We have combined a versatile and powerful route to deliver nucleic acids with peptide-based cell-specific targeting. siRNA targeting the polo-like kinase gene is in clinical trials for cancer treatment, and here we deliver this RNA selectively to cancer cells displaying the neuropilin-1 epitope using gold nanoshells. Release of the siRNA from the nanoparticles results from irradiation with a pulsed near-infrared laser, which also provides efficient endosomal escape within the cell. As a result, our approach requires 10-fold less material than standard nucleic acid transduction materials and is significantly more efficient than other particle-based methods. We also describe a particle–nucleic acid design that does not rely on modified RNA, thereby making the preparation of these materials more efficient and much less expensive. These improvements, when combined with control over when and where the siRNA is released, could provide the basis for diverse cell biological studies.
Advances in automation and data analytics can aid exploration of the complex chemistry of nanoparticles. Lead halide perovskite colloidal nanocrystals provide an interesting proving ground: there are reports of many different phases and transformations, which has made it hard to form a coherent conceptual framework for their controlled formation through traditional methods. In this work, we systematically explore the portion of Cs-Pb-Br synthesis space in which many optically distinguishable species are formed using high-throughput robotic synthesis to understand their formation reactions. We deploy an automated method that allows us to determine the relative amount of absorbance that can be attributed to each species in order to create maps of the synthetic space. These in turn facilitate improved understanding of the interplay between kinetic and thermodynamic factors that underlie which combination of species are likely to be prevalent under a given set of conditions. Based on these maps, we test potential transformation routes between perovskite nanocrystals of different shapes and phases. We find that shape is determined kinetically, but many reactions between different phases show equilibrium behavior. We demonstrate a dynamic equilibrium between complexes, monolayers and nanocrystals of lead bromide, with substantial impact on the reaction outcomes. This allows us to construct a chemical reaction network that qualitatively explains our results as well as previous reports and can serve as a guide for those seeking to prepare a particular composition and shape. File list (2) download file view on ChemRxiv perovskite synthesis SI revision submission.pdf (12.37 MiB) download file view on ChemRxiv perovskite synthesis paper revision submission.pdf (2.42 MiB) S 1 Supporting Information for: Elucidating the weakly reversible Cs-Pb-Br perovskite nanocrystal reaction network with high-throughput maps and transformations
While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.
Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, we developed biocompatible immune cell engaging particles (ICEp) that use synthetic short DNA as scaffolds for efficient and tunable protein loading. To improve the safety of chimeric antigen receptor (CAR) T cell therapies, micron-sized ICEp were injected intratumorally to present a priming signal for systemically administered AND-gate CAR-T cells. Locally retained ICEp presenting a high density of priming antigens activated CAR-T cells, driving local tumor clearance while sparing uninjected tumors in immunodeficient mice. The ratiometric control of costimulatory ligands (anti-CD3 and anti-CD28 antibodies) and the surface presentation of a cytokine (IL-2) on ICEp were shown to significantly impact human primary T cell activation phenotypes. This modular and versatile biomaterial functionalization platform can provide new opportunities for immunotherapies.
a b s t r a c tWe describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laseractivated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine.Published by Elsevier Ltd.
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