derived hMSCs were purchased from Lonza Walkersville Inc. (Cat. #: PT-2501; Walkersville, MD, USA). The cells were subcultured using a conventional method for 5− 7 passages. 2.5. Cellular Uptake. hMSCs were treated with green fluorescent protein (GFP)-tagged BMP2 pDNA-coated red fluorescent protein (RFP)-loaded particles in serum-free media for 24, 48, and 72 h at 37 °C. Cells were rinsed with buffer to remove free particles in the media and trypsinized to measure cellular uptake. Cells were cultured with GFP-tagged BMP2 pDNA-coated RFPloaded NPs for 4, 48, and 72 h at 37 °C. After cultivation, the uptake of NPs was visualized by acquiring fluorescence images (LSM 880 META, Zeiss).

on the membrane of hMSCs, triggering Smad signaling. This codelivery system has advantages because the transfected PLGA NPs released RUNX2 protein and BMP2 into the cytosol. However, the timing of their actions differed. RUNX2 protein triggered osteogenesis initially. Then, the BMP2 gene moved into the nucleus and triggered osteogenesis of hMSCs by the Smad signaling pathway. The study procedures are illustrated in Scheme 1.

2. MATERIALS AND METHODS 2.1. Materials. The biodegradable polymer PLGA, with a molecular weight of 33 000, was purchased from Boehringer-Ingelheim (Petersburg, VA, USA). PEI (25 kDa, branched) was purchased from Polysciences (Warrington, PA, USA). Recombinant human RUNX2 protein (10 μg; Cat. #: ab112259) was obtained from Abcam (Cambridge, MA, USA). Phosphate-buffered saline (PBS), fetal bovine serum (FBS), and alpha minimum essential medium were purchased from Life Technologies Corporation (Carlsbad, CA, USA). The Smad2/3 inhibitor SB431542 and the transforming growth factor-βactivated kinase 1 (TAK1) inhibitor 5Z-7-oxo-zeaenol were purchased from Cell Signaling Technology (Beverly, MA, USA). 2.2. Preparation of BMP2 pDNA Expression Vector. The vectors used in this study were fabricated by recombinant PCR methods and confirmed by nucleotide sequencing. BMP2 cDNA was obtained by culture of the SW1353 cell line. The pEGFP-tagged BMP2 vector was obtained by ligating human BMP2 into the multiple cloning site of pEGFP-C1. 2.3. Preparation of BMP2 pDNA-Coated RUNX2 ProteinLoaded PLGA NPs. RUNX2 protein-loaded NPs and BMP2 pDNAB


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Figure 1. Expression of RFP, GFP, and Cy5.5 in hMSCs following internalization of GFP-tagged BMP2 pDNA-coated RFP-loaded PLGA NPs. (A) FACS analysis of RFP, GFP, and Cy5.5 expression in hMSCs over time. a−d: RFP expression, e−h: GFP expression, and i−l: Cy5.5 expression. (B) mRNA and protein levels of RFP, BMP2, and GFP in hMSCs over time. (C) Visualization of hMSCs that have internalized GFP-tagged BMP2 pDNA-coated RFP-loaded PLGA NPs. Blue, DAPI; green, GFP-tagged BMP2; red, RFP. Scale bar, 20 μm. a: 0 h, b: 24 h, c: 48 h, and d: 72 h. 2.6. In Vitro Smad2/3 and TAK1 Inhibition Studies. hMSCs transfected with BMP2 pDNA-coated RUNX2 protein-loaded NPs were cultured for 0, 1, 1.5, 2, 6, and 12 h. Before transfection, subcultured cells were pretreated with 10 μM SB431542 (Smad2/3 inhibitor) or 5Z-7-oxo-zeaenol (TAK1 inhibitor) for 1 h. Cells were harvested after 0, 1, 1.5, 2, 6, and 12 h, washed with PBS and either lysed or cultured for an additional 1 or 4 h. Lysates were subjected to SDS-PAGE analysis and immunoblotted with anti-p-Smad2, anti-pP38, anti-P38, or anti-Smad2/3 antibodies. 2.7. Transfection. Cells transfected with PLGA NPs carrying BMP2 pDNA and RUNX2 protein were incubated for 1, 3, 6, 9, 12, 24, 48, and 72 h. Thereafter, the cellular internalization of NPs was measured by FACS and visualized by fluorescence confocal microscopy (LSM 880 META, Zeiss). The protein levels were detected by Western blotting. 2.8. Cell Mass Culture. For detection of osteogenesis of stem cells, the cells were pelleted in a conical tube by centrifugation at 1200 rpm. Cell masses were divided into three and subcultured under the same conditions for 28 days. 2.9. mRNA and Protein Detection. After 4 weeks of mass cell cultivation, mRNAs and proteins were extracted. Osteoblast-related marker gene and protein expression was detected via real-time PCR and Western blot analyses, respectively. The detailed methods were described in previous studies.32,33 2.10. Implantation of PLGA NPs into Nude Mice. Nude 6week-old mice were divided into four groups (n = 10 per group) for transplantation of transfected hMSCs. Nontreated hMSCs, hMSCs that had internalized PLGA NPs coated with BMP2 pDNA, hMSCs that had internalized PLGA NPs loaded with RUNX2 protein, or hMSCs that had internalized PLGA NPs loaded with the RUNX2

