Three-dimensional in vitro co-culture model of breast tumor using magnetic levitation.

In this study, we investigate a novel in vitro model to mimic heterogeneous breast tumors without the use of a scaffold while allowing for cell-cell and tumor-fibroblast interactions. Previous studies have shown that magnetic levitation system under conventional culturing conditions results in the formation of three-dimensional (3D) structures, closely resembling in vivo tissues (fat tissue, vasculature, etc.). Three-dimensional heterogeneous tumor models for breast cancer were designed to effectively model the influences of the tumor microenvironment on drug efficiency. Various breast cancer cells were co-cultured with fibroblasts and then magnetically levitated. Size and cell density of the resulting tumors were measured. The model was phenotypically compared to in vivo tumors and examined for the presence of ECM proteins. Lastly, the effects of tumor stroma in the 3D in vitro model on drug transport and efficiency were assessed. Our data suggest that the proposed 3D in vitro breast tumor is advantageous due to the ability to: (1) form large-sized (millimeter in diameter) breast tumor models within 24 h; (2) control tumor cell composition and density; (3) accurately mimic the in vivo tumor microenvironment; and (4) test drug efficiency in an in vitro model that is comparable to in vivo tumors.

Design and in vitro evaluation of layer by layer siRNA nanovectors targeting breast tumor initiating cells.

Efficient therapeutics and early detection has helped to increase breast cancer survival rates over the years. However, the recurrence of breast cancer remains to be a problem and this may be due to the presence of a small population of cells, called tumor initiating cells (TICs). Breast TICs are resistant to drugs, difficult to detect, and exhibit high self-renewal capabilities. In this study, layer by layer (LBL) small interfering RNA (siRNA) nanovectors (SNVs) were designed to target breast TICs. SNVs were fabricated using alternating layers of poly-L-lysine and siRNA molecules on gold (Au) nanoparticle (NP) surfaces. The stability, cell uptake, and release profile for SNVs were examined. In addition, SNVs reduced TIC-related STAT3 expression levels, CD44+/CD24-/EpCAM+ surface marker levels and the number of mammospheres formed compared to the standard transfection agent. The data from this study show, for the first time, that SNVs in LBL assembly effectively delivers STAT3 siRNA and inhibit the growth of breast TICs in vitro.

Biocompatibility assessment of Si-based nano- and micro-particles.

Silicon is one of the most abundant chemical elements found on the Earth. Due to its unique chemical and physical properties, silicon based materials and their oxides (e.g. silica) have been used in several industries such as building and construction, electronics, food industry, consumer products and biomedical engineering/medicine. This review summarizes studies on effects of silicon and silica nano- and micro-particles on cells and organs following four main exposure routes, namely, intravenous, pulmonary, dermal and oral. Further, possible genotoxic effects of silica based nanoparticles are discussed. The review concludes with an outlook on improving and standardizing biocompatibility assessment for nano- and micro-particles.

Detecting DNA methylation through changes in transverse proton relaxation.

We present a facile, simple method to detect DNA methylation by measuring the transverse proton relaxation behaviour. Positively charged nanoparticles are arranged along the negatively charged backbone of DNA strands through electrostatic interactions. The arrangement of NPs along DNA strands aids to amplify and compare the transverse proton relaxation signal for un-cut versus cut DNA strands cleaved by sequence specific restriction enzymes. Results from this study suggest that the presence of methylation on DNA can be detected using superparamagnetic NPs using NMR.

Examining MRI contrast in three-dimensional cell culture phantoms with DNA-templated nanoparticle chains.

DNA-templated nanoparticle (NP) chains were examined as potential magnetic resonance imaging (MRI) contrast agents using in vitro environments of the extracellular matrix and tissue. A 3-T clinical MRI scanner was utilized to examine and compare image contrast enhanced by dispersed NPs, DNA-templated NP chains, gold-superparamagnetic multicomponent NP chains, and polyelectrolyte encapsulated, multicomponent NP chains in both T(1)-weighted and T(2)-weighted images. In addition, the longitudinal and transverse relaxivity (r(1) and r(2)) changes were measured both in the basement membrane, using Matrigel, and in the tissue environment, using in vitro 3D cell culture scaffolds. Results suggest that MRI contrast was significantly enhanced from NP chains compared to dispersed NPs in the basement membrane and polyelectrolyte encapsulation for NP chains produced similar relaxivity to nonencapsulated NP chains due to the enhanced cell uptake of encapsulated NP chains.

