Heather Kalish

Comparison of Transfection Agents in Forming Complexes with Ferumoxides, Cell Labeling Efficiency, and Cellular Viability

By complexing ferumoxides or superparamagnetic iron oxide (SPIO) to transfection agents (TAs), it is possible to magnetically label mammalian cells. There has been no systematic study comparing TAs complexed to SPIO as far as cell labeling efficiency and viability. This study investigates the toxicity and labeling efficiency at various doses of FEs complexed to different TAs in mammalian cells. Different classes of TAs were used, such as polycationic amines, dendrimers, and lipid-based agents. Cellular toxicity was measured using doses of TAs from 1 to 50 μg/mL in incubation media. Iron incorporation efficiency was measured by combining various amounts of FEs and different doses of TAs. Lipofectamine2000 showed toxicity at lowest dose (1 μg/mL), whereas FuGENE6 and low molecular weight poly-L-lysine (PLI.) showed the least toxicity. SPIO labeling efficiency was similar with high-molecular-weight PIX (388.1 kDa) and superfect, whereas FuGENE6 and low-molecular-weight PLL were inefficient in labeling cells. Concentrations of 25 to 50 μg/mL of FEs complexed to TAs in media resulted in sufficient endocytosis of the SPIO into endosomes to detect cells on cellular magnetic resonance imaging.

COMBINATION OF TRANSFECTION AGENTS AND MAGNETIC RESONANCE CONTRAST AGENTS FOR CELLULAR IMAGING: RELATIONSHIP BETWEEN RELAXIVITIES, ELECTROSTATIC FORCES, AND CHEMICAL COMPOSITION

The purpose of this study was to investigate the changes in electrostatic and magnetic resonance (MR) properties observed when MR contrast agents (CAs) (Feridex®, MION‐46L, or G5‐dendrimer‐DOTA‐Gd) are combined with transfection agents (TAs) under various conditions for use as a CA‐TA complex basis for cellular labeling and MRI. CAs were incubated with various classes of TAs for 0–48 hr in solutions of varying concentrations and pH values. NMR relaxation rates (1/T1, 1/T2), MRI and zeta potential (ZP) of CA‐TA solutions were measured. TAs decreased the 1/T1 and 1/T2 of G5‐DOTA‐Gd, Feridex®, and MION‐46L by 0–95%. Altering the pH of G5‐DOTA‐Gd‐TA decreased the T1‐weighted signal intensity (SI) on MRI from 0 to 78%. Measured ZP values for G5‐DOTA‐Gd, Feridex®, and MION‐46L were −51, −41, and −2.0 mV, respectively. The TA LV had a negative ZP, while the other TAs had ZPs ranging from +20 to +65 mV. The alteration of the ZP and NMR relaxivities of the MR CAs, Feridex®, MION‐46L, and G5‐DOTA‐Gd by TAs has been demonstrated. These results enhance our understanding of the relationship between electrostatic and MR properties. Magn Reson Med 50:275–282, 2003. Published 2003 Wiley‐Liss, Inc.

Analysis of neurotrophins in human serum by immunoaffinity capillary electrophoresis (ICE) following traumatic head injury.

Neurotrophins, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and beta-nerve growth factor (beta-NGF), play an active role in the development, maintenance and survival of cells of the central nervous system (CNS). Previous research has indicated that a decrease in concentrations of these neurotrophins is often associated with cell death and ultimately patient demise. However, much of the research conducted analyses of samples taken directly from the CNS, i.e., of samples that are not readily available in clinical trauma centers. In an attempt to obtain a method for evaluating neurotrophins in a more readily accessible matrix, i.e., serum, a precise and accurate immunoaffinity capillary electrophoresis (ICE) method was developed and applied to measure neurotrophins in serum from patients with various degrees of head injury. The five neurotrophins of interest were extracted and concentrated by specific immunochemically immobilized antibodies, bound directly to the capillary wall, and eluted and separated in approximately 10min. NT-3, BDNF, CNTF and beta-NGF showed a marked decrease in concentration as the severity of the head injury increased: mild versus severe: 91% decrease for NT-3; 93 % decrease for BDNF; 93 % decrease for CNTF; and a 87 % decrease for beta-NGF. This decrease in concentration is consistent with the neuro-protective roles that neurotrophins play in the maintenance and survival of neuronal cells. The results obtained by the ICE method were closely comparable with those generated by a commercially available ELISA method.

Magneto‐Plasmonic Janus Vesicles for Magnetic Field‐Enhanced Photoacoustic and Magnetic Resonance Imaging of Tumors

Magneto-plasmonic Janus vesicles (JVs) integrated with gold nanoparticles (AuNPs) and magnetic NPs (MNPs) were prepared asymmetrically in the membrane for in vivo cancer imaging. The hybrid JVs were produced by coassembling a mixture of hydrophobic MNPs, free amphiphilic block copolymers (BCPs), and AuNPs tethered with amphiphilic BCPs. Depending on the size and content of NPs, the JVs acquired spherical or hemispherical shapes. Among them, hemispherical JVs containing 50 nm AuNPs and 15 nm MNPs showed a strong absorption in the near-infrared (NIR) window and enhanced the transverse relaxation (T2 ) contrast effect, as a result of the ordering and dense packing of AuNPs and MNPs in the membrane. The magneto-plasmonic JVs were used as drug delivery vehicles, from which the release of a payload can be triggered by NIR light and the release rate can be modulated by a magnetic field. Moreover, the JVs were applied as imaging agents for in vivo bimodal photoacoustic (PA) and magnetic resonance (MR) imaging of tumors by intravenous injection. With an external magnetic field, the accumulation of the JVs in tumors was significantly increased, leading to a signal enhancement of approximately 2-3 times in the PA and MR imaging, compared with control groups without a magnetic field.