protein and coated with BMP2 pDNA were subcutaneously injected into the backs of the mice. These transplantations of transfected hMSCs were approved by the Animal Care Committee of CHA University. 2.11. Histology and Immunohistochemistry. Briefly, samples from nude mice transplanted with stem cells were placed in optimum cutting temperature material (TISSUE-TEK 4583; Sakura Finetek USA, Inc.) for freezing. The frozen samples were sliced into sections (5−10 μm thick) at −20 °C, and hematoxylin and eosin staining was performed. Moreover, histological analyses of the samples were performed to observe calcium and inorganic materials. Immunohistochemical analyses were conducted to identify RUNX2 (Abcam, Cambridge, UK) and collagen type I (COL I; Millipore, Temecula, CA, USA) by incubation with specific antibodies in humidified conditions. Samples were then stained with fluorescently labeled secondary antibodies (1:500; Thermo Scientific, PT, USA). Thereafter, sliced sections were incubated with DAPI to stain nuclei. 2.12. Statistical Analysis. A paired t test was performed to compare the data gained with PLGA NPs carrying BMP2 pDNA and/ or RUNX2 protein.

3. RESULTS AND DISCUSSION The sizes and shapes of the biodegradable carriers were first measured (Figure S1). The size of the biodegradable carriers was increased by complexation with BMP2 pDNA. This was explained by the ζ-potential assay (Figure S2). The charge of PLGA NPs became positive upon addition of positively charged PEI and was slightly shifted to a negative charge when they were complexed with negatively charged BMP2 pDNA. To C


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Figure 2. Expression of Smad signaling pathway factors in cells following internalization of PLGA NPs carrying GFP-tagged BMP2 pDNA and RUNX2 protein. (A) Release profiles and expression levels of RUNX2, GFP, and BMP2 following addition of GFP-tagged BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs. (B) Expression of Smad signaling pathway factors following addition of GFP-tagged BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs. (C) Expression of non-Smad signaling factors over time following addition of GFP-tagged BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs.

