Kyuri Lee

Hyaluronic Acid-Paclitaxel Conjugate Micelles : Synthesis, Characterization, and Antitumor Activity

Chemical conjugates of paclitaxel and hyaluronic acid (HA) were synthesized by utilizing a novel HA solubilization method in a single organic phase. Hydrophilic HA was completely dissolved in anhydrous DMSO with addition of poly(ethylene glycol) (PEG) by forming nanocomplexes. Paclitaxel was then chemically conjugated to HA in the DMSO phase via an ester linkage without modifying extremely hydrophilic HA. A series of HA-paclitaxel conjugates with different conjugation percentages were synthesized and characterized. HA-paclitaxel conjugates self-assembled in aqueous solution to form nanosized micellar aggregates, as characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). An intact form of paclitaxel was regenerated from HA-paclitaxel conjugate micelles at acidic pH conditions. HA-paclitaxel conjugate micelles exhibited more pronounced cytotoxic effect for HA receptor overexpressing cancer cells than for HA receptor deficient cells, suggesting that they can be potentially utilized as tumor-specific nanoparticulate therapeutic agents.

Synthesis, characterization, and in vivo diagnostic applications of hyaluronic acid immobilized gold nanoprobes

Herein we describe a new class of multifunctional gold nanoprobes for ultra-sensitive optical detection of reactive oxygen species (ROS) and hyaluronidase (HAdase). The nanoprobes were fabricated by end-immobilizing near-infrared fluorescence (NIRF) dye labeled hyaluronic acid (HA) onto the surface of gold nanoparticles (AuNPs). The nanoprobes effectively induced nanoparticle surface energy transfer (NSET) between NIRF dyes and AuNPs. When the surface immobilized HA was cleaved by ROS and HAdase, strong fluorescence recovery signals were attained with extreme sensitivity. In live animal models of rheumatoid arthritis (RA) and metastatic tumor, local arthritic inflammation and tumor sites were clearly identified upon systemic injection of the nanoprobes. These results suggest that the gold nanoprobes can be exploited not only as in vitro molecular and cellular imaging sensors for ROS and HAdase, but also as in vivo optical imaging agents for detection of local HA degrading diseases such as RA and tumor.

Surface functionalized hollow manganese oxide nanoparticles for cancer targeted siRNA delivery and magnetic resonance imaging

Multifunctional hollow manganese oxide nanoparticles (HMON) were produced by a bio-inspired surface functionalization approach, using 3,4-dihydroxy-L-phenylalanine (DOPA) as an adhesive moiety, for cancer targeted delivery of therapeutic siRNA and simultaneous diagnosis via magnetic resonance imaging (MRI). Cationic polyethylenimine-DOPA conjugates were stably immobilized onto the surface of HMON due to the strong binding affinity of DOPA to metal oxides, as examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. These nanoparticles were subsequently functionalized with a therapeutic monoclonal antibody, Herceptin, to selectively target cancer cells. Confocal microscopy and MR imaging studies revealed that the surface functionalized HMON enabled the targeted detection of cancer cells in T(1)-weighted MRI as well as the efficient intracellular delivery of siRNA for cell-specific gene silencing. These nanomaterials are expected to be widely exploited as multifunctional delivery vehicles for cancer therapy and imaging applications.

Heparin immobilized gold nanoparticles for targeted detection and apoptotic death of metastatic cancer cells

In the present study, heparin immobilized, multifunctional gold nanoparticles (AuNPs) were developed as a new class of theragnostic nanomaterials for metastatic cancer cell imaging and apoptosis. AuNPs were surface modified with fluorescent dye labeled heparin molecules to detect a metastatic stage of cancer cells that over-express heparin-degrading enzymes. The heparin immobilized AuNPs exhibited enhanced fluorescence signals by specific cleavage of heparin molecules from the surface of AuNPs by heparinase or heparanase secreted from metastatic cancer cells. In addition, heparin immobilized AuNPs that were additionally tethered with RGD peptides on the surface demonstrated highly specific apoptotic activities for selective cancer cells over-expressing RGD receptors on the membrane, revealing that internalized heparin within cells clearly triggered an apoptotic event. These results suggest that heparin immobilized AuNPs can be usefully exploited for optical imaging agents for metastatic tumors as well as therapeutic cancer treatment.

Pluronic/polyethylenimine shell crosslinked nanocapsules with embedded magnetite nanocrystals for magnetically triggered delivery of siRNA.

