Gadolinium-based nanoparticles for highly efficient T1-weighted magnetic resonance imaging.

We developed Pyrene-Gadolinium (Py-Gd) nanoparticles as pH-sensitive magnetic resonance imaging (MRI) contrast agents capable of showing a high-Mr signal in cancer-specific environments, such as acidic conditions. Py-Gd nanoparticles were prepared by coating Py-Gd, which is a complex of gadolinium with pyrenyl molecules, with pyrenyl polyethyleneglycol PEG using a nano-emulsion method. These particles show better longitudinal relaxation time (T1) MR signals in acidic conditions than they do in neutral conditions. Furthermore, the particles exhibit biocompatibility and MR contrast effects in both in vitro and in vivo studies. From these results, we confirm that Py-Gd nanoparticles have the potential to be applied for accurate cancer diagnosis and therapy.

Targetable gold nanorods for epithelial cancer therapy guided by near-IR absorption imaging.

Well-designed nanoparticle-mediated, image-guided cancer therapy has attracted interest for increasing the efficacy of cancer treatment. A new class of smart theragnostic nanoprobes employing cetuximab (CET)-conjugated polyethylene glycol (PEG)ylated gold nanorods (CET-PGNRs) is presented; these nanoprobes target epithelial cancer cells using near-infrared light. The cetyltrimethylammonium bromide bilayer on GNRs is replaced with heterobifunctional PEG (COOH-PEG-SH) to serve as a biocompatible stabilizer and to increase specificity. The carboxylated GNRs are further functionalized with CET using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry. To assess the potential of such GNRs, their optical properties, biocompatibility, colloidal stability, in vitro/in vivo binding affinities for cancer cells, absorption imaging, and photothermal therapy effects are investigated. CET-PGNRs exhibit excellent tumor targeting ability and strong potential for simultaneous absorption imaging and photothermal ablation of epithelial cancer cells.

Highly selective CD44-specific gold nanorods for photothermal ablation of tumorigenic subpopulations generated in MCF7 mammospheres.

Heterogeneous stem-like populations within tumor tissues are the primary suspects in causing cancer recurrence and malignancy. It is essential to selectively kill these tumorigenic populations. We created a novel system for photothermally ablating specific cells from three-dimensional mammospheres. A CD44-positive subpopulation, with tumorigenic and self-renewal potential, spontaneously arises in MCF7 breast cancer cell-engineered mammospheres. Using anti-CD44 antibody-linked gold nanorods, which strongly absorb near infrared light and increase local temperature, we effectively targeted and photo-ablated atypical cells. This biomarker-specific photothermal ablation model, using a smart nanoplatform, is a promising new strategy for selectively killing cancer cells, while sparing normal tissues.

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.

Thiolated dextran-coated gold nanorods for photothermal ablation of inflammatory macrophages.

Thiolated dextran-coated gold nanorods (DEX-GNRs) were synthesized for targeted delivery to inflammatory macrophages and their photothermal ablation under near-infrared (NIR) light irradiation. Successful synthesis of DEX-GNRs was achieved using thiolated dextran, generated by applying mercaptopropionic acid to transform a hydroxyl group of dextran into a thiol group which has strong binding affinity with surfaces of GNRs. We confirmed both the existence of a thiol group in the functionalized dextran using Ellman's reagent in a thiol group assay and the characteristic band of DEX-GNRs using FT-IR spectrum. Furthermore, a cellular uptake study revealed that dextran showed a superior ability to bind the GNRs surface against macrophages compared to those of PEGylated GNRs with various molecular weights of polyethyleneglycol (PEG). Consequently, an in vitro photothermal irradiation experiment using NIR light indicated that DEX-GNRs exhibited a significant cell-killing efficacy, even with a lower concentration of Au and a low-power light source.

Synthesis of gold nanorod-embedded polymeric nanoparticles by a nanoprecipitation method for use as photothermal agents.