expression level was coincident with the BMP2 expression level because BMP2 was tagged with GFP. PLGA NPs internalized by hMSCs were imaged (Figure 1C). BMP2 pDNA-coated RFP protein-loaded PLGA NPs were well-visualized in hMSCs. This means that RFP was released from PLGA NPs and was located in the cytosol of hMSCs. In addition, green fluorescence, indicating GFP-tagged BMP2, was present in the cytosol. This means that not only GFP but also BMP2 was translated. The concentration of RUNX2 protein, which was encapsulated by PLGA NPs, was measured (Figure 2A, a). Release of RUNX2 was high at 3−6 h, after which it decreased. This means that the loaded protein was readily released from PLGA NPs following internalization. PLGA NPs carrying BMP2 pDNA and RUNX2 protein were tested. Components of the Smad signaling pathway were detected to assess the effects of BMP2 pDNA and RUNX2 protein on osteogenesis of hMSCs. GFP-tagged BMP2 was clearly expressed after 48 h. By contrast, endogenous BMP2 protein was not clearly detected in hMSCs (Figure 2A, b). Stimulation of the Smad signaling pathway by BMP2 was confirmed by Western blot analysis. hMSCs transfected with PLGA NPs carrying BMP2 pDNA and RUNX2 protein demonstrated altered expression of BMP2-Smad signaling components over time. Expression of BMPR-1A and BMPR1B increased. Expression of the signaling factors ERK1/2, p-

investigate the complexation of BMP2 pDNA with PEI-coated PLGA NPs, a gel retardation test was performed (Figure S3). BMP2 pDNA was tightly complexed with PEI-coated PLGA NPs, meaning that BMP2 pDNA would be safely delivered into the desired cells. To investigate the uptake of BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs by hMSCs, the model RFP was first used instead of RUNX2 protein. BMP2 pDNA was fused with GFP and PEI was conjugated with Cy5.5 so they could be detected in hMSCs following internalization. The uptake of PLGA NPs coated with BMP2 pDNA and loaded with RFP was detected (Figure 1A). The percentage of RFP-positive cells was increased due to internalization of the particles (Figure 1A, a−d). Moreover, BMP2 pDNA, indicated by green fluorescence, and PEI, indicated by emerald fluorescence, were highly internalized at 48 h (Figure 1A, e−l). This means that PLGA NPs entered hMSCs and that RFP, GFP, and Cy5.5 were then released into the cytosol and detected. To confirm internalization of the carriers, mRNA and protein levels were determined (Figure 1B). RFP and GFP-tagged BMP2 were detected for 48 h. These results are coincident with the FACS analysis, which showed that PLGA NPs were highly internalized at 48 h. This means that PLGA NPs entered hMSCs, after which RFP was located in the cytosol and GFPtagged BMP2 pDNA was released. Moreover, the GFP D


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Figure 3. Inhibition of the Smad and non-Smad signaling pathways in hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. (A) Inhibition of the Smad and non-Smad pathways using SB431542 and 5Z-7-oxo-zeaenol in hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. (B) RUNX2 induction using SB431542 and 5Z-7-oxo-zeaenol in hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein.

oxo-zeaenol) showed no differences in blocking of BMP2. From these results, BMP2 produced by transfection of BMP2 pDNA-coated PLGA NPs bound to its receptor on the cell membrane and activated Smad and P38 signaling. Inhibition of the Smad and non-Smad signaling pathways will decrease production of RUNX2 by hMSCs. From this viewpoint, suppression of RUNX2 production was evaluated by inhibiting the Smad and non-Smad signaling pathways using two types of inhibitors (Figure 3B, a,b). Inhibition of Smad and non-Smad signaling reduced the level of RUNX2 in hMSCs. Addition of SB431542 and 5Z-7-oxo-zeaenol, inhibitors of Smad and non-Smad signaling factors, respectively, demonstrated that Smad and non-Smad signaling were stimulated upon addition of BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs. Inhibition of RUNX2 production was observed at 6, 12, 18, 24, and 48 h. In Smad signaling pathway, the induction of RUNX2 production in hMSCs were started by transfection of BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs for 48 h (red arrow) (Figure 3B, a). However, the inhibition of RUNX2 produced from hMSCs was stopped for 72 h, and recovering of the induction of RUNX2 in hMSCs in non-Smad signaling pathway was started (Figure 3B, b). The release, denaturation, and bioactivity of RUNX2 protein loaded in PLGA NPs were tested. After PLGA NPs were deposited in distilled water, the amount of RUNX2 released was highly increased after 6 h and then decreased (Figure S4).

