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

Dye-Condensed Biopolymeric Hybrids: Chromophoric Aggregation and Self-Assembly toward Fluorescent Bionanoparticles for Near Infrared Bioimaging

Novel fluorescent bionanoparticles (GC3CNx) were prepared by self-assembly of biopolymeric amphiphiles that are composed of hydrophilic glycol chitosan (GC) as a biopolymeric backbone and a dipolar tricyanostilbene derivative (3CN) as a densely conjugated hydrophobic pendant. In the molecular design of 3CN, a dipolar electronic structure was introduced to spectrally shift the aggregation-induced enhanced fluorescence of the α-cyanostilbene skeleton toward the near-infrared (NIR) useful for bioimaging. It has been found that the fluorescence of GC3CNx nanoparticles is red-shifted and intensified by increasing the 3CN content in the biopolymeric amphiphile. By cellular and in vivo imaging experiments, we demonstrate that GC3CNx nanoparticles have great potential for NIR bioimaging, with attractive properties including enhanced fluorescence signal, efficient cellular uptake, and better spectral coincidence with the in vivo transparent window

Physicochemical Characterizations of Self-Assembled Nanoparticles of Glycol Chitosan−Deoxycholic Acid Conjugates

Physicochemical Characterizations of Self-Assembled Nanoparticles of Glycol Chitosan−Deoxycholic Acid Conjugate

Bioreducible Block Copolymers Based on Poly(Ethylene Glycol) and Poly(γ-Benzyl l-Glutamate) for Intracellular Delivery of Camptothecin

Poly(ethylene glycol)-b-poly(γ-benzyl l-glutamate)s bearing the disulfide bond (PEG-SS-PBLGs), which is specifically cleavable in intracellular compartments, were prepared via a facile synthetic route as a potential carrier of camptothecin (CPT). Diblock copolymers with different lengths of PBLG were synthesized by ring-opening polymerization of benzyl glutamate N-carboxy anhydride in the presence of a PEG macroinitiator (PEG-SS-NH2). Owing to their amphiphilic nature, the copolymers formed spherical micelles in an aqueous condition, and their particle sizes (20–125 nm in diameter) were dependent on the block length of PBLG. Critical micelle concentrations of the copolymers were in the range 0.005–0.065 mg/mL, which decreased as the block length of PBLG increased. CPT, chosen as a model anticancer drug, was effectively encapsulated up to 12 wt % into the hydrophobic core of the micelles by the solvent casting method. It was demonstrated by the in vitro optical imaging technique that the fluorescence signal of doxorubicin, quenched in the PEG-SS-PBLG micelles, was highly recovered in the presence of glutathione (GSH), a tripeptide reducing disulfide bonds in the cytoplasm. The micelles released CPT completely within 20 h under 10 mM GSH, whereas only 40% of CPT was released from the micelles in the absence of GSH. From the in vitro cytotoxicity test, it was found that CPT-loaded PEG-SS-PBLG micelles showed higher toxicity to SCC7 cancer cells than CPT-loaded PEG-b-PBLG micelles without the disulfide bond. Microscopic observation demonstrated that the disulfide-containing micelle could effectively deliver the drug into nuclei of SCC7 cells. These results suggest that PEG-SS-PBLG diblock copolymer is a promising carrier for intracellular delivery of CPT

Caspase Sensitive Gold Nanoparticle for Apoptosis Imaging in Live Cells

We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications

Biolighted Nanotorch Capable of Systemic Self-Delivery and Diagnostic Imaging

Sensitive imaging of inflammation with a background-free chemiluminescence (CL) signal has great potential as a clinically relevant way of early diagnosis for various inflammatory diseases. However, to date, its feasibility has been limitedly demonstrated <i>in vivo</i> with locally induced inflammation models by <i>in situ</i> injection of CL probes. To enable systemic disease targeting and imaging by intravenous administration of CL probes, hurdles need to be overcome such as weak CL emission, short glowing duration, or inability of long blood circulation. Here, we report a CL nanoprobe (BioNT) that surmounted such limitations to perform precise identification of inflammation by systemic self-delivery to the pathological tissues. This BioNT probe was engineered by physical nanointegration of multiple kinds of functional molecules into the ultrafine nanoreactor structure (∼15 nm in size) that combines solid-state fluorescence-induced enhanced peroxalate CL and built-in machinery to control the intraparticle kinetics of CL reaction. Upon intravenous injection into a normal mouse, BioNT showed facile blood circulation and generated a self-lighted strong CL torchlight throughout the whole body owing to the tiny colloidal structure with an antifouling surface as well as high CL sensitivity toward endogenous biological hydrogen peroxide (H₂O₂). In mouse models of local and systemic inflammations, blood-injected BioNT visualized precise locations of inflamed tissues with dual selectivity (selective probe accumulation and selective CL reaction with H₂O₂ overproduced by inflammation). Even a tumor model that demands a long blood circulation time for targeting (>3 h) could be accurately identified by persistent signaling from the kinetics-tailored BioNT with a 65-fold slowed CL decay rate. We also show that BioNT exhibits no apparent toxicity, thus holding potential for high-contrast diagnostic imaging

Dye/Peroxalate Aggregated Nanoparticles with Enhanced and Tunable Chemiluminescence for Biomedical Imaging of Hydrogen Peroxide

