Dan Heo

Role of surface charge in cytotoxicity of charged manganese ferrite nanoparticles towards macrophages

Amphiphilic surfactants have been used to disperse magnetic nanoparticles in biological media, because they exhibit a dual hydrophobic/hydrophilic affinity that facilitates the formation of a nanoemulsion, within which nanoparticle surfaces can be modified to achieve different physicochemical properties. For the investigation of the interactions of cells with charged magnetic nanoparticles in a biological medium, we selected the nanoemulsion method to prepare water-soluble magnetic nanoparticles using amphiphilic surfactant (polysorbate 80). The hydroxyl groups of polysorbate 80 were modified to carboxyl or amine groups. The chemical structures of carboxylated and aminated polysorbate 80 were confirmed, and water-soluble manganese ferrite nanoparticles (MFNPs) were synthesized with three types of polysorbate 80. Colloidal size, morphology, monodispersity, solubility and T2 relaxivity were found to be similar between the three types of MFNP. However, cationic MFNPs exhibited greater cytotoxicity in macrophages (RAW264.7 cells) and lower cellular membrane effective stiffness than anionic and non-ionic MFNPs. Moreover, cationic MFNPs exhibited large uptake efficiency for RAW264.7 cells compared with anionic or non-ionic MFNPs under the same conditions. Therefore, we propose that surface charge should be a key consideration factor in the design of magnetic nanoparticles for theragnostic applications.

In vivo sensing of proteolytic activity with an NSET-based NIR fluorogenic nanosensor.

Biomedical in vivo sensing methods in the near-infrared (NIR) range, which that provide relatively high photon transparency, separation from auto-fluorescence background, and extended sensitivity, are being used increasingly for non-invasive mapping and monitoring of molecular events in cancer cells. In this study, we fabricated an NIR fluorogenic nanosensor based on the nanoparticle surface energy transfer effect, by conjugation of fluorescent proteolytic enzyme-specific cleavable peptides with gold nanorods (GNRs). Membrane-anchored membrane type 1-matrix metalloproteinases (MT1-MMPs), a family of zinc-dependent proteolytic enzymes, can induce the metastatic potential of cancer cells by promoting degradation of the extracellular matrix. Therefore, sensitive detection of MT1-MMP activity can provide essential information in the clinical setting. We have applied in vivo NIR sensing to evaluate MT1-MMP activity, as an NIR imaging target, in an MT1-MMP-expressing metastatic tumor mouse model.

Galactosylated manganese ferrite nanoparticles for targeted MR imaging of asialoglycoprotein receptor

Cancer cells can express specific biomarkers, such as cell membrane proteins and signaling factors. Thus, finding biomarkers and delivering diagnostic agents are important in the diagnosis of cancer. In this study, we investigated a biomarker imaging agent for the diagnosis of hepatic cancers. The asialoglycoprotein receptor (ASGPr) was selected as a biomarker for hepatoma cells and the ASGPr-targetable imaging agent bearing a galactosyl group was prepared using manganese ferrite nanoparticles (MFNP) and galactosylgluconic acid. The utility of the ASGPr-targetable imaging agent, galactosylated MFNP (G-MFNP) was assessed by several methods in ASGPr-expressing HepG2 cells as target cells and ASGPr-deficient MCF7 cells. Physical and chemical properties of G-MFNP were examined using Fourier-transform infrared spectroscopy, dynamic light scattering, zeta potential analysis, and transmission electron microscopy. No significant cytotoxicity was observed in either cell line. Targeting ability was assessed using flow cytometry, magnetic resonance imaging, inductively coupled plasma atomic emission spectroscopy, absorbance analysis, dark-field microscopy, Prussian blue staining, and transmission electron microscopy. We demonstrated that G-MFNP target successfully and bind to ASGPr-expressing HepG2 cells specifically. We suggest that these results will be useful in strategies for cancer diagnoses based on magnetic resonance imaging.