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CVs below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.

Application of immunoaffinity capillary electrophoresis to the measurements of secreted cytokines by cultured astrocytes

The ability of the central nervous system (CNS) to act in conjunction with the immune system has been of great interest to both neurobiologists and immunologists. Previous studies have shown that astrocytes can be stimulated, by various peptides, to act as immune regulators within the CNS and release cytokines and chemokines. However, the regulatory mechanism of astrocytes is still poorly understood. Our present study describes a micro-device capable of monitoring the growth and stimulation of 20 astrocytes by vasoactive intestinal peptide. A microdialysis needle was used to collect the secretion by products, which were analyzed by immunoaffinity capillary electrophoresis (ICE) for the secretion of pro-inflammatory cytokines, IL-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha; hemopoietic cytokines, IL-3, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF); and chemokines; regulated upon activation normal T-cell expression sequence (RANTES), macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Vasoactive intestinal peptide stimulated astrocytes showed an almost immediate release of pro-inflammatory cytokines and chemokines, with an increase over baseline ranging from 3 to 15 fold, while no substantial increase over baseline was observed for hemopoietic cytokines. This system demonstrates the ability to isolate individual cells in a closely controlled environment and identify and quantify their analytes.

A chip-based immunoaffinity capillary electrophoresis assay for assessing hormones in human biological fluids

A chip‐based capillary electrophoresis system has been designed for assessing the concentrations of four hormones in whole human blood, saliva, and urine. The desired analytes were isolated by immunoextraction using a panel of four analyte‐specific antibodies immobilized onto a glass fiber insert within the injection port of the chip. Following extraction, the captured analytes were labeled prior to electro‐elution into the chip separation channel, where they were resolved into four individual peaks in circa 2 min. Quantification of each peak was achieved by on‐line LIF detection and integration of the area under each peak. Comparison to commercial high‐sensitivity immunoassays demonstrated that the chip‐based assay provided fast, accurate, and precise measurements for the analytes under investigation. As the availability of commercially available antibodies rapidly expands, the application of this system will greatly increase. Chip‐based CE separations of multiple analytes from a single sample also provide a significant advantage in the analysis of small samples.

Color Transformation and Fluorescence of Prussian Blue- Positive Cells: Implications for Histologic Verification of Cells Labeled with Superparamagnetic Iron Oxide Nanoparticles

Superparamagnetic iron oxide (SPIO) nanoparticles, either modified or in combination with other macromolecules, are being used for magnetic labeling of stem cells and other cells to monitor cell trafficking by magnetic resonance imaging (MRI) in experimental models. The correlation of histology to MRI depends on the ability to detect SPIO-labeled cells using Prussian blue (PB) stain and fluorescent tags to cell surface markers. Exposure of PB-positive sections to ultraviolet light at a wavelength of 365 nm commonly used fluorescence microscopy can result in color transformation of PB-positive material from blue to brown. Although the PB color transformation is primarily an artifact that may occur during fluorescence microscopy, the transformation can be manipulated using imaging process software for the detection of low levels of iron labeled cells in tissues samples

Suppressing Nanoparticle-Mononuclear Phagocyte System Interactions of Two-Dimensional Gold Nanorings for Improved Tumor Accumulation and Photothermal Ablation of Tumors

The clearance of nanoparticles (NPs) by mononuclear phagocyte system (MPS) from blood leads to high liver and spleen uptake and negatively impacts their tumor delivery efficiency. Here we systematically evaluated the in vitro and in vivo nanobio interactions of a two-dimensional (2D) model, gold (Au) nanorings, which were compared with Au nanospheres and Au nanoplates of similar size. Among different shapes, Au nanorings achieved the lowest MPS uptake and highest tumor accumulation. Among different sizes, 50 nm Au nanorings showed the highest tumor delivery efficiency. In addition, we demonstrated the potential use of Au naonrings in photoacoustic imaging and photothermal therapy. Thus, engineering the shape, surface area, and size of Au nanostructures is important in controlling NP-MPS interactions and improving the tumor uptake efficiency.

Receptor affinity CE for measuring bioactive inflammatory cytokines in human skin biopsies.

A chip‐based receptor affinity CE system has been employed to measure the concentrations of bioactive pro‐inflammatory cytokines in biopsy materials obtained from human atopic skin lesions. The device employs a replaceable affinity disk to which recombinant cytokine receptors have been chemically immobilized. Homogenates obtained from micro‐dissected human skin samples were injected into the system where the bioactive cytokines were captured in the receptor affinity port and labeled in situ with a laser dye. The captured cytokines were released and separated by CE, the resolved peaks being detected and measured by laser‐induced fluorescence. When compared with conventional cell‐based bioassays, the affinity receptor chip showed reasonable correlation with r2 values of 0.998 for interferon gamma, 0.994 for IL‐6 and 0.991 for tumor necrosis factor alpha. The complete process including cytokine capture, labeling, and analysis took approximately 12.5 min with intra‐ and inter‐assay CVs below 5.3% and recoveries of 84.9–98.4% at the 100 pg/mL concentration in buffer solutions and 84.5–95% in normal human tissue extract. The system could indicate clear differences between the various clinical stages of atopic dermatitis in human patients and could run 4–6 samples per hour. This system, like previous chip‐based systems designed in our laboratory, holds the potential for being modified to be a portable unit that could be used in clinics and other biomedical screening studies.