Fluorochrome-Functionalized Nanoparticles for Imaging DNA in Biological Systems

Attaching DNA binding fluorochromes to nanoparticles (NPs) provides a way of obtaining NPs that bind to DNA through fluorochrome mediated interactions. To obtain a nanoparticle (NP) that bound to the DNA in biological systems, we attached the DNA binding fluorochrome, TO-PRO 1 (TO), to the surface of the Feraheme (FH) NP, to obtain a fluorochrome-functionalized NP denoted TO-FH. When reacted with DNA <i>in vitro</i>, TO-FH formed microaggregates that were characterized by fluorescence, light scattering, and T2 changes. The formation of DNA/TO-FH microaggregates was also characterized by AFM, with microaggregates exhibiting a median size of 200 nm, and consisting of DNA and multiple TO-FH NPs whose individual diameters were only 25–35 nm. TO-FH failed to bind normal cells in culture, but treatment with chemotherapeutic agents or detergents yielded necrotic cells that bound TO-FH and vital fluorochromes similarly. The uptake of TO-FH by HT-29 xenografts (treated with 5-FU and oxaliplatin) was evident by surface fluorescence and MRI. Attaching multiple DNA binding fluorochromes to magnetic nanoparticles provides a way of generating DNA binding NPs that can be used to detect DNA detection by microaggregate formation <i>in vitro</i>, for imaging the DNA of necrotic cells in culture, and for imaging the DNA of a tumor treated with a chemotherapeutic agent. Fluorochrome functionalized NPs are a multimodal (magnetic and fluorescent), highly multivalent (<i>n </i>≈ 10 fluorochromes/NP) nanomaterials useful for imaging the DNA of biological systems

Cell patterning and aggregate formation inside microwells.

<p><b>A)</b> Cell patterning. Cells were localized inside the microwells. <b>B)</b> After cell seeding, the cells in the microwell array were cultured in a petri dish and aggregates formed within 24 h. <b>C)</b> Once the aggregate formation is complete inside the microwells, they can be stained. <b>D)</b> Aggregates can be imaged inside microwells. <b>E)</b> Aggregates can be easily released from the microwells by gentle flushing with media for other applications.</p

CSP cell survival <i>in vivo</i> following cardiac injury.

<p><b>A)</b> Protocol to measure the <i>in vivo</i> survival of CSP aggregates and suspensions. <b>B)</b> Representative serial bioluminescence images (BLI) of mice injected with CSP cell aggregates and CSP single cell suspensions. <b>C)</b> Percentage of CSP cell survival measured with BLI.</p

Aggregate integrity and survival in fluidic manipulations.

<p><b>A)</b> Aggregates formed in microwells can be easily flushed out from the microwell and centrifuged while remaining intact. <b>B)</b> Aggregate can be easily passed through a 30G needle without loosing integrity. <b>C)</b> A representative DAPI/EthD fluorescent image of aggregates before injection. <b>D)</b> A representative DAPI/EthD fluorescent image of aggregates after injection. <b>E)</b> Quantification of dead CSP cells in aggregates passing a 30G needle using EthD/DAPI fluorescent intensity ratio. (All bars represent 100 µm).</p

Aggregate survival tests <i>in vitro</i>.

<p><b>A)</b> Subsets of microwell arrays with 2D monolayer of cell culture (2D) and aggregates of three sizes (S, M, and L). Hydrogen peroxide and anoxia/reoxygenation treatments were employed to induce cell death. EthD (red) and DAPI (blue) staining were performed for the determination of cell death. <b>B)</b> Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to 200 µM-hydrogen peroxide treatment using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes. <b>C)</b> Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to anoxia/reoxygenation using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes.</p