Photothermal ablation of cancer cells using self-doped polyaniline nanoparticles.

Water-stable confined self-doping polyaniline nanocomplexes are successfully fabricated by nano-assembly using lauric acid both as a stabilizer and as a localized dopant. In particular, the colloidal stability of the polyaniline nanocomplexes in neutral pH and the photothermal potential by near-infrared light irradiation are characterized. We demonstrate that confined self-doping polyaniline nanocomplexes as a photothermal nanoagent are preserved in the doped state even at a neutral pH. Finally, confined self-doping polyaniline nanocomplexes aided by lauric acid are successfully applied for the photothermal ablation of cancer cells.

Molecular recognition of proteolytic activity in metastatic cancer cells using fluorogenic gold nanoprobes

We describe the development of biomarker-sensitive nanoprobes based on nanoparticle surface energy transfer (NSET) effect that enabling recognition of the expression of membrane type-1 matrix metalloproteinase (MT1-MMP) anchored on invasive cancer cells and its proteolytic activity simultaneously. First of all, we confirmed invasiveness of cancer cell lines (HT1080 and MCF7) via migration and invasion assay. We also prepared gold nanoparticle (GNP) acts as a quencher for fluorescein isothiocyanate (FITC). This FITC is conjugated in end-terminal of activatable fluorogenic peptide (ActFP). The ActFP attach to surface of GNP (GNP-ActFP) for a targeting moiety and proteolytic activity ligand toward MT1-MMP. The GNP-ActFP can generate fluorescence signal when ActFP is cleaved by proteolytic activity after targeting toward MT1-MMP. In order to study specificity for MT1-MMP, GNP-ActFP is treated to HT1080 and MCF7 cells, and then, we determine the in vitro targeting potential and fluorogenic activity of GNP-ActFP for MT1-MMP via fluorescence multi-reader. We also confirmed fluorogenic activity of GNP-ActFP via confocal microscopic imaging, and finally, endocytosis of GNP-ActFP is observed via cellular transmission electron microscopic imaging.

Maleimidyl magnetic nanoplatform for facile molecular MRI

In this study, we developed the maleimidyl magnetic nanoplatform, which enables functional targeting of a biomarker-specific moiety for molecular imaging via MRI. The maleimide group of the maleimidyl magnetic nanoplatform is conjugated with a thiol group without additional crosslinkers and side products. A physicochemical analysis was conducted to verify the effectiveness of the maleimidyl magnetic nanoplatform, and the existence of the maleimidyl group was investigated using the platform. To prepare biomarker-specific MRI probes, a thiolated aptamer and peptide were immobilized onto the maleimidyl group of the maleimidyl magnetic nanoplatform. The fabricated MRI probes were applied to four cancer cell lines: HT1080, MCF7, MKN45, and HEK293T. To investigate the potential of the molecular MRI probe, the target-biomarker specificity was confirmed without serious cytotoxicity, and in vivo MRI analysis using a xenograft mouse model was demonstrated. We believe these results will be useful for fabricating molecular MRI probes for the diagnosis of cancer.

T 2 - and T*2-weighted MRI of rat glioma using polysorbate-coated magnetic nanocrystals as a blood-pool contrast agent