Mechanism of proton relaxation for enzyme-manipulated, multicomponent gold-magnetic nanoparticle chains.

Longitudinal and transverse relaxation times of multicomponent nanoparticle (NP) chains are investigated for their potential use as multifunctional imaging agents in magnetic resonance imaging (MRI). Gold NPs (ca. 5 nm) are arranged linearly along double-stranded DNA, creating gold NP chains. After cutting gold NP chains with restriction enzymes (EcoRI or BamHI), multicomponent NP chains are formed through a ligation reaction with enzyme-cut, superparamagnetic NP chains. We evaluate the changes in relaxation times for different constructs of gold-iron oxide NP chains and gold-cobalt iron oxide NP chains using 300 MHz (1)H NMR. In addition, the mechanism of proton relaxation for multicomponent NP chains is examined. The results indicate that relaxation times are dependent on the one-dimensional structure and the amount of superparamagnetic NP chains present in the multicomponent constructs. Multicomponent NP chains arranged on double-stranded DNA provide a feasible method for fabrication of multifunctional imaging agents that improve relaxation times effectively for MRI applications.

Characterizing proton relaxation times for metallic and magnetic layer-by-layer-coated, DNA-templated nanoparticle chains.

Metallic and superparamagnetic DNA-templated nanoparticle (NP) chains are examined as potential imaging agents. Proton relaxation times (T(1) and T(2)) are measured for DNA nanostructures using nuclear magnetic resonance (NMR) spectroscopy. The layer-by-layer (LBL) method was used to encapsulate the DNA-templated NP chains and demonstrated a change in proton relaxation times. Results from this study suggest that LBL-coated, DNA-templated nanostructures can serve as effective imaging agents for magnetic resonance imaging (MRI) applications.

In vitro cytotoxic evaluation of metallic and magnetic DNA-templated nanostructures.

We evaluate the potential in vitro cytotoxicity that may arise from metallic and magnetic DNA-templated nanostructures. By using a fluorescence-based assay, the viability of cells was examined after treatment with DNA-templated nanostructures. Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the amount of nanoparticles internalized by the cells. Cell uptake of DNA-templated nanostructures was enhanced after encapsulating the nanostructure with layers of polyelectrolytes (PSS and PAH) and targeting ligands. Transmission electron microscope (TEM) images provided evidence that the nanostructures were localized in vesicles in the cytoplasm of the cells. The results from this study suggest that gold, iron oxide, and cobalt iron oxide DNA-templated nanostructures do not induce in vitro toxicity.

Serial and parallel dip-pen nanolithography using a colloidal probe tip.

AFM tips terminated with PMMA colloids are used to pattern molecules in both serial and parallel modes by allowing the polymer on the tip to swell under different humidity conditions. This extension of the dip-pen nanolithography technique provides an easy methodology to place inks on different substrates without the need to perform specialized tip alignment.

Circular dichroism study of enzymatic manipulation on magnetic and metallic DNA template nanowires.

Circular dichroism spectroscopy (CD) was used to examine the mechanism of endonuclease clipping and ligation of the DNA template nanowires. The biomolecular manipulation of the DNA template is compared for both metallic (Au) and magnetic (Fe(2)O(3) and CoFe(2)O(4)) nanowires. The dependence of nanoparticle (NP) concentration on enzymatic clipping and DNA ligation was studied, in addition to performing absorbance and thermal melting experiments. Low-NP concentration preserved and digested the DNA template structure. Yet, at higher NP concentrations, the DNA template began to denature before enzyme addition. It was also observed that ligation of the digested DNA occurred more efficiently at low-NP concentrations. These results provide significant information on structural alteration and biorecognition effectiveness of the DNA template after enzymatic manipulation.

Circular dichroism study of the mechanism of formation of DNA templated nanowires.

In order to control the fabrication method, the mechanism used in the formation of DNA templated nanowires is investigated through circular dichroism (CD) spectroscopy. Metallic (Au) and magnetic (Fe(2)O(3) and CoFe(2)O(4)) nanoparticles (NP) are aligned along the DNA strand at various mass ratios. The DNA templated nanowires are compared to the structure of B-form dsDNA through CD experiments. Absorbance and thermal melting tests are performed to verify the structural changes of DNA templated nanowires. Low concentrations of nanoparticles preserve the DNA B-form through electrostatic interactions. Conversely, at higher concentrations of nanoparticles aligned along the DNA strand, the template is denatured. Information on the mode of nanoparticle binding and DNA helix alterations are explored for metallic and magnetic nanowires based upon the results.