Masao Kamimura

Design of Poly(ethylene glycol)/Streptavidin Coimmobilized Upconversion Nanophosphors and Their Application to Fluorescence Biolabeling

Infrared-to-visible upconversion phosphors (i.e., rare earth ion-doped Y2O3 nanoparticles (UNPs)) were synthesized by the homogeneous precipitation method. Because the charge on the erbium (Er) ion-doped Y2O3 (Y2O3:Er) NP (UNP1) surface is positive under neutral conditions, the UNP1 surface was electrostatically PEGylated using negatively charged poly(ethylene glycol)- b-poly(acrylic acid) (PEG- b-PAAc). The adsorption of PEG- b-PAAc was confirmed by Fourier transform infrared (FT-IR) measurements and thermal gravimetric analysis (TGA). The surface charge of the PEGylated UNP1s (PEG-UNP1s) was effectively shielded by the PEGylation. The dispersion stability of the UNP1s was also significantly improved by the PEGylation. The PEG-UNP1s were dispersed over 1 week under physiological conditions as a result of the steric repulsion between the PEG chains on the UNP1 surface. The upconversion emission spectrum of PEG-UNP1s was observed under physiological conditions and was confirmed by near-infrared excited fluorescence microscope observation. Streptavidin (SA)-installed ytterbium (Yb) and Er ion-codoped Y2O3 (Y2O3:Yb,Er) NPs (UNP2s) were prepared by the coimmobilization of PEG- b-PAAc and streptavidin. The PEG/SA coimmobilized UNP2s (PEG/SA-UNP2s) specifically recognized biotinylated antibodies and emitted strong upconversion luminescence upon near-infrared excitation. The obtained PEG/streptavidin coimmobilized UNPs are promising as high-performance near-infrared biolabeling materials.

Near-infrared (1550 nm) in vivo bioimaging based on rare-earth doped ceramic nanophosphors modified with PEG-b-poly(4-vinylbenzylphosphonate)

A novel poly(ethylene glycol) (PEG)-based block copolymer possessing a 4-vinylbenzylphosphonate repeating unit in another segment (PEG-block-poly(4-vinylbenzylphosphonate)) (PEG-b-PVBP) was designed and successfully synthesized. As a control, an end-functionalized PEG possessing a mono-phosphonate group (PEG-PO(3)H(2)) was also synthesized. The surface of near-infrared (NIR) phosphors (i.e., ytterbium (Yb) and erbium (Er) ion-codoped Y(2)O(3) nanoparticles (YNPs)) were modified with PEG-b-PVBP (PEG-YNP(b)s) and PEG-PO(3)H(2) (PEG-YNP(1)s). The adsorption of PEG-b-PVBP and PEG-PO(3)H(2) was estimated by Fourier transform infrared (FT-IR) measurements and thermal gravimetric analysis (TGA). The physicochemical characteristics of the obtained YNP samples were analyzed by ζ-potential and dynamic light scattering (DLS) measurements. The ζ-potentials of YNPs modified by these polymers were close to zero, indicating the effective coverage of the YNP surface by our new PEG derivatives. However, the dispersion stability of the PEGylated YNPs was strongly affected by the structure of the PEG terminus. The average diameter of the PEG-YNP(1)s increased, and aggregates precipitated after less than 1 h in phosphate buffer saline (PBS). In contrast, the size did not change at all in the case of PEG-YNP(b)s and the dispersion in PBS was stable for over 1 week. PEG-YNP(b)s also showed high erosion resistance under acidic conditions. The multiple coordinated PVBP segment of the block copolymer on the YNP surface plays a substantial role in improving such dispersion stability. The excellent dispersion stability and strong NIR luminescence of the obtained PEG-YNP(b)s were also confirmed in fetal bovine serum (FBS) solution over 1 week. Furthermore, in vivo NIR imaging of live mice was performed, and the 1550 nm NIR emission of PEG-YNP(b)s from the organ of live mice was confirmed without dissection.

Block ionomer complexes of PEG-block-poly(4-vinylbenzylphosphonate) and cationic surfactants as highly stable, pH responsive drug delivery system

A new family of block ionomer complexes (BIC) formed by poly(ethylene glycol)-block-poly(4-vinylbenzylphosphonate) (PEG-b-PVBP) and various cationic surfactants was prepared and characterized. These complexes spontaneously self-assembled in aqueous solutions into particles with average size of 40-60nm and remained soluble over the entire range of the compositions of the mixtures including stoichiometric electroneutral complexes. Solution behavior and physicochemical properties of such BIC were very sensitive to the structure of cationic surfactants. Furthermore, such complexation was used for incorporation of cationic anti-cancer drug, doxorubicin (DOX), into the core of BIC with high loading capacity and efficiency. The DOX/PEG-b-PVBP BIC also displayed high stability against dilution, changes in ionic strength. Furthermore, DOX release at the extracellular pH of DOX/PEG-b-PVBP BIC was slow. It was greatly increased at the acidic pH mimicking the endosomal/lysosomal environment. Confocal fluorescence microscopy using live MCF-7 breast cancer cells suggested that DOX/PEG-b-PVBP BICs are transported to lysosomes. Subsequently, the drugs are released and exert cytotoxic effect killing these cancer cells. These findings indicate that the obtained complexes can be attractive candidates for delivery of cationic drugs to tumors.

