Tumor-Homing Glycol Chitosan-Based Optical/PET Dual Imaging Nanoprobe for Cancer Diagnosis

Imaging techniques including computed tomography, magnetic resonance imaging, and positron emission tomography (PET) offer many potential benefits to diagnosis and treatment of cancers. Each method has its own strong and weak points. Therefore, multimodal imaging techniques have been highlighted as an alternative method for overcoming the limitations of each respective imaging method. In this study, we fabricated PET/optical activatable imaging probe based on glycol chitosan nanoparticles (CNPs) for multimodal imaging. To prepare the dual PET/optical probes based on CNPs, both ⁶⁴Cu radiolabeled DOTA complex and activatable matrix metalloproteinase (MMP)-sensitive peptide were chemically conjugated onto azide-functionalized CNPs via bio-orthogonal click chemistry, which was a reaction between azide group and dibenzyl cyclooctyne. The PET/optical activatable imaging probes were visualized by PET and optical imaging system. Biodistribution of probes and activity of MMP were successfully measured in tumor-bearing mice

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

Precise Targeting of Liver Tumor Using Glycol Chitosan Nanoparticles: Mechanisms, Key Factors, and Their Implications

Herein, we elucidated the mechanisms and key factors for the tumor-targeting ability of nanoparticles that presented high targeting efficiency for liver tumor. We used several different nanoparticles with sizes of 200–300 nm, including liposome nanoparticles (LNPs), polystyrene nanoparticles (PNPs) and glycol chitosan-5β-cholanic acid nanoparticles (CNPs). Their sizes are suitable for the enhanced permeation and retention (EPR) effect in literature. Different <i>in vitro</i> characteristics, such as the particle structure, stability, and bioinertness, were carefully analyzed with and without serum proteins. Also, pH-dependent tumor cell uptakes of nanoparticles were studied using fluorescence microscopy. Importantly, CNPs had sufficient stability and bioinertness to maintain their nanoparticle structure in the bloodstream, and they also presented prolonged circulation time in the body (blood circulation half-life <i>T</i>_1/2 = about 12.2 h), compared to the control nanoparticles. Finally, employing liver tumor bearing mice, we also observed that CNPs had excellent liver tumor targeting ability <i>in vivo</i>, while LNPs and PNPs demonstrated lower tumor-targeting efficiency due to the nonspecific accumulation in normal liver tissue. Liver tumor models were produced by laparotomy and direct injection of HT29 tumor cells into the left lobe of the liver of athymic nude mice. This study provides valuable information concerning the key factors for the tumor-targeting ability of nanoparticles such as stability, bioinertness, and rapid cellular uptake at targeted tumor tissues

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (-N₃) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac₄ManNAz and DBCO-650 to the cell culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled chondrocytes (1 × 10⁶ cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>

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

Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal Click Reaction for Improving Active-Targeting Efficiency of Tumor

Biological ligands such as aptamer, antibody, glucose, and peptide have been widely used to bind specific surface molecules or receptors in tumor cells or subcellular structures to improve tumor-targeting efficiency of nanoparticles. However, this active-targeting strategy has limitations for tumor targeting due to inter- and intraheterogeneity of tumors. In this study, we demonstrated an alternative active-targeting strategy using metabolic engineering and bioorthogonal click reaction to improve tumor-targeting efficiency of nanoparticles. We observed that azide-containing chemical reporters were successfully generated onto surface glycans of various tumor cells such as lung cancer (A549), brain cancer (U87), and breast cancer (BT-474, MDA-MB231, MCF-7) via metabolic engineering in vitro. In addition, we compared tumor targeting of artificial azide reporter with bicyclononyne (BCN)-conjugated glycol chitosan nanoparticles (BCN–CNPs) and integrin αvβ3 with cyclic RGD-conjugated CNPs (cRGD–CNPs) in vitro and in vivo. Fluorescence intensity of azide-reporter-targeted BCN–CNPs in tumor tissues was 1.6-fold higher and with a more uniform distribution compared to that of cRGD–CNPs. Moreover, even in the isolated heterogeneous U87 cells, BCN–CNPs could bind artificial azide reporters on tumor cells more uniformly (∼92.9%) compared to cRGD–CNPs. Therefore, the artificial azide-reporter-targeting strategy can be utilized for targeting heterogeneous tumor cells via bioorthogonal click reaction and may provide an alternative method of tumor targeting for further investigation in cancer therapy

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (-N₃) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac₄ManNAz and DBCO-650 to the cell culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled chondrocytes (1 × 10⁶ cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac₄ManNAz) to generate unnatural azide groups (-N₃) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac₄ManNAz and DBCO-650 to the cell culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled chondrocytes (1 × 10⁶ cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>

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