Biocompatible glycol chitosan-coated gold nanoparticles for tumor-targeting CT imaging.

PURPOSE: The application of gold nanoparticles (AuNPs) in biomedical field was limited due to the low stability in the biological condition. Herein, to enhance stability and tumor targeting ability of AuNPs, their surface was modified with biocompatible glycol chitosan (GC) and the in vivo biodistribution of GC coated AuNPs (GC-AuNPs) were studied through computed tomography (CT). METHODS: Polymer-coated gold nanoparticles were produced using GC as a reducing agent and a stabilizer. Their feasibility in biomedical application was explored through CT in tumor-bearing mice. RESULTS: Stability of gold nanoparticles increased in the physiological condition due to the GC coating layer on the surface. Tomographic images of tumor were successfully obtained in the tumor-xenografted animal model when the GC-AuNPs were used as a CT contrast agent. The tumor targeting property of the gold nanoparticles was due to the properties of GC because GC-AuNPs were accumulated in the tumor, while most of heparin-coated nanoparticles were found in the liver and spleen. CONCLUSIONS: The polymer properties on the surface played an important role in the behavior of gold nanoparticles in the biological condition and the enhanced stability and tumor targeting property of nanoparticles were inherited from GC on the surface.

Nano-sized metabolic precursors for heterogeneous tumor-targeting strategy using bioorthogonal click chemistry in vivo.

Herein, we developed nano-sized metabolic precursors (Nano-MPs) for new tumor-targeting strategy to overcome the intrinsic limitations of biological ligands such as the limited number of biological receptors and the heterogeneity in tumor tissues. We conjugated the azide group-containing metabolic precursors, triacetylated N-azidoacetyl-d-mannosamine to generation 4 poly(amidoamine) dendrimer backbone. The nano-sized dendrimer of Nano-MPs could generate azide groups on the surface of tumor cells homogeneously regardless of cell types via metabolic glycoengineering. Importantly, these exogenously generated 'artificial chemical receptors' containing azide groups could be used for bioorthogonal click chemistry, regardless of phenotypes of different tumor cells. Furthermore, in tumor-bearing mice models, Nano-MPs could be mainly localized at the target tumor tissues by the enhanced permeation and retention (EPR) effect, and they successfully generated azide groups on tumor cells in vivo after an intravenous injection. Finally, we showed that these azide groups on tumor tissues could be used as 'artificial chemical receptors' that were conjugated to bioorthogonal chemical group-containing liposomes via in vivo click chemistry in heterogeneous tumor-bearing mice. Therefore, overall results demonstrated that our nano-sized metabolic precursors could be extensively applied to new alternative tumor-targeting technique for molecular imaging and drug delivery system, regardless of the phenotype of heterogeneous tumor cells.

Predicting the in vivo accumulation of nanoparticles in tumor based on in vitro macrophage uptake and circulation in zebrafish.

Nanoparticles have resulted in great progress in biomedical imaging and targeted drug delivery in cancer theranostics. To develop nanoparticles as an effective carrier system for therapeutics, chemical structures and physicochemical properties of nanoparticle may provide a reliable means to predict the in vitro characteristics of nanoparticles. However, in vivo fates of nanoparticles, such as pharmacokinetics and tumor targeting efficiency of nanoparticles, have been difficult to predict beforehand. To predict the in vivo fates of nanoparticles in tumor-bearing mice, differences in physicochemical properties and in vitro cancer cell/macrophage uptake of 5 different nanoparticles with mean diameter of 200-250nm were comparatively analyzed, along with their circulation in adult zebrafish. The nanoparticles which showed favorable cellular uptake by macrophages indicated high unintended liver accumulation in vivo, which is attributed to the clearance by the reticuloendothelial system (RES). In addition, blood circulation of nanoparticles was closely correlated in adult zebrafish and in mice that the zebrafish experiment may elucidate the in vivo behavior of nanoparticles in advance of the in vivo experiment using mammal animal models. This comparative study on various nanoparticles was conducted to provide the basic information on predicting the in vivo fates of nanoparticles prior to the in vivo experiments.

DNA amplification in neutral liposomes for safe and efficient gene delivery.

In general, traditional gene carriers contain strong cationic charges to efficiently load anionic genes, but this cationic character also leads to destabilization of plasma membranes and causes severe cytotoxicity. Here, we developed a PCR-based nanofactory as a safe gene delivery system. A few template plasmid DNA can be amplified by PCR inside liposomes about 200 nm in diameter, and the quantity of loaded genes highly increased by more than 8.8-fold. The liposome membrane was composed of neutral lipids free from cationic charges. Consequently, this system is nontoxic, unlike other traditional cationic gene carriers. Intense red fluorescent protein (RFP) expression in CHO-K1 cells showed that the amplified genes could be successfully transfected to cells. Animal experiments with the luciferase gene also showed in vivo gene expression by our system without toxicity. We think that this PCR-based nanofactory system can overcome the toxicity problem that is the critical limitation of current gene delivery to clinical application.

Blood-pool multifunctional nanoparticles formed by temperature-induced phase transition for cancer-targeting therapy and molecular imaging.

