Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

BACKGROUND: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. METHODS: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. RESULTS: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. CONCLUSIONS: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

Self-fluorescence property of octa-arginine functionalized hydroxyapatite nanoparticles aids in studying their intracellular fate in R1 ESCs.

Deciphering the endocytosis mechanisms of nanoparticle entry in cells is crucial to understand the fate of nanoparticles and the biological activity of the transported cargo. Such studies require the use of reporter agents such as fluorescent markers. Previously, we have reported the synthesis of self-fluorescent HAp nanoparticles as efficient nucleic acid delivery agents in prokaryotic and eukaryotic cells. Here, we show the application of biocompatible self-fluorescent nano delivery vehicle based on HAp and CPP- octa-arginine as an efficient system to study the mechanisms of intracellular fate of a gene delivery agent. The pathway of octa-arginine functionalized HAp NP (R8HNP) and HAp NP uptake in R1 ESCs was elucidated using confocal microscopy with the help of endocytic inhibitors. The NPs mainly enter R1 ESCs by clathrin mediated and macropinocytosis pathways. It was established that the NPs escape endosomal degradation by proton sponge effect owing to their ability to buffer the pH and trigger osmotic rupture. The functionalization of CPP, effectively enhanced the internalization and endosomal escape in R1 ESCs. The detailed results of these studies are outlined in this manuscript.

Design of a Biocompatible Hydroxyapatite-Based Nanovehicle for Efficient Delivery of Small Interference Ribonucleic Acid into Mouse Embryonic Stem Cells.

The small interference RNA (siRNA)-assisted RNA interference approach in stem cells for differentiating into cell-specific lineages is gaining importance for its therapeutic potential. An effective gene delivery platform is crucial to achieve this goal. In this context, self-fluorescent, cell-penetrating peptide (CPP)-functionalized hydroxyapatite nanoparticles (R8HNPs) were synthesized by a modified sol gel technique. R8HNPs were crystalline, displayed characteristic bands, and exhibited broad emission spectra from 350 to 750 nm corresponding to green and red fluorescence. The biocompatible R8HNPs displayed robust binding with siRNA and excellent uptake in R1 ESCs. This was attributed to functionalization with CPP. Moreover, the R8HNP-complexed siRNA exhibited excellent serum and room temperature stability. The NPs protected the siRNA from sonication, pH, and temperature-induced stress and efficiently delivered siRNA to trigger 80% silencing of a pluripotency marker gene, Oct4, in R1 ESCs at 48 h. The transient downregulation was also observed at the protein level. Our findings demonstrate R8HNPs as a promising delivery agent for siRNA therapeutics with the potential for lineage-specific differentiation and future applications in regenerative medicine.

Nitrogen-Doped Carbon Dots via Hydrothermal Synthesis: Naked Eye Fluorescent Sensor for Dopamine and Used for Multicolor Cell Imaging.

The development of a novel uncomplicated sensor for sensitive and selective detection in biomedical analysis is necessary. This study demonstrates the preparation of nitrogen-doped carbon dots (N-C dots) via hydrothermal synthesis as a naked eye fluorescent probe for selective and sensitive detection of dopamine (DA). The successful synthesis of N-C dots was confirmed using various microscopic and spectroscopic techniques. In addition, N-C-dot-incorporated gel strips were prepared through a simple approach for naked eye detection of dopamine. The linear range for detection of dopamine was 2-20 µg/mL with a detection limit as low as 1.97 µg/mL. Moreover, N-C dots showed a low toxicity toward HeLa cells, demonstrating their relevance in multicolor cell imaging. The developed probe was also utilized for quantitative determination of dopamine in a commercial pharmaceutical sample with adequate results.