Iodinated Photosensitizing Chitosan: Self-Assembly into Tumor-Homing Nanoparticles with Enhanced Singlet Oxygen Generation

A novel iodinated chitosan-backboned conjugate (GC–I–Ce6) was designed and prepared to fabricate self-assembled biopolymeric nanoparticles with heavy atom-effected enhanced singlet oxygen generation as well as biological merits. The heavy atom-rich nature of the hydrophobic particle interior was characterized with X-ray absorption and the modified photophysical properties of a chemically embedded photosensitizer, chlorin e6 (Ce6). From the comparative spectroscopic studies as well as cellular and animal experiments, it has been shown that the self-assembled GC–I–Ce6 nanoparticles have enhanced capability of singlet oxygen generation by the intraparticle heavy-atom effect, along with high tumor targetability in vitro and in vivo thanks to the glycol chitosan-surfaced exterior with biocompatible, positively charged and tumor-homing characteristics. Actual efficacy improvement in the photodynamic therapy of a human breast cancer cell line (MDA-MB-231) demonstrates potential of our photophysically and pharmaceutically motivated hybrid bioconjugate approach for nanomedicine applications

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 <i>in vivo</i> 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

Tumor-Targeting Transferrin Nanoparticles for Systemic Polymerized siRNA Delivery in Tumor-Bearing Mice

Transferrin (TF) is widely used as a tumor-targeting ligand for the delivery of anticancer drugs because the TF receptor is overexpressed on the surface of various fast-growing cancer cells. In this article, we report on TF nanoparticles as an siRNA delivery carrier for in vivo tumor-specific gene silencing. To produce siRNA carrying TF nanoparticles (NPs), both TF and siRNA were chemically modified with sulfhydryl groups that can build up self-cross-linked siRNA-TF NPs. Self-polymerized 5′-end thiol-modified siRNA (poly siRNA, psi) and thiolated transferrin (tTF) were spontaneously cross-linked to form stable NPs (psi-tTF NPs) under optimized conditions, and they could be reversibly degraded to release functional monomeric siRNA molecules under reductive conditions. Receptor-mediated endocytosis of TF induced rapid tumor-cell-specific uptake of the psi-tTF NPs, and the internalized NPs resulted in a downregulation of the target protein in red-fluorescent-protein-expressing melanoma cancer cells (RFP/B16F10) with negligible cytotoxicity. After systemic administration, the psi-tTF NPs showed marked accumulation at the tumor, leading to successful target-gene silencing in vivo. This psi-tTF NP system provided a safe and effective strategy for in vivo systemic siRNA delivery for cancer therapy

pH-Controlled Gas-Generating Mineralized Nanoparticles: A Theranostic Agent for Ultrasound Imaging and Therapy of Cancers

We report a theranostic nanoparticle that can express ultrasound (US) imaging and simultaneous therapeutic functions for cancer treatment. We developed doxorubicin-loaded calcium carbonate (CaCO₃) hybrid nanoparticles (DOX-CaCO₃-MNPs) through a block copolymer templated <i>in situ</i> mineralization approach. The nanoparticles exhibited strong echogenic signals at tumoral acid pH by producing carbon dioxide (CO₂) bubbles and showed excellent echo persistence. <i>In vivo</i> results demonstrated that the DOX-CaCO₃-MNPs generated CO₂ bubbles at tumor tissues sufficient for echogenic reflectivity under a US field. In contrast, the DOX-CaCO₃-MNPs located in the liver or tumor-free subcutaneous area did not generate the CO₂ bubbles necessary for US contrast. The DOX-CaCO₃-MNPs could also trigger the DOX release simultaneously with CO₂ bubble generation at the acidic tumoral environment. The DOX-CaCO₃-MNPs displayed effective antitumor therapeutic activity in tumor-bearing mice. The concept described in this work may serve as a useful guide for development of various theranostic nanoparticles for US imaging and therapy of various cancers

Chemical Tumor-Targeting of Nanoparticles Based on Metabolic Glycoengineering and Click Chemistry

Tumor-targeting strategies for nanoparticles have been predominantly based on optimization of physical properties or conjugation with biological ligands. However, their tumor-targeting abilities remain limited and insufficient. Furthermore, traditional biological binding molecules have intrinsic limitations originating from the limited amount of cellular receptors and the heterogeneity of tumor cells. Our two-step <i>in vivo</i> tumor-targeting strategy for nanoparticles is based on metabolic glycoengineering and click chemistry. First, an intravenous injection of precursor-loaded glycol chitosan nanoparticles generates azide groups on tumor tissue specifically by the enhanced permeation and retention (EPR) effect followed by metabolic glycoengineering. These ‘receptor-like’ chemical groups then enhance the tumor-targeting ability of drug-containing nanoparticles by copper-free click chemistry <i>in vivo</i> during a second intravenous injection. The advantage of this protocol over traditional binding molecules is that there are significantly more binding molecules on the surface of most tumor cells regardless of cell type. The subsequent enhanced tumor-targeting ability can significantly enhance the cancer therapeutic efficacy in animal studies

Facile Method To Radiolabel Glycol Chitosan Nanoparticles with ⁶⁴Cu via Copper-Free Click Chemistry for MicroPET Imaging

An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the <i>in vivo</i> biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with ⁶⁴Cu via a strain-promoted azide–alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N₃) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with ⁶⁴Cu radionuclide. Following incubation with the ⁶⁴Cu-radiolabeled DBCO complex (DBCO-PEG₄-Lys-DOTA-⁶⁴Cu with high specific activity, 18.5 GBq/μmol), the azide-functionalized CNPs were radiolabeled successfully with ⁶⁴Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the ⁶⁴Cu-radiolabeled CNPs (⁶⁴Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After ⁶⁴Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, <i>in vivo</i> biodistribution and tumor-targeting ability of ⁶⁴Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time <i>in vivo</i> biodistribution of nanoparticles