ERK1/2, Smad1, Smad5, p-Smad1/5/8, Smad4, Smad2/3, and p-Smad2 was observed following internalization of PLGA NPs carrying BMP2 pDNA and RUNX2 protein. Expression of some factors increased over time, while expression of others decreased. Importantly, internalization of PLGA NPs carrying BMP2 pDNA and RUNX2 protein changed expression of these signaling factors. This means that a combination of BMP2 pDNA and RUNX2 protein can stimulate the Smad signaling pathway to induce osteogenesis of hMSCs (Figure 2B). p-P38 and P38 were also detected following internalization of PLGA NPs carrying BMP2 pDNA and RUNX2 protein (Figure 2C). Upon addition of these PLGA NPs, p-P38 was expressed and then its level decreased. By contrast, P38 expression increased over time. To certify the effect of BMP2 pDNA-coated RUNX2-loaded PLGA NPs on Smad and non-Smad signaling activation, expression of phosphorylated Smad2 and P38 was investigated, and Smad2/3 and TAK1 were inhibited using specific inhibitors (Figure 3A). In the group not treated with the inhibitor, Smad2/3 was phosphorylated by BMP2 signaling at 1.5, 2, and 6 h. However, the group treated with the Smad2/3 inhibitor (SB431542) showed no differences in blocking of BMP2. The same results were obtained when cells were treated with the TAK1 inhibitor. In the group not treated with the inhibitor, P38 was phosphorylated by BMP2 signaling at 1.5, 2, and 6 h. However, the group treated with the TAK1 inhibitor (5Z-7E


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Figure 4. Osteogenesis of hMSCs in a 3D culture system following internalization of various types of PLGA NPs, as determined by assessment of mRNA and protein levels. (A) mRNA levels of chondrocyte- and osteoblast-related specific markers. a: control, b: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, c: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and d: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. (B) qPCR analysis. a: control, b: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, c: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and d: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. *, P < 0.01. (C) Histological analysis of osteogenesis. a and e: control, b and f: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, c and g: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and d and h: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. Scale bar, 100 μm.

This was due to degradation of PLGA NPs. Coomassie blue staining was used to determine the stability of the released protein (Figure S5). The released RUNX2 protein migrated at 55 kDa, demonstrating that it was stable. This means that RUNX2 protein maintained its properties when loaded into PLGA NPs and after its release. The α-helix secondary structure of released RUNX2 protein was tested by circular dichroism (Figure S6). This demonstrated that the released RUNX2 protein maintained its secondary structure for 24 h, meaning it was functional. We evaluated the uptake of BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs (Figure S7). Significant cell death was not induced upon addition of these PLGA NPs, meaning tthat hey did not cause cytotoxicity. They are biocompatible and nontoxic materials when used in gene delivery carriers. To confirm osteogenesis of nondifferentiated stem cells transfected with PLGA NPs carrying BMP2 pDNA and RUNX2 protein, expression of matured osteoblast-related marker genes, including RUNX2, bone sialoprotein (BSP), COL I, and osteocalcin (OCN), was detected after 4 weeks of in vitro culture (Figure 4A). Expression of these markers, which

are related to osteogenesis, was increased when PLGA NPs carrying BMP2 pDNA and RUNX2 protein were used compared with when PLGA NPs carrying only BMP2 pDNA or RUNX2 protein were used (Figure 4A). Specifically, OCN, which is a marker gene of the final stages of osteogenesis, was highly expressed in hMSCs. mRNA levels were quantified to compare osteogenesisrelated gene expression (Figure 4B). Expression of the marker genes was more than 20-fold higher in cells transfected with PLGA NPs carrying BMP2 pDNA and RUNX2 protein than that in cells transfected with only BMP2 pDNA or RUNX2 protein. These results indicate that PLGA NPs carrying BMP2 pDNA and RUNX2 protein increased osteogenesis of hMSCs. Specific extracellular matrix (ECM) marker proteins were observed by histological analyses. Alizarin red S and von Kossa staining demonstrated a high level of calcium deposition (Figure 4C). Vivid red and black labeling, which represented the calcification of bone tissue, was observed in hMSCs transfected with BMP2 pDNA-loaded RUNX2 protein-loaded PLGA NPs (Figure 4C, d,h). Alizarin red S also binds to metals and thereby labels calcium. The vivid red color implied ECM F