Hydrogen peroxide (H₂O₂) is an endogenous molecule that plays diverse physiological and pathological roles in living systems. Here we report multimolecule integrated nanoprobes with the enhanced chemiluminescence (CL) response to H₂O₂ that is produced in cells and <i>in vivo</i>. This approach is based on the nanoscopic coaggregation of a dye exhibiting aggregation-enhanced fluorescence (AEF) with a H₂O₂-responsive peroxalate that can convert chemical reaction energy into electronic excitation. The coaggregated CL nanoparticles (FPOA NPs) with an average size of ∼20 nm were formulated by aqueous self-assembly of a ternary mixture of a surfactant (Pluronic F-127) and concentrated hydrophobic dye/peroxalte payloads. Spectroscopic studies manifest that FPOA NPs as a reagent-concentrated nanoreactor possess the signal enhancement effect by AEF, as well as the optimized efficiencies for H₂O₂ peroxalate reaction and subsequent intraparticle energy transfer to the dye aggregates, to yield greatly enhanced CL generation with a prolonged lifetime. It is shown that the enhanced CL signal thereby is capable of detecting intracellular H₂O₂ overproduced during immune response. We also demonstrate that the densely integrated nature of FPOA NPs facilitates further intraparticle CL energy transfer to a low-energy dopant to red shift the spectrum toward the biologically more transparent optical window, which enables the high-sensitivity <i>in vivo</i> visualization of H₂O₂ associated with early stage inflammation

Heparin/Poly(l-lysine) Nanoparticle-Coated Polymeric Microspheres for Stem-Cell Therapy

Three-dimensional microspheres have been used extensively in several biomaterials fields, in applications such as tissue-regeneration scaffolds and drug delivery systems. To apply these biomaterials as novel cell therapeutic agents, we have devised a novel method for the fabrication of nanostructured 3D scaffolds consisting of heparin/poly(l-lysine) nanoparticles on the surface of polymeric microspheres, attached via a layer-by-layer (LbL) system. The initial step of this strategy involves the creation of heparin/poly(l-lysine) nanoparticles, which were simply produced as polyion complex micelles (PICM) with diameters of 200−500 nm. In the second step, the heparin/poly(l-lysine) nanoparticles were coated onto positively charged poly(lactic-co-glycolic acid) (PLGA) pretreated with polyethyleneimine (PEI). The production of the heparin/poly(l-lysine) nanoparticles and their subsequent coating onto PLGA microspheres represents a novel method for the functionalization of the polymeric matrix, which requires the cellular active surfaces of nanoscaled heparinized surfaces as 3D scaffolds, thus creating a better microenvironment of cell adhesion and growth for use in cell therapy applications

Dark Quenched Matrix Metalloproteinase Fluorogenic Probe for Imaging Osteoarthritis Development <i>in Vivo</i>

The early detection of osteoarthritis (OA) is currently a key challenge in the field of rheumatology. Biochemical studies of OA have indicated that matrix metalloproteinase-13 (MMP-13) plays a central role in cartilage degradation. In this study, we describe the potential use of a dark-quenched fluorogenic MMP-13 probe to image MMP-13 in both in vitro and rat models. The imaging technique involved using a MMP-13 peptide substrate, near-infrared (NIR) dye, and a NIR dark quencher. The results from this study demonstrate that the use of a dark-quenched fluorogenic probe allows for the visual detection of MMP-13 in vitro and in OA-induced rat models. In particular, by targeting this OA biomarker, the symptoms of the early and late stages of OA can be readily monitored, imaged, and analyzed in a rapid and efficient fashion. We anticipate that this simple and highly efficient fluorogenic probe will assist in the clinical management of patients with OA, not only for early diagnosis but also to assess individual patient responses to new drug treatments

Glycol Chitosan/Heparin Immobilized Iron Oxide Nanoparticles with a Tumor-Targeting Characteristic for Magnetic Resonance Imaging

We described the preparation of the glycol chitosan/heparin immobilized iron oxide nanoparticles (composite NPs) as a magnetic resonance imaging agent with a tumor-targeting characteristic. The iron oxide nanoseeds used clinically as a magnetic resonance imaging agent were immobilized into the glycol chitosan/heparin network to form the composite NPs. To induce the ionic interaction between the iron oxide nanoseeds and glycol chitosan, gold was deposited on the surface of iron oxide nanoseeds. After the immobilization of gold-deposited iron oxide NPs into the glycol chitosan network, the NPs were stabilized with heparin based on the ionic interaction between cationic glycol chitosan and anionic heparin. FE-SEM (field emission-scanning electron microscopy) and a particle size analyzer were used to observe the formation of the stabilized composite NPs, and a Jobin-Yvon Ultima-C inductively coupled plasma-atomic emission spectrometer (ICP-AES) was used to measure the contents (%) of formed iron oxide nanoseeds as a function of reaction temperature and formed gold deposited on the iron oxide nanoparticles. We also evaluated the time-dependent excretion profile, in vivo biodistribution, circulation time, and tumor-targeting ability of the composite NPs using a noninvasive NIR fluorescence imaging technology. To observe the MRI contrast characteristic, the composite NPs were injected into the tail veins of tumor-bearing mice to demonstrate their selective tumoral distribution. The MR images were collected with conventional T2-weighted spin echo acquisition parameters