Modulating cell activity by opto-nanomedicine

Abstract

Event Abstract Back to Event Modulating cell activity by opto-nanomedicine Miguel M. Lino1, 2, Susana Simões1, 2, Sónia Pinho1, 2 and Lino Ferreira1, 2 1 Center for Neurosciences and Cell Biology, Portugal 2 Biocant, Center of Innovation in Biotechnology, Portugal Introduction: Intracellular delivery of proteins is extremely useful for the manipulation of cellular processes and cell reprogramming[1]. Although some formulations have been described in the last years for the successful intracellular delivery of proteins[2], so far no formulation has the capacity to orchestrate the delivery of multiple proteins. This is an important issue in many biological applications. For example, lineage-switching experiments in the hematopoietic system have shown that the order in which two transcription factors become expressed in a progenitor can decide lineage outcome and thus timing is of utmost importance[3]. Here we describe a formulation that can orchestrate the delivery of multiple proteins by light. The user can control the delivery of a specific protein immobilized to a nanoparticle by controlling the power of a near-infrared light source. The system is compatible with cells and further experiments are being performed to extend the potential of the system in vivo. Materials and Methods: Our formulation is based on gold nanorods (NR) conjugated with more than one protein using different DNA oligonucleotides as linkers. The release kinetics of proteins was done using gold nanorods conjugated with Dylight-BSA (NR-BSA-DyLight). The same was done for NR conjugated with β-Gal (NR- β-Gal) and the enzymatic activity of the supernatant was measured. Mouse fibroblasts were incubated with NR-β-Gal or NR-BSA-Dylight and irradiated with a 785 nm laser for 2 min with different laser powers. The activity of β-Gal was detected using a commercial Kit and the intracellular localization of the protein was detected by immunocytochemistry. Results and Discussion: Proof of concept experiments were performed with BSA and β-galactosidase proteins. NR-BSA-Dylight were used to test the sequential release of fluorescent proteins with three laser powers at 785nm. In order to release both proteins independently from the same nanorod, the samples were irradiated at 570 mW/cm2, releasing only one of the proteins and then at 780 mW/cm2, releasing the other protein. In cells incubated with NR-DNA-βGal, the fluorescence from XGal staining increased 1.2 and 1.5 fold when the cells were irradiated at 0.57 and 1.25W/cm2, respectively. The protein was distributed homogeneously in the cytosol and the colocalization with NR decreased. The same level of activity was achieved when the photo-activation was done up to six hours post incubation. In the case of NR-BSA-DyLight, the fluorescence increased, the signal was more diffuse and the colocalization with NR decreased significantly in the irradiated samples, indicating a displacement of the protein from the NR. Our results are the first experimental evidence for the spatio-temporal release of multiple proteins. The authors would like to thank the financial support of Fundação para a Ciência e a Tecnologia (SFRH/BD/81705/2011, SFRH/BPD/105327/2014 and PTDC/CTM-NAN/120552/2010), EC funding (ERC project nº 307384, “Nanotrigger”), and COMPETE funding (Project “Stem cell based platforms for Regenerative and Therapeutic Medicine”, Centro-07-ST24-FEDER-002008).