Co-delivery of Cbfa-1-targeting siRNA and SOX9 protein using PLGA nanoparticles to induce chondrogenesis of human mesenchymal stem cells

Abstract

During stem cell differentiation, various cellular responses occur that are mediated by transcription factors and proteins. This study evaluated the abilities of SOX9, a crucial protein during the early stage of chondrogenesis, and siRNA targeting Cbfa-1, a transcription factor that promotes osteogenesis, to stimulate chondrogenesis. Non-toxic poly-(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. Coomassie blue staining and circular dichroism revealed that the loaded SOX9 protein maintained its stability and bioactivity. These NPs easily entered human mesenchymal stem cells (hMSCs) in vitro and caused them to differentiate into chondrocytes. Markers that are typically expressed in mature chondrocytes were examined. These markers were highly expressed at the mRNA and protein levels in hMSCs treated with PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. By contrast, these cells did not express osteogenesis-related markers. hMSCs were injected into mice following internalization of PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. When the injection site was excised, markers of chondrogenesis were found to be highly expressed at the mRNA and protein levels, similar to the in vitro results. When hMSCs internalized these NPs and were then cultured in vitro or injected into mice, chondrogenesis-related extracellular matrix components were highly expressed.

Introduction

Various cell lines have been proposed as candidates with which to develop cell therapies for cartilage tissue engineering [1], [2], [3]. Such cell lines include stem cells, embryonic stem cells (ESCs), and adult mesenchymal stem cells (MSCs) [4], [5]. In recent years, induced pluripotent stem cells (iPSCs) have gained increasing attention in stem cell therapy owing to the ethical concerns associated with ESCs [6], [7]. Although iPSCs have the potential to be used for cell therapy, various obstacles remain, including their potential immunogenicity and the use of genome-integrating viral vectors [8], [9].

MSCs are a potential alternative to ESCs and iPSCs owing to their multipotency, which allows them to differentiate into various cell types, including chondrocytes, osteoblasts, and adipose cells [10], [11], [12]. MSCs can be easily obtained from cord blood, bone marrow, and adipose tissue [13], [14]. In the case of chondrogenesis, MSCs transplanted into degenerated cartilage cannot easily differentiate into chondrocytes. To overcome this problem, MSCs must give rise to chondrogenic progenitor cells.

Several factors are likely able to induce the generation of progenitor chondrocytes from MSCs. The first candidate is growth factors that are related to chondrogenesis. Members of the transforming growth factor-β families have been used to induce chondrogenesis in MSCs [15], [16], [17]. The second candidate is small molecules such as dexamethasone. However, dexamethasone is involved in osteogenesis, adipogenesis, and myogenesis, as well as chondrogenesis [18], [19], [20], [21]. Thus, the use of dexamethasone is not the best approach to induce chondrogenesis. The third candidate is proteins that are implicated in chondrogenesis. Several proteins are involved in chondrogenesis and influence chondrogenic differentiation [22], [23], [24], [25]. SOX9 is an essential chondrogenic differentiation-related protein in the cytosol [26]. Once translated in the nucleus [27], SOX9 triggers the expression of aggrecan and collagen type-II (COL II), which are important for chondrogenic differentiation [28], [29], [30]. Therefore, SOX9 protein could induce stem cells to differentiate into chondrocytes. However, simply adding SOX9 protein to the culture medium is not a viable approach because the protein will be degraded before it can stimulate the differentiation of stem cells into chondrocytes. To prevent degradation of SOX9 protein, well-designed delivery vehicles are necessary.

Expression of Cbfa-1, an osteogenesis-related transcription factor, inhibits chondrogenesis of MSCs [31]. Therefore, expression of Cbfa-1 must be silenced during chondrogenesis. Transfection of Cbfa-1-targeting siRNA promotes chondrogenic differentiation and suppresses osteogenic differentiation of MSCs.

Several kinds of nanoparticles have been applied for gene and drug delivery vehicles, such as cationic nanoparticles [32], [33], lipid-like oligonucleotide nanoparticle [34], inorganic nanoparticle [35], and organic nanoparticles [36]. Among them, nanoparticles were modified with molecules for upgraded cellular uptaking into specific cells [37]. These modified nanoparticles were used as candidates for gene delivery into stem cells instead of viral vectors [38]. By internalization into stem cells, the stem cell differentiation was accelerated by delivered specific genes such as chondrogenesis [39].

In this study, we fabricated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) containing SOX9 protein and coated with Cbfa-1-targeting siRNA to enhance chondrogenesis and block osteogenesis of human MSCs (hMSCs) (Scheme 1). As a concept of co-delivery of protein and siRNA, the SOX9 proteins were easily encapsulated in PLGA NPs and siRNAs were coated outer part of them by layer-by layer method mediated by polyethyleneimine (PEI).