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Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

ABSTRACT: Achieving a controlled and reproducible means to direct stem cell differentiation is the single most critical concern scientists have been trying to address since the discovery of stem cells. In this regard, the use of small molecules and RNA interference offers unique advantages by targeting different cellular mechanisms. Our cyclodextrin-modified dendritic polyamine construct (termed DexAM) combines the unique properties of two distinct chemical moieties in a single delivery vehicle. DexAM is a single vehicle that not only solubilizes hydrophobic small molecules in physiological solutions but also forms complexes with siRNA molecules, making it an attractive delivery system for controlling stem cell differentiation. Herein, we report the synthesis and application of DexAM to simultaneously deliver hydrophobic small molecules and siRNA into neural stem cells to significantly enhance their neuronal differentiation.

SUBMITTER: Shah S

PROVIDER: S-EPMC3841293 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

Shah Shreyas S Solanki Aniruddh A Sasmal Pijus K PK Lee Ki-Bum KB

Journal of the American Chemical Society 20131011 42

Achieving a controlled and reproducible means to direct stem cell differentiation is the single most critical concern scientists have been trying to address since the discovery of stem cells. In this regard, the use of small molecules and RNA interference offers unique advantages by targeting different cellular mechanisms. Our cyclodextrin-modified dendritic polyamine construct (termed DexAM) combines the unique properties of two distinct chemical moieties in a single delivery vehicle. DexAM is ...[more]

PMID: 24106916

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Project description:The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy.Statement of significanceApplication of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment.

Project description:siRNA treatment has great promise to specifically control gene expression and select cell behaviors but has delivery challenges limiting its use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effectively deliver siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA. This is vastly superior to a commercially available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), was cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT/siRNA treatment resulted in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There was no statistically significant differences on cell surface markers of diblock copolymer/siRNA or DharmaFECT/siRNA-treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there was no discernible differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitated uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC survival or differentiation capacity and therefore show great promise for use within regenerative medicine applications.

Dataset Information

Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

Publications

Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

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