Highlight
Nanoparticle Films for Novel Gene Delivery to Stem Cells
Gene Delivery to Stem Cells via Matrix-Based Nanoparticles
Achievement/Results
Rutgers IGERT PI: Prabhas Moghe May 2012
IGERT Trainee: Shreyas Shah
In recent years, RNA interference (RNAi) has become a major area of interest for directing stem cell fate. This methodology involves using small interfering RNA (siRNA) to selectively silence (or knockdown) genes or pathways to control stem cell differentiation. However, a major challenge in this area is developing a means to efficiently deliver siRNA into stem cells. The most common methods include solution-based delivery using viruses, non-viral cationic lipids, nanoparticles and polyplexes. However, when treated with exogenous materials, stem cells tend to die or undergo undesired differentiation patterns. Therefore, there are concerns associated with the introduction of viruses, nanoparticles, and other exogenous materials into stem cells. From the vast repertoire of techniques that can be used to deliver siRNA into stem cells, methods based on substrate-mediated delivery, where cells uptake siRNA from their microenvironments, are extremely advantageous as they provide a way to improve the efficiency of siRNA delivery by simply changing the cellular microenvironments. However, the fact that nanotopographical features of the extracellular microenvironment can be used to deliver siRNA into stem cells efficiently remained to be explored.
Trainee Shreyas Shah of the Rutgers NSF funded IGERT Program on Integrated Science and Engineering of Stem Cells has pioneered an extracellular matrix (ECM)-based nanotechnology platform in the laboratory of Professor KiBum Lee to successfully control gene expression. A simple technique was developed to deliver siRNA into NSCs using nanoparticle films coated with a mixture of ECM proteins and the desired siRNA of interest. In particular, this siRNA delivery platform was used manipulate gene expression within neural stem cells with relative ease, to enhance neuronal differentiation.
The experimental conditions were initially optimized using siRNA targeting endogenously expressed GFP in neural stem cells, wherein in the expression level of GFP was used probe the delivery efficiency (see Figure). Various conditions were optimized including the size and composition of the nanoparticle, the concentration of the mixture of ECM protein and siRNA and the temporal exposure on the substrates. Additionally, the mechanism of siRNA delivery from the ECM-based nanotechnology platform was also explored to provide support for the highly efficient siRNA delivery.
Having demonstrated the efficiency of the system by performing GFP knockdown, this substrate-mediated delivery platform was utilized to enhance the neuronal differentiation of neural stem cells by suppressing the expression of a specific proteins and transcription factors. The differentiation of the neural stem cells was quantified by immunostaining and PCR assays for neuronal and glial markers. As compared to control substrates, a remarkably high percentage of NSCs differentiated into neurons on ECM-based nanotechnology platform.
The IGERT program has catalyzed cross-disciplinary interactions between the Lee laboratory and the Keck Center for Collaborative Neuroscience (Professors Martin Grumet; Bonnie Firestein), which enabled the advancement of this unique siRNA delivery platform. A proof of concept was demonstrated by manipulating gene expression within neural stem cells, a cell line which has traditionally proven to be difficult to transfect with current delivery platforms. This system relies primarily on the ability of cells to sense the nanotopographical features and take up only the siRNA from its microenvironment, and it does not require the use of cationic polymers, viruses, or nanowires that may mechanically disrupt and perturb the cells.
Address Goals
This IGERT project at Rutgers addresses the DISCOVERY of a new platform of gene delivery from nanoparticle-based substrates to stem cells. New RESEARCH INFRASTRUCTURE advances have also been put in place to fabricate the nanoparticles and create spatially engineered substrates to enable both culture and genetic engineering of stem cells. These platforms will enable longer term reprogramming of stem cells, which may open new vistas for testing new drug candidates and culture reagents on disease-specific stem cells.
In recent years, RNA interference (RNAi) has become a major area of interest for directing stem cell fate. This methodology involves using small interfering RNA (siRNA) to selectively silence (or knockdown) genes or pathways to control stem cell differentiation. However, a major challenge in this area is developing a means to efficiently deliver siRNA into stem cells. The most common methods include solution-based delivery using viruses, non-viral cationic lipids, nanoparticles and polyplexes. However, when treated with exogenous materials, stem cells tend to die or undergo undesired differentiation patterns. Therefore, there are concerns associated with the introduction of viruses, nanoparticles, and other exogenous materials into stem cells. From the vast repertoire of techniques that can be used to deliver siRNA into stem cells, methods based on substrate-mediated delivery, where cells uptake siRNA from their microenvironments, are extremely advantageous as they provide a way to improve the efficiency of siRNA delivery by simply changing the cellular microenvironments. However, the fact that nanotopographical features of the extracellular microenvironment can be used to deliver siRNA into stem cells efficiently remained to be explored. This IGERT project addresses this specific gap in the field and thus meets the criteria of both discovery (advancing the frontiers of knowledge, emphasizing areas of greatest opportunity and potential benefit) and research infrastructure (bionanotechnology, stem cell sourcing).