For the synthesis of biocompatible photothermal agents, gold nanorod-embedded polymeric nanoparticles (GPNs) were synthesized using a nanoprecipitation method. Uniform gold nanorods (GNRs), which are sensitive to a photothermal effect by near-infrared (NIR) light, with an aspect ratio of 4.0 were synthesized by a seed-mediated growth method. The hydroxyl groups of polycaprolactone diol (PCL diOH) were modified by esterification with mercaptopropionic acid to give a dithiol (polycaprolactone dithiol, PCL diSH) as a phase transfer and capping agent. Subsequently, hexadecyltrimethylammonium bromide (CTAB), a stabilizer of GNRs, was exchanged and/or removed by PCL diSH. PCL diSH-coated GNRs were further wrapped in a hydrophilic polymer, Pluronic F127, as a stabilizer. These newly formulated GPNs exhibit excellent stability in water and a maximum absorbance in the NIR region indicating a highly efficient surface plasmon resonance effect, phenomena useful for photothermal agents.

Fluorescent magnetic nanohybrids as multimodal imaging agents for human epithelial cancer detection.

Cetuximab conjugated fluorescent magnetic nanohybrids (CET-FMNHs) were synthesized for detection of human epithelial cancer via magnetic resonance (MR) and optical imaging. Spherical FMNHs consist of MnFe(2)O(4) magnetic nanocrystals encapsulated in pyrene-labeled PCL-b-PMAA as a surfactant prepared by a nano-emulsion method. FMNHs demonstrated excellent colloidal stability and biocompatibility for biomedical application. Antibody conjugated fluorescent magnetic nanohybrids (CET-FMNHs) served as effective agents for both magnetic resonance (MR) and fluorescence optical imaging of cancer cell lines.

Enhancement of cellular binding efficiency and cytotoxicity using polyethylene glycol base triblock copolymeric nanoparticles for targeted drug delivery.

Folate (FA) conjugated tri-block copolymers were prepared by bioconjugation of poly epsilon-caprolactonediol and various molecular weights of diamine polyethylene glycol. The synthetic tri-block copolymers were characterized by 1H-NMR. Three types of nanoparticles were prepared by nanoprecipitation. Their size and morphology were verified by laser scattering and transmission electron microscopy, respectively. The colloidal stability of the nanoparticles was evaluated by turbidity test. The anticancer drug doxorubicin (DOX) was encapsulated in the nanoparticles during preparation. Drug loading amounts and release behavior from prepared nanoparticles were investigated. Fluorescent-activated cell sorting analysis and epi-fluorescencic microscopic imaging of prepared nanoparticles exhibited good cellular uptake against target cells. FA receptor expressed OVCAR3 cells that showed higher mean fluorescence intensity than FA receptor defect A549 cells at specific polyethylene glycol chain lengths. The cell cytotoxicity of prepared nanoparticles was evaluated for receptor mediated drug delivery.

Synthesis of water soluble PEGylated magnetic complexes using mPEG-fatty acid for biomedical applications.

We report the successful fabrication of the various types of water soluble PEGylated magnetic complexes (PMCs) for magnetism-related biomedical applications. Various types of PMCs were synthesized and tested to accomplish phase transfer from organic to aqueous phase using monomethoxy polyethylene glycol (mPEG)-fatty acid amphiphilic block copolymers (PFs) through conjugation of the hydroxyl group of mPEG with the carboxyl group of fatty acids. We also carefully investigate their colloidal stabilities in aqueous phase according to the ratio of hydrophilic and hydrophobic lengths relying on different types of fatty acids. Synthesized PMCs clearly demonstrated high magnetic sensitivity under magnetic field as magnetic resonance (MR) contrast agents. Furthermore, PMCs exhibited sufficient cell viabilities and excellent cell affinities in an in vitro model. Our results demonstrated that our PMCs possessed the potential for highly efficient magnetism-related biomedical applications such as MR image agents, drug delivery and tracking of cells.

