The temporal and spatial control over the delivery of materials such as siRNA into cells remains a significant technical challenge. We demonstrate the pulsed near-infrared (NIR) laser-dependent release of siRNA from coated 40 nm gold nanoshells. Tat-lipid coating mediates the cellular uptake of the nanomaterial at picomolar concentration, while spatiotemporal silencing of a reporter gene (green fluorescence protein) was studied using photomasking. The NIR laser-induced release of siRNA from the nanoshells is found to be power- and time-dependent, through surface-linker bond cleavage, while the escape of the siRNA from endosomes occurs above a critical pulse energy attributed to local heating and cavitation. NIR laser-controlled drug release from functional nanomaterials should facilitate more sophisticated developmental biology and therapeutic studies.

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The extracellular environment is a complex medium in which cells secrete and consume metabolites. Molecular gradients are thereby created near cells, triggering various biological and physiological responses. However, investigating these molecular gradients remains challenging because the current tools are ill-suited and provide poor temporal and special resolution while also being destructive. Herein, we report the development and application of a machine learning approach in combination with a surface-enhanced Raman spectroscopy (SERS) nanoprobe to measure simultaneously the gradients of at least eight metabolites in vitro near different cell lines. We found significant increase in the secretion or consumption of lactate, glucose, ATP, glutamine, and urea within 20 μm from the cells surface compared to the bulk. We also observed that cancerous cells (HeLa) compared to fibroblasts (REF52) have a greater glycolytic rate, as is expected for this phenotype. Endothelial (HUVEC) and HeLa cells exhibited significant increase in extracellular ATP compared to the control, shining light on the implication of extracellular ATP within the cancer local environment. Machine-learningdriven SERS optophysiology is generally applicable to metabolites involved in cellular processes, providing a general platform on which to study cell biology.

Self-assembly of peptide amphiphiles into nanostructures makes them attractive for a variety of applications in drug and peptide delivery. We here report on the interactions of micelles composed of a palmitoylated, pro-apoptotic peptide derived from p53 tumor suppressor protein with a human cancer cell line. Characterization of self-assembly in aqueous buffered solutions revealed formation of elongated rod-like micelles above a critical micelle concentration. Our results however demonstrate that monomers instead of micelles are internalized, a finding which correlates with the dynamic nature of the assemblies and the non-covalent interactions that hold them together. Internalization is shown to occur via adsorption-mediated, energy-dependent pathways, resulting in accumulation of the material in endocytic vesicles. We conclude that palmitoylation of peptides is an efficient way to increase peptide permeability inside SJSA-1 cells and that increased micelle stability would be required for intact micelle internalization.Synthetic peptides derived from human proteins, or identified via powerful new technologies, have emerged as potential therapeutics for various diseases, acting as signals, promoters or inhibitors of cellular functions. Frequently, their site of action is intracellular and therefore they are required to cross cell membranes in order to reach their target. Among the strategies explored in order to attain this goal, the one receiving the most attention is modification with peptide sequences that are able to translocate inside cells, widely referred to as cell penetrating peptides (CPPs) 1, 2 . A variety of different CPPs have been identified, ranging in activity and mechanism of action. An alternative strategy has been the covalent attachment of a hydrophobic tail onto the peptide of interest 3-6 . Borrowed from nature, where cells link hydrophobic moieties onto proteins to efficiently localize them on cellular membranes 7 , this mechanism has proven a simple, yet effective way to transport peptides inside the cell and thus enhance their activity 8, 9 . Peptide amphiphiles or lipopeptides, as the resulting constructs are usually termed, have a higher affinity for lipid bilayers than the parent peptides 9, 10 and are able to insert into the cell membrane by virtue of their hydrophobic tail. Following this first step toward internalization, the subsequent uptake mechanism and the final destination of lipopeptides remain a subject of investigation. Attaching a hydrophobic tail onto a peptide additionally has implication on its biodistribution. Depending on the type of lipid tail, binding to certain biomolecules in vivo can be promoted in order to aid their transport to sites of interest 11 or act as a drug reservoir in the blood 12 . Moreover, lipopeptides exhibit improved stability against As an example of a pro-apoptotic signal we here selected a peptide derived from the binding site of tumor suppressor p53 to the MDM2 protein 26 . The 16-mer peptide from the N-terminus of p53 (p53 [14][15][16...