Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics.

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

Controlling Cell Organization in 3D Coculture Spheroids Using DNA Interactions.

The role of stromal and immune cells in transforming the tumor microenvironment is a key consideration in understanding tumor cell behavior and anticancer drug development. To better model these systems in vitro, 3D coculture tumor spheroids have been engineered using a variety of techniques including centrifugation to microwells, hanging drop, low adhesion cultures, and culture of cells in a microfluidic platform. Aside from using bioprinting, however, it has remained more challenging to direct the spatial organization of heterotypic cells in standalone 3D spheroids. To address this, we present an in vitro 3D coculture tumor model where we modulated the interactions between cancer cells and fibroblasts through DNA hybridization. When native heterotypic cells are simply mixed, the cell aggregates typically show cell sorting behavior to form phase separated structures composed of single cell types. In this work, we demonstrate that when MDA-MB-468 breast cancer and NIH/3T3 fibroblasts are directed to associate via complementary DNA, a uniform distribution of the two cell types within a single spheroid was observed. In contrast, in the absence of specific DNA interactions between the cancer cells and fibroblasts, individual clusters of the NIH/3T3 cells formed in each spheroid due to cell sorting. To better understand the effect of heterotypic cell organization on either cell-cell contacts or matrix protein production, the spheroids were further stained with anti-E-cadherin and antifibronectin antibodies. While the amounts of E-cadherin appeared to be similar between the spheroids, a significantly higher amount of fibronectin secretion was observed in the coculture spheroids with uniform mixing of two cell types. This result showed that different heterotypic cell distributions within 3D architecture can influence the ECM protein production that can again alter the properties of the tumor or tumor microenvironment. The present study thus describes the use of DNA templating to direct the organization of cells in coculture spheroids, which can provide mechanistic biological insight into how heterotypic distribution in tumor spheroids can influence tumor progression, metastasis, and drug resistance.

Silica-coated gold nanorods with hydrophobic modification show both enhanced two-photon fluorescence and ultrasound drug release.

Hydrophobically-modified silica-coated gold nanorods are presented here as multifunctional theranostic agents. A single modification both increases two-photon fluorescence and promotes cavitation-based acoustic signal for imaging. A two-fold greater release of small molecule drugs was observed under ultrasound-mediated conditions as compared to passive release without ultrasound.

Generation of 3D cellular spheroids using DNA modified cell receptors and programmable DNA interactions.

3D culture is known to provide more faithful tissue models than 2D culture, and thus it is a valuable tool for in vitro evaluation of biological models. However, many cell lines are unable to form desired 3D spheroids by traditional methods because the naturally occurring cell-cell adhesion is too weak. Here, we present a method to produce 3D cell spheroids by using DNA-mediated assembly. We first demonstrate an Affinity Mediated Photoconjugation Approach (AMCP) to covalently modify cell receptors with affibody-streptavidin fusion proteins, where the affibody chemically crosslinks to cell expressed EGFR and the streptavidin is used to attach DNA strands. The DNA conjugated cells were then mixed with complementary DNA 'linker strands' to impart cell-cell interactions. When incubated in wells coated with non-adhesive polymers, cells formed dense spherical aggregates larger than 500 microns in diameter. Each of these studies was carried out using human breast cancer cells (MBA-MB-468), aneuploid human keratinocytes (HaCaT), and human colon cancer cells (Caco-2). Without either DNA on the cells or in solution as linkers, no cell spheroids were observed. After 96 h of incubation, the cultured DNA assembled spheroids were found to be mechanically stable enough to be handled easily for further analysis and confocal imaging. The findings suggest that the proposed DNA assembly method can be considered as an attractive strategy for assembling cells into stable spheroids.

Investigating the use of conducting oligomers and redox molecules in CdS-MoFeP biohybrids.

In this work we report the effect of incorporating conducting oligophenylenes and a cobaltocene-based redox mediator on photodriven electron transfer between thioglycolic acid (TGA) capped CdS nanorods (NR) and the native nitrogenase MoFe protein (MoFeP) by following the reduction of H+ to H2. First, we demonstrate that the addition of benzidine-a conductive diphenylene- to TGA-CdS and MoFeP increased catalytic activity by up to 3-fold as compared to CdS-MoFeP alone. In addition, in comparing the use of oligophenylenes composed of one (p-phenylenediamine), two (benzidine) or three (4,4''-diamino-p-terphenyl)phenylene groups, the largest gain in H2 was observed with the addition of benzidine and the lowest with phenylenediamine. As a comparison to the conductive oligophenylenes, a cobaltocene-based redox mediator was also tested with the TGA-CdS NRs and MoFeP. However, adding either cobaltocene diacid or diamine caused negligible gains in H2 production and at higher concentrations, caused a significant decrease. Agarose gel electrophoresis revealed little to no detectable interaction between benzidine and TGA-CdS but strong binding between cobaltocene and TGA-CdS. These results suggest that the tight binding of the cobaltocene mediator to CdS may hinder electron transfer between CdS and MoFe and cause the mediator to undergo continuous reduction/oxidation events at the surface of CdS.

Hydrophobically Modified Silica-Coated Gold Nanorods for Generating Nonlinear Photoacoustic Signals.

In this work, we report that gold nanorods coated with hydrophobically-modified mesoporous silica shells not only enhance photoacoustic (PA) signal over unmodified mesoporous silica coated gold nanorods, but that the relationship between PA amplitude and input laser fluence is strongly nonlinear. Mesoporous silica shells of ~14 nm thickness and with ~3 nm pores were grown on gold nanorods showing near infrared absorption. The silica was rendered hydrophobic with addition of dodecyltrichlorosilane, then re-suspended in aqueous media with a lipid monolayer. Analysis of the PA signal revealed not only an enhancement of PA signal compared to mesoporous silica coated gold nanorods at lower laser fluences, but also a nonlinear relationship between PA signal and laser fluence. We attribute each effect to the entrapment of solvent vapor in the mesopores: the vapor has both a larger expansion coefficient and thermal resistance than silica that enhances conversion to acoustic energy, and the hydrophobic porous surface is able to promote phase transition at the surface, leading to a nonlinear PA response even at fluences as low as 5 mJ cm-2. At 21 mJ cm-2, the highest laser fluence tested, the PA enhancement was >12-fold over mesoporous silica coated gold nanorods.

Formation of Silver Nanostructures by Rolling Circle Amplification Using Boranephosphonate-Modified Nucleotides.

We investigate the efficiency of incorporation of boranephosphonate-modified nucleotides by phi29 DNA polymerase and present a simple method for forming large defined silver nanostructures by rolling circle amplification (RCA) using boranephosphonate internucleotide linkages. RCA is a linear DNA amplification technique that can use specifically circularized DNA probes for detection of target nucleic acids and proteins. The resulting product is a collapsed single-stranded DNA molecule with tandem repeats of the DNA probe. By substituting each of the natural nucleotides with the corresponding 5'-(α-P-borano)deoxynucleosidetriphosphate, only a small reduction in amplification rate is observed. Also, by substituting all four natural nucleotides, it is possible to enzymatically synthesize a micrometer-sized, single-stranded DNA molecule with only boranephosphonate internucleotide linkages. Well-defined silver particles are then readily formed along the rolling circle product.