Magnetic nanoclusters engineered by polymer-controlled self-assembly for the accurate diagnosis of atherosclerotic plaques via magnetic resonance imaging.

Oleyl dextran-coated magnetic nanoclusters (ODMCs) are fabricated for the accurate detection of macrophage-rich atherosclerotic plaques using magnetic resonance (MR) imaging. Dextran is introduced to the cluster surface of magnetic nanocrystals (MNCs) through self-assembly using amphiphilic oleic acid-conjugated dextran (ODex) to provide not only hydrophilicity but also a high affinity to macrophages. Enhanced magnetism of the ODMCs is engineered by optimizing the degree of substitution (DS) of the oleyl group in ODex and the concentration of ODex used for the synthesis of ODMC. Consequently, ODMCs exhibit significantly increased T2 relaxivity and excellent macrophage-targeting ability without cytotoxicity, in vitro. Moreover, in vivo T2-weighted MR imaging after intravenous injection of ODMCs into a rat artery balloon injury model demonstrates considerable MR contrast strength efficacy in the plaques of the injured carotid artery. These novel ODMCs may offer a highly efficient MR imaging nanoprobes for the selective diagnosis of atherosclerosis.

A biodegradable polymersome containing Bcl-xL siRNA and doxorubicin as a dual delivery vehicle for a synergistic anticancer effect.

Combined cancer treatment via co-delivery of siRNAs and an anticancer drug can be a promising strategy due to the synergistic effect of simultaneously minimizing gene/drug administration. In this study, Bcl-xL siRNA and doxorubicin (DOX) are encapsulated into designed methoxy-poly(ethylene glycol)-block-poly(D,L-lactic acid) (mPEG-b-PLA) block copolymer polymersomes (PSomes). A study of the cytotoxicity of Bcl-xL siRNA and DOX co-encapsulated PSomes (CPSomes) shows more inhibited proliferation of MKN-45 and MKN-28 human gastric cancer cell lines than only gene- and drug-loaded ones. Consequently, these results demonstrate that co-delivery of genes and drugs using PSomes results in a synergistic efficacy and indicates the potential of PSomes as efficient nanocarriers for combined cancer therapy.

Self-assembled fluorescent magnetic nanoprobes for multimode-biomedical imaging.

We fabricated multimode nanoprobes for acquisition of biological information at different object levels, i.e., in vivo detection and ex vivo validation for characterizing tumor angiogenesis. Fluorescent magnetic nanoprobes (FMNPs) were synthesized by using amphiphilic pyrenyl polyethylene glycol (Py-PEG) and superparamagnetic MnFe(2)O(4) nanocrystals (MNCs). Py-PEG, which is synthesized by conjugation of hydrophilic PEG with hydrophobic and fluorescent 1-pyrenebutyric acid through an esterification process, is capable of self-assembly and maintaining a high UV fluorescent intensity in aqueous phase. Py-PEG can be used as a fluorescent surfactant that simultaneously and efficiently encapsulates MNCs to exhibit fluorescent and magnetic properties as well as maintaining high water-solubility. Consequently, we proved that our biologically non-toxic FMNPs were prominent multimode imaging probes by showing not only excellent MR sensitivity but also high illumination intensity with strong signal strength under short exposure time of UV light from the extensive imaging studies of in vitro/vivo and ex vivo using orthotopic and xenograft mice models.

Synthesis of aminated polysorbate 80 for polyplex-mediated gene transfection.

To develop novel gene delivery carriers, aminated polysorbate 80 (P80-NH(2)) was synthesized with strong positively charged properties through the introduction of three amine groups. The resulting P80-NH(2) and DNA polyplex exhibited superb condensation abilities due to the high densities of positively charged amines groups. Size and surface charge of polyplex were shown to be well suited for cellular internalization. In addition, the P80-NH(2) /DNA polyplex demonstrated acceptable transfection efficiency in HeLa cells and was nontoxic relative to the conventional 25-kDa polyethyleneimine system.

Prostate cancer cell death produced by the co-delivery of Bcl-xL shRNA and doxorubicin using an aptamer-conjugated polyplex.

We investigated the synergism between shRNAs against Bcl-xL and doxorubicin (DOX) using aptamer-conjugated polyplexes (APs) in combination cancer therapy. Synergistic and selective cancer cell death was achieved by AP-mediated co-delivery of very small amounts of DOX and Bcl-xL-specific shRNA, which simultaneously activated an intrinsic apoptotic pathway. A branched polyethyleneimine (PEI) was grafted to polyethylene glycol (PEI-PEG) to serve as a vehicle for shRNA delivery, and its surface was further conjugated with an anti-PSMA aptamer (APT) for the selective delivery of APs to prostate cancer cells that express prostate-specific membrane antigens (PSMA) on their cell surface. The APs were finally obtained after intercalation of DOX to form shRNA/PEI-PEG-APT/DOX conjugates. Cell viability assays and FACS analysis of GFP expression against PC3 (PSMA deficient) and LNCaP (PSMA overexpressed) cells demonstrated that the synthesized APs inhibited the growth of PSMA-abundant prostate cancer cells with strong cell selectivity. Consequently, IC(50) values of APs loaded with both DOX and shRNA were approximately 17-fold less than those for the simple mixture of shRNA plus drug (shRNA/Lipofectamine + DOX). These results suggest that AP-mediated co-delivery of an anti-cancer drug and shRNA against Bcl-xL may widen the therapeutic window and allow for the selective destruction of cancer cells.

Synthesis and characterization of fluorescent magneto polymeric nanoparticles (FMPNs) for bimodal imaging probes.

Novel bifunctional fluorescent magneto polymeric nanoprobes (FMPNs) were synthesized to provide simultaneous diagnostic information via magnetic resonance imaging (MRI) and optical imaging. FMPNs consist of ultra-sensitive magnetic nanocrystals that function as MR probes combined with Nile Red, which functions as a fluorescent probe. FMPNs were encapsulated by a nano-emulsion method in polyvinyl alcohol (PVA, 87-89% hydrolyzed) through a matrix of polymethyl methacrylate (PMMA). FMPNs exhibited excellent colloidal stability and monodispersity. The production of MR and optical images demonstrated that FMPNs have potential as dual-mode imaging agents.

Nanomechanical in situ monitoring of proteolysis of peptide by Cathepsin B.

Characterization and control of proteolysis of peptides by specific cellular protease is a priori requisite for effective drug discovery. Here, we report the nanomechanical, in situ monitoring of proteolysis of peptide chain attributed to protease (Cathepsin B) by using a resonant nanomechanical microcantilever immersed in a liquid. Specifically, the detection is based on measurement of resonant frequency shift arising from proteolysis of peptides (leading to decrease of cantilever's overall mass, and consequently, increases in the resonance). It is shown that resonant microcantilever enables the quantification of proteolysis efficacy with respect to protease concentration. Remarkably, the nanomechanical, in situ monitoring of proteolysis allows us to gain insight into the kinetics of proteolysis of peptides, which is well depicted by Langmuir kinetic model. This implies that nanomechanical biosensor enables the characterization of specific cellular protease such as its kinetics.

Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging.

Since the use of magnetic nanocrystals as probes for biomedical system is attractive, it is important to develop optimal synthetic protocols for high-quality magnetic nanocrystals and to have the systematic understanding of their nanoscale properties. Here we present the development of a synthetically controlled magnetic nanocrystal model system that correlates the nanoscale tunabilities in terms of size, magnetism, and induced nuclear spin relaxation processes. This system further led to the development of high-performance nanocrystal-antibody probe systems for the diagnosis of breast cancer cells via magnetic resonance imaging.