Photothermal-Enhanced Modulation of Cellular Membrane Potential Using Long-Wavelength-Activated Gold Nanoflowers.

In mammalian cells, plasma membrane potential plays vital roles in both physiology and pathology and it is controlled by a network of membrane-resident ion channels. There is considerable interest in the use of nanoparticles (NPs) to control biological functions, including the modulation of membrane potential. The photoexcitation of gold NPs (AuNPs) tethered close to the plasma membrane has been shown to induce membrane depolarization via localized heating of the AuNP surface coupled with the opening of voltage-gated sodium channels. Previous work has employed spherical AuNPs (AuNS) with absorption in the 500-600 nm range for this purpose. However, AuNP materials with absorption at longer wavelengths [e.g., near-infrared (NIR)] would enable greater tissue penetration depth in vivo. We show here the use of new anisotropic-shaped AuNPs [gold nanoflowers (AuNFs)] with broad absorption spanning into the NIR part of the spectrum (∼650-1000 nm). The AuNFs are directly synthesized with bidentate thiolate ligands, which preserves the AuNF's shape and colloidal stability, while facilitating conjugation to biomolecules. We describe the characterization of the AuNF particles and demonstrate that they adhere to the plasma membrane when bioconjugated to PEGylated cholesterol (PEG-Chol) moieties. The AuNF-PEG-Chol mediated the depolarization of rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells more effectively than AuNS-PEG-Chol and unconjugated AuNS and AuNF when photoexcited at ∼561 or ∼640 nm. Importantly, AuNF induction of depolarization had no impact on cellular viability. This work demonstrates anisotropic AuNFs as an enabling nanomaterial for use in cellular depolarization and the spatiotemporal control of cellular activity.

Multivalent Display of Erythropoietin on Quantum Dots Enhances Aquaporin-4 Expression and Water Transport in Human Astrocytes In Vitro.

In mammalian cells, growth factor-induced intracellular signaling and protein synthesis play a critical role in cellular physiology and homeostasis. In the brain's glymphatic system (GS), the water-conducting activity of aquaporin-4 (AQPN-4) membrane channels (expressed in polarized fashion on astrocyte end-feet) mediates the clearance of wastes through the convective transport of fluid and solutes through the perivascular space. The glycoprotein erythropoietin (EPO) has been shown to induce the astrocyte expression of AQPN-4 via signaling through the EPO receptor and the JAK/STAT signaling pathway. Here, we self-assemble EPO in a multivalent fashion onto the surface of semiconductor quantum dots (QDs) (driven by polyhistidine-based self-assembly) to drive the interaction of the bioconjugates with EPOR on human astrocytes (HA). This results in a 2-fold augmentation of JAK/STAT signaling activity and a 1.8-fold enhancement in the expression of AQPN-4 in cultured primary HA compared to free EPO. This translates into a 2-fold increase in the water transport rate in HA cells as measured by the calcein AM water transport assay. Importantly, EPO-QD-induced augmented AQPN-4 expression does not elicit any deleterious effect on the astrocyte viability. We discuss our results in the context of the implications of EPO-nanoparticle (NP) bioconjugates for use as research tools to understand the GS and their potential as therapeutics for the modulation of GS function. More generally, our results illustrate the utility of NP bioconjugates for the controlled modulation of growth factor-induced intracellular signaling.

Determining the Cytosolic Stability of Small DNA Nanostructures In Cellula.

DNA nanostructures have proven potential in biomedicine. However, their intracellular interactions─especially cytosolic stability─remain mostly unknown and attempts to discern this are confounded by the complexities of endocytic uptake and entrapment. Here, we bypass the endocytic uptake and evaluate the DNA structural stability directly in live cells. Commonly used DNA structures─crosshairs and a tetrahedron─were labeled with a multistep Förster resonance energy transfer dye cascade and microinjected into the cytosol of transformed and primary cells. Energy transfer loss, as monitored by fluorescence microscopy, reported the structure's direct time-resolved breakdown in cellula. The results showed rapid degradation of the DNA crosshair within 20 min, while the tetrahedron remained consistently intact for at least 1 h postinjection. Nuclease assays in conjunction with a current understanding of the tetrahedron's torsional rigidity confirmed its higher stability. Such studies can inform design parameters for future DNA nanostructures where programmable degradation rates may be required.

Recent Progress in Bioconjugation Strategies for Liposome-Mediated Drug Delivery.

In nanoparticle (NP)-mediated drug delivery, liposomes are the most widely used drug carrier, and the only NP system currently approved by the FDA for clinical use, owing to their advantageous physicochemical properties and excellent biocompatibility. Recent advances in liposome technology have been focused on bioconjugation strategies to improve drug loading, targeting, and overall efficacy. In this review, we highlight recent literature reports (covering the last five years) focused on bioconjugation strategies for the enhancement of liposome-mediated drug delivery. These advances encompass the improvement of drug loading/incorporation and the specific targeting of liposomes to the site of interest/drug action. We conclude with a section highlighting the role of bioconjugation strategies in liposome systems currently being evaluated for clinical use and a forward-looking discussion of the field of liposomal drug delivery.

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration.

BACKGROUND: Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. METHODS: For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. RESULTS: Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. CONCLUSION: Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration.

Sensing Nitric Oxide in Cells: Historical Technologies and Future Outlook.

Nitric oxide (NO) is a critical cell signaling molecule with important roles in both normal cellular physiology and pathology. Over the past 20 years, multiple sensing modalities have been developed for the intracellular synthesis (endogenous) and release (exogenous) of NO. In this review, we survey the historical progression of NO sensing platforms, highlight the current state of the art, and offer a forward-looking view of how we expect the field of NO sensing to develop in the context of recent advances in bio-nanotechnology and nanoscale cellular biosensors.