In this study, T2- and T*2-weighted imaging potential of polysorbate-coated magnetic nanocrystals (P-MNCs) was investigated as a blood-pool contrast agent using a 9L-rat glioma model after intravenous injection via 3.0T MRI. Magnetic nanocrystals (MNCs, Fe3O4) synthesized by the thermal decomposition method were coated with polysorbate 80 using a nanoemulsion method to generate a water-stable MRI contrast agent. The physical properties and MR imaging capability of P-MNCs were verified. The orthotopic tumor models were established by implanting 9L-rat glioma cells into the rat brain. After tail-vein injection of P-MNCs, T2- and T*2-weighted imaging of tumor sites was performed. Blood clearance and biodistribution studies were also performed. The hydrodynamic diameter of P-MNCs was 10.5 ± 0.8 nm and a spherical magnetic core was confirmed. The r2 value of P-MNCs was calculated to be 114.1 mM−1 s−1. Heterogeneous contrast T2-weighted MRI images of the 9L-rat glioma model were visualized at the tumor site before injecting the MRI contrast agent. In particular, T*2-weighted images demonstrated more obvious signal intensity changes than did T2-weighted images. Neovasculature in the tumor tissue was clearly observed in T*2 images compared with T2-weighted images. The blood half-life of P-MNCs was 2 h and the Fe ion concentration of blood had returned to the baseline after 16 h. Well-tailored P-MNCs can be effectively used as a novel MRI contrast agent for visualizing of vasculatures for solid tumors via T2- and T*2-weighted imaging.

Galactosylated magnetic nanovectors for regulation of lipid metabolism based on biomarker-specific RNAi and MR imaging

The specific delivery of ribonucleic acid (RNA) interfering molecules to disease-related cells is still a critical blockade for in vivo systemic treatment. Here, this study suggests a robust delivery carrier for targeted delivery of RNA-interfering molecules using galactosylated magnetic nanovectors (gMNVs). gMNVs are an organic-inorganic polymeric nanomaterial composed of polycationics and magnetic nanocrystal for delivery of RNA-interfering molecules and tracking via magnetic resonance (MR) imaging. In particular, the surface of gMNVs was modified by galactosylgluconic groups for targeted delivering to asialoglycoprotein receptor (ASGPR) of hepatocytes. Moreover, the small interfering RNAs were used to regulate target proteins related with low-density lipoprotein level and in vivo MR imaging was conducted for tracking of nanovectors. The obtained results show that the prepared gMNVs demonstrate potential as a systemic theragnostic nanoplatform for RNA interference and MR imaging.

Compensatory UTE/T2W Imaging of Inflammatory Vascular Wall in Hyperlipidemic Rabbits.

Objectives To obtain compensatory ultra-short echo time (UTE) imaging and T2-weighted (T2W) imaging of Watanabe heritable hyperlipidemic (WHHL) rabbits following dextran-coated magnetic nanocluster (DMNC) injection for the effective in vivo detection of inflammatory vascular wall. Methods Magnetic nanoparticle was synthesized by thermal decomposition and encapsulated with dextran to prepare DMNC. The contrast enhancement efficiency of DMNC was investigated using UTE (repetition time [TR] = 5.58 and TE = 0.07 ms) and T2W (TR = 4000 and TE = 60 ms) imaging sequences. To confirm the internalization of DMNC into macrophages, DMNC-treated macrophages were visualized by cellular transmission electron microscope (TEM) and magnetic resonance (MR) imaging. WHHL rabbits expressing macrophage-rich plaques were subjected to UTE and T2W imaging before and after intravenous DMNC (120 μmol Fe/kg) treatment. Ex vivo MR imaging of plaques and immunostaining studies were also performed. Results Positive and negative contrast enhancement of DMNC solutions with increasing Fe concentrations were observed in UTE and T2W imaging, respectively. The relative signal intensities of the DMNC solution containing 2.9 mM Fe were calculated as 3.53 and 0.99 in UTE and T2W imaging, respectively. DMNC uptake into the macrophage cytoplasm was visualized by electron microscopy. Cellular MR imaging of DMNC-treated macrophages revealed relative signals of 3.00 in UTE imaging and 0.98 in T2W imaging. In vivo MR images revealed significant brightening and darkening of plaque areas in the WHHL rabbit 24 h after DMNC injection in UTE and T2W imaging, respectively. Ex vivo MR imaging results agreed with these in vivo MR imaging results. Histological analysis showed that DMNCs were localized to areas of inflammatory vascular wall. Conclusions Using compensatory UTE and T2W imaging in conjunction with DMNC is an effective approach for the noninvasive in vivo imaging of atherosclerotic plaque.