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Figure 5. Osteogenesis of hMSCs in a 3D culture system following internalization of various types of PLGA NPs, as assessed by immunohistochemical analysis of osteogenesis-related ECM proteins. a−d: control, e−h: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, i−l: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and m−p: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. p−s: magnifications of d, h, l, and p. a, e, i, and m: DAPI staining; b, f, j, and n: COL I staining; c, g, k, and o: RUNX2 staining; and d, h, l, and p: merged images. Scale bars, 200 and 50 μm.

transfected with PLGA NPs carrying both BMP2 pDNA and RUNX2 protein (Figure 5n,o). This means that hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein produced specific ECM and osteogenesis proteins and differentiated into matured osteoblasts. Quantification of the fluorescence intensities was evaluated (Figure S9). This demonstrated that production of RUNX2 and COL I proteins was much higher in hMSCs pretreated with PLGA NPs carrying both BMP2 pDNA and RUNX2 than in the other groups. Osteogenesis of hMSCs cultured in vivo was also tested. The expression of specific matured osteoblast-related marker genes demonstrated that transfection of BMP2 pDNA-coated RUNX2 protein-loaded PLGA NPs had the most drastic effect on osteogenesis of hMSCs (Figure 6). Similar to the in vitro culture system, cells pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein expressed typical matured osteoblast related marker genes such as RUNX2, BSP, and COL I after 4 weeks of in vivo culture, as determined by RTPCR (Figure 6A). Some differences between in vitro culture system, above-mentioned matured osteoblast cell-related marker genes released from hMSCs transfected with PLGA NPs which coated and loaded the BMP2 pDNA and RUNX2 proteins were only well expressed (Figure 6A). Specifically, OCN, a final stage marker protein, and COL I, a typical ECM

calcification by hMSCs. von Kossa staining, which labels calcium, was used for histological visualization of calcium accumulation in hMSCs transfected with different carriers (Figure 4C, e−h). Although this method does not confirm calcium deposition in hMSCs, differentiated hMSCs appear metallic silver. Thus, the black colors were replaced the calcium reducing of transfected cells. hMSCs that had internalized PLGA NPs carrying BMP2 pDNA and RUNX2 protein demonstrated black labeling (Figure 4C, h), meaning they differentiated into matured osteoblasts. Cells pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein differentiated into matured osteoblasts even in a 2D culture system (Figure S8). Nontreated hMSCs cultured in the 2D system did not differentiate. Histological analyses demonstrated that hMSCs transfected with PLGA NPs carrying both BMP2 pDNA and RUNX2 protein were highly differentiated (Figure S8d,i,h,j). Immunofluorescence staining was performed to confirm osteogenesis of hMSCs in vitro (Figure 5). Specific ECM and osteogenesis marker proteins were detected in various types of cells. hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein exhibited green and red fluorescence, representing COL I and RUNX2 proteins, respectively. hMSCs transfected with only BMP2 pDNA or RUNX2 protein also exhibited green and red fluorescence. However, the fluorescence intensity and distribution differed from those in hMSCs G


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Figure 6. Osteogenesis of hMSCs following internalization of various types of PLGA NPs and transplantation into mice, as determined by assessment of mRNA and protein levels. (A) mRNA levels of chondrocyte- and osteoblast-related specific markers. a: control, b: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, c: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and d: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. (B) qPCR analysis. a: control, b: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, c: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and d: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. *, P < 0.01. (C) Protein levels of chondrocyte- and osteoblast-related specific markers. a: control, b: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, c: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and d: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein.