Enhanced intracellular drug delivery of pH-sensitive doxorubicin /poly(ethylene glycol)- block -poly(4-vinylbenzylphosphonate) nanoparticles in multi- drug resistant human epidermoid KB carcinoma cells

A pH-sensitive nanoparticle was prepared using our original amphiphilic block copolymer, poly(ethylene glycol)-b-poly(4-vinylbenzylphosphonate) (PEG-b-PVBP), which possesses phosphate groups as a side chain of its hydrophobic segment (termed here, phosphate nanoparticle (PNP)). The cationic anticancer drug, doxorubicin (DOX) was incorporated into a PNP (DOX@PNP), and its loading capacity was 320 mg g-1-PNP. Electrostatic and hydrophobic interactions in the core of the PNP might act synergistically to significantly improve its loading capacity. The cytotoxicity of the DOX@PNP was examined using the drug-sensitive human epidermoid KB carcinoma cell line (KB-3-1) and two different multi-drug resistance (MDR) KB cell lines (P-glycoprotein (P-gp) overexpressed (KB-C-2) and multidrug resistance protein 1 (MRP1) overexpressed (KB/MRP) cell lines). The DOX@PNP displayed a lower cytotoxic activity than free DOX against KB-3-1 cells. In contrast, the DOX@PNP showed a higher cytotoxic activity than free DOX against MDR cells. Of particular note, the cytotoxicity of the DOX@PNP against KB-C-2 cells was much higher than that against KB/MRP cells, suggesting that different mechanisms of drug reflux via the ATP binding cassette (ABC) transporting system play an important role in nanoparticle-assisted chemotherapy. Observation with confocal laser scanning microscopy (CLSM) indicated that the DOX@PNP was taken up by cells via the endocytosis pathway. The DOX@PNP was initially localized in the late endosome and lysosome, with the subsequent release of DOX from the DOX@PNP in response to the acidic pH of the late endosome and lysosome. Quantitative analysis using flow cytometry confirmed that the uptake of the DOX@PNP into KB-C-2 cells was much higher than that into KB/MRP cells, which was one of the reasons for the enhanced toxicity of the DOX@PNP against KB-C-2 cells compared to that against KB/MRP cells. Reflux of the liberated free DOX in the cytosol, via an endosomal membrane transporter, is considered one of the reasons for the low efficiency of DOX@PNP chemotherapy against KB/MRP cells. However, compared to the free DOX dose, a high dose of the DOX@PNP was effectively delivered to the nuclei of the KB/MRP cells. On the basis of these results, the pH-sensitive DOX@PNP is anticipated as one of the effective chemotherapeutic drugs with enhanced cytotoxicity for multiple types of MDR cancer cells.

Ratiometric near-infrared fluorescence nanothermometry in the OTN-NIR (NIR II/III) biological window based on rare-earth doped β-NaYF4 nanoparticles

A novel nanothermometer based on over-1000 nm (OTN) near-infrared (NIR) emission of rare-earth doped ceramic nanophosphors (RED-CNPs) was developed for temperature measurement in deep tissue. Hexagonal-phase β-NaYF4 nanoparticles co-doped with Yb3+, Ho3+, and Er3+ (NaYF4:Yb3+,Ho3+,Er3+ NPs) were synthesized and used as a nanothermometer. The NaYF4:Yb3+,Ho3+,Er3+ NPs displayed two OTN-NIR emission peaks in the second (NIR-II) (at 1150 nm of Ho3+) and third (NIR-III) (at 1550 nm of Er3+) biological window regions under NIR (980 nm) excitation in the first (NIR-I) biological window region. Oleic acid (OA) capped NaYF4:Yb3+,Ho3+,Er3+ NPs were dispersed in non-polar media, i.e., cyclohexane, and showed a temperature-dependent intensity ratio of the emission peaks of Ho3+ and Er3+ (IHo/IEr). The temperature-dependent IHo/IEr of the OA-NaYF4:Yb3+,Ho3+,Er3+ NPs was also evident through imitation tissue. The surfaces of the NaYF4:Yb3+,Ho3+,Er3+ NPs were modified with a poly(ethylene glycol) (PEG)-based block copolymer. The PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs were dispersed in water and emitted strong NIR-II and III emissions under NIR-I excitation. The PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs were injected into mice via the tail vein, and the OTN-NIR emissions of the PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs from the mouse blood vessels were clearly observed using an OTN-NIR fluorescence in vivo imaging system. In a polar media, water, the IHo/IEr of PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs was inversely related to the temperature. In both non-polar and polar media, the IHo/IEr values of the NaYF4:Yb3+,Ho3+,Er3+ NPs were almost linearly dependent on the temperature. The obtained NaYF4:Yb3+,Ho3+,Er3+ NPs are promising as a novel fluorescent nanothermometer for deep tissue.

Targeted Delivery of Functionalized Upconversion Nanoparticles for Externally Triggered Photothermal/Photodynamic Therapies of Brain Glioblastoma

Therapeutic efficacy of glioblastoma multiforme (GBM) is often severely limited by poor penetration of therapeutics through blood-brain barrier (BBB) into brain tissues and lack of tumor targeting. In this regard, a functionalized upconversion nanoparticle (UCNP)-based delivery system which can target brain tumor and convert deep tissue-penetrating near-infrared (NIR) light into visible light for