Multifunctional nanoparticles (NPs) were prepared based on temperature-induced phase transition in a molten mixture of Lipiodol(®), Tween 80, paclitaxel (PTX), and Pluronic F-68, wherein the Lipiodol(®)/Tween 80 mixture is used as a solubilizer for PTX, and Pluronic F-68 is used for the stabilization of the molten mixture. The morphology and size distribution of optimized multifunctional NPs were observed using transmittance electron microscopy (TEM) and a particle size analyzer. In the optical imaging of tumor-bearing mice using a near-infrared fluorescence (NIRF) imaging system, the multifunctional NPs were evaluated in terms of a time-dependent excretion profile, in vivo biodistribution and tumor-targeting capability compared to free fluorescence dye. In addition, the prolonged circulation of multifunctional NPs was confirmed by enhancement of the blood-pool in live animals using a micro-CT imaging system, because iodine-containing Lipiodol(®) has an X-ray enhancement property. Finally, the anti-tumor efficacy of multifunctional NPs was monitored by injecting the multifunctional NPs into the tail veins of tumor-bearing mice. The multifunctional NPs showed excellent tumor targetability and anti-tumor efficacy in tumor-bearing mice, caused by the enhanced permeation and retention (EPR) effect.

In vivo NIRF imaging of tumor targetability of nanosized liposomes in tumor-bearing mice.

To optimize tumor targetability of nanosized liposomes for application as drug carriers, various liposomes are prepared by incorporating different amounts (10, 30, and 50 wt%) of cationic, anionic, and PEGylated lipids into neutral lipid. In vivo near-infrared fluorescence images reveal that PEG-PE/PC liposomes display high tumor accumulation in tumor-bearing mice, while large amounts of DOTAP/PC liposomes are rapidly captured in the liver, resulting in poor tumor accumulation. These results demonstrate that optimization of the surface properties of liposomes is very important for their tumor targetability, and that in vivo imaging techniques are useful in developing and optimizing nanosized liposome-based drug carriers.

Theranostic nanoparticles based on PEGylated hyaluronic acid for the diagnosis, therapy and monitoring of colon cancer.

Colon cancer is the second leading cause of cancer-related death in the United States. The considerable mortality from colon cancer is due to metastasis to other organs, mainly the liver. In the management of colon cancer, early detection and targeted therapy are crucial. In this study, we aimed to establish a versatile theranostic system for early tumor detection and targeted tumor therapy by using poly(ethylene glycol)-conjugated hyaluronic acid nanoparticles (P-HA-NPs) which can selectively accumulate in tumor tissue. For the diagnostic application, a near-infrared fluorescence (NIRF) imaging dye (Cy 5.5) was chemically conjugated onto the HA backbone of P-HA-NPs. After intravenous injection of Cy5.5-P-HA-NPs into the tumor-bearing mice, small-sized colon tumors as well as liver-implanted colon tumors were effectively visualized using the NIRF imaging technique. For targeted therapy, we physically encapsulated the anticancer drug, irinotecan (IRT), into the hydrophobic cores of P-HA-NPs. Owing to their notable tumor targeting capability, IRT-P-HA-NPs exhibited an excellent antitumor activity while showing a reduction in undesirable systemic toxicity. Importantly, we demonstrated the theranostic application using Cy5.5-P-HA-NPs and IRT-P-HA-NPs in orthotopic colon cancer models. Following the systemic administration of Cy5.5-P-HA-NPs, neoplasia was clearly visualized, and the tumor growth was effectively suppressed by intravenous injection of IRT-P-HA-NPs. It should be emphasized that the therapeutic responses could be simultaneously monitored by Cy5.5-P-HA-NPs. Our results suggest that P-HA-NPs can be used as a versatile theranostic system for the early detection, targeted therapy, and therapeutic monitoring of colon cancer.

A monitoring method for Atg4 activation in living cells using peptide-conjugated polymeric nanoparticles.

To date, several principal methods are presently used to monitor the autophagic process, but they have some potential experimental pitfalls or limitations that make them not applicable to living cells. In order to improve on the currently developed detection methods for autophagy, we report here fluorescent peptide-conjugated polymeric nanoparticles loaded with a lysosome staining dye in their core. The fluorescent peptide is designed to be specifically cleaved by the Atg4 cysteine protease, which plays a crucial role in autophagy activation. In this study, we demonstrate that peptide-conjugated polymeric nanoparticles can be used to visualize Atg4 activity in both cell-free and cell culture systems. The fluorescence imaging of cells incubated with nanoparticles demonstrates that Atg4 activity is activated in the autophagy-induced conditions, but suppressed in the autophagy-inhibited conditions. These results indicate that Atg4 activity is correlated with autophagic flux through its own regulatory pathway. Therefore, our strategy provides an alternative detection method that can clearly distinguish between an "autophagy active" and "autophagy inactive" state in cultured cells. As our nanoparticles are highly cell-permeable and biocompatible, this detection system has general applicability to living cells and can be extended to cell-based screening to evaluate newly developed compounds.