Figure 7. Chondrogenesis of hMSCs following internalization of various types of PLGA NPs and transplantation into mice, as assessed by histological analysis of osteogenesis. a and e: control, b and f: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, c and g: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and d and h: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. Scale bar, 100 μm.

marker protein, were highly expressed, indicative of osteogenesis. qPCR was performed to measure and directly compare expression of marker genes in hMSCs transfected with different

carriers (Figure 6B). There were remarkable differences between hMSCs transfected with different carriers. Expression was 30-fold higher in cells transfected with both BMP2 pDNA and RUNX2 protein than that in cells transfected with only H


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Figure 8. Osteogenesis and chondrogenesis of hMSCs in a 3D culture system following internalization of various types of PLGA NPs, as assessed by histology and immunohistochemical analysis of osteogenesis-related ECM proteins. a−d: control, e−h: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA, i−l: hMSCs pretreated with PLGA NPs carrying RUNX2 protein, and m−p: hMSCs pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. a, e, i, and m: DAPI staining; b, f, j, and n: COL I staining; c, g, k, and o: RUNX2 staining; and d, h, l, and p: merged images. Scale bar, 100 μm.

mentioned above, red and black labeling of accumulated calcium represents osteogenesis. Thus, the red and black colors replaced the calcium reducing of hMSCs. Cells pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein exhibited black staining, meaning they differentiated into matured osteoblasts. To confirm osteogenesis of hMSCs at 28 days after injection into animals, samples of transfected cells were extracted from sacrificed mice and subjected to immunofluorescence staining (Figure 8). Similar to in vitro cultures of hMSCs, specific ECM and marker proteins were detected in several types of hMSCs. Cells pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein exhibited green and red fluorescence, which represented COL I and RUNX2 proteins, respectively. hMSCs transfected with PLGA NPs carrying only BMP2 pDNA or RUNX2 protein also exhibited green and red fluorescence. However, the fluorescence intensity and distribution differed from those in hMSCs transfected with PLGA NPs carrying

BMP2 pDNA or RUNX2 protein. COL I and OCN expression was increased 50-fold in cells transfected with both BMP2 pDNA and RUNX2 protein. These results indicate that PLGA NPs carrying both BMP2 pDNA and RUNX2 protein induced osteogenesis of hMSCs more potently in vivo than in vitro. Expression of the marker proteins was higher in hMSCs transfected with PLGA NPs carrying both BMP2 pDNA and RUNX2 protein than that in hMSCs transfected with the other carriers (Figure 6C). OCN, COL I, and BSP proteins were highly produced when cells were pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein. This result is coincident with the findings obtained in the in vitro culture system. Following transplantation of hMSCs, histological analysis was performed to detect calcium accumulation (Figure 7). Red and black staining in the mass of hMSCs represented calcification and was clearly observed in cells pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein (Figure 7d,h). As I


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both BMP2 pDNA and RUNX2 protein (Figure 8, n and o). This means that cells pretreated with PLGA NPs carrying BMP2 pDNA and RUNX2 protein produced specific ECM and osteogenesis proteins and differentiated into matured osteoblasts.

4. CONCLUSIONS Internalization by stem cells of PLGA NPs carrying BMP2 pDNA and RUNX2 protein induced differentiation into matured osteoblasts. Stem cells pretreated with these NPs had characteristics of matured osteoblasts both in vitro and in vivo. Codelivery of BMP2 pDNA and RUNX2 affected cell differentiation and bone formation. Codelivery of this gene and protein induced osteogenesis of hMSCs not only in vitro but also in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b08343. Details on preparation and characterization of nanoparticles, experimental methods, DLS and SEM analyses, ζ-potentials results, and assay results (gel retardation, RUNX2 release, SOX9 release/CD analysis, PLGA nanoparticle release of FITC-BSA, cell viability, histological analysis, and quantification of fluoresecence intensity of RUNX2 and COL I proteins) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

J.S.P. and S.W.Y. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF2014R1A2A1A09002838).

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Regulation of Cell Signaling Factors Using PLGA Nanoparticles

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