Pluronic/polyethylenimine shell crosslinked nanocapsules with embedded magnetite nanocrystals (PPMCs) were developed for magnetically triggered delivery of siRNA. The positively charged PPMCs formed stable nanosized polyelectrolyte complexes via electrostatic interactions with negatively charged siRNA-polyethylene glycol conjugate (siRNA-s-s-PEG) that was linked via a cleavable disulfide linkage. PPMC/siRNA-s-s-PEG polyelectrolyte complexes were efficiently taken up by cancer cells upon exposure to a magnet, thereby enhancing intracellular uptake and silencing effect of siRNA. The present study suggests that these novel nanomaterials could be potentially utilized for magnetically triggered delivery of various nucleic acid-based therapeutic agents.

Optical imaging of intracellular reactive oxygen species for the assessment of the cytotoxicity of nanoparticles

The generation of intracellular reactive oxygen species (ROS) was optically monitored using ROS-sensitive gold nanoprobes in response to an exposure of nanoparticles (NPs). Fluorescent dye-labeled hyaluronic acid was grafted onto the surface of gold nanoparticles (HF-AuNPs) for imaging intracellular ROS. The ultrasensitive detection of intracellular ROS was utilized as a powerful analytical tool to assess early cellular toxicities of monodisperse polystyrene (PS) particles with different sizes and different functional groups on the surface. The effect of PEGylation on the surface of PS NPs was also investigated by evaluating intracellular ROS generation. For various PS NPs, the extent of intracellular ROS was well correlated with cellular uptake, apoptosis inducing activity, and cytotoxic effect of NPs. In addition to the nanoparticles, commonly used polymeric gene carriers such as linear and branched polyethylenimine (PEI) were tested to analyze their extent of intracellular ROS generation related to cellular toxicity. This study demonstrated that sensitive and optical detection of intracellular ROS generation can provide a valuable toxicity index value for a wide range of NPs as an early indicator for cellular responses.

Pluronic/chitosan shell cross-linked nanocapsules encapsulating magnetic nanoparticles

We have developed novel Pluronic/chitosan nanocapsules encapsulating iron oxide nanoparticles. These nanocapsules were produced by dispersing hydrophobically-modified iron oxide nanoparticles and amine-reactive Pluronic derivatives in an organic solvent, and subsequently emulsification in an aqueous chitosan solution by ultrasonication. The resultant shell cross-linked nanocapsules had a unique core/shell type nanoreservoir architecture: an inner core encapsulating magnetic nanoparticles and a hydrophilic Pluronic/chitosan polymer shell layer, as confirmed by thermogravimetric analysis and transmission electron microscopy. Confocal laser scanning microscopy revealed that the rhodamine-labeled nanocapsules were efficiently internalized by human lung carcinoma cells upon exposure to an external magnetic field. The present study suggested that these novel nanomaterials could be dually utilized for the magnetically-triggered delivery of various anti-cancer agents and for cancer diagnosis with magnetic resonance imaging.

Ischemic brain imaging using fluorescent gold nanoprobes sensitive to reactive oxygen species.

Fluorescein-labeled hyaluronic acids (HA) were immobilized on gold nanoparticles for reactive oxygen species (ROS) detection. The efficacy of HA immobilized gold nanoparticles (HHAuNPs) was evaluated in a stroke animal model. The stroke rat model was produced by transient middle cerebral artery occlusion (MCAO), which induced transient ischemia and reperfusion (I/R) in the brain. The increase of ROS in the I/R brain was confirmed by TBARS assay with the brain extracts. For brain imaging, HHAuNPs were injected into the rat brain 1 h before transient MCAO. Five hours after the injection, the rats were sacrificed and the brains were subjected to imaging analysis. The results showed that stronger signals were detected in the I/R brains than in the normal brains. To identify the time window for effective detection of ROS, HHAuNPs were injected into the post-ischemic rat brains at various time points. The results showed that ROS level reached a maximum at 24 h after the transient MCAO. Also, a live imaging study was performed with HHAuNPs in the normal and I/R animals. The results confirmed that ROS level increased in the I/R animal group with time, while the signal was decreased in the normal animal group. Together, our results suggest that HHAuNPs may be useful to monitor ROS level in the ischemic brain and to identify the infarct areas in ischemic brains for the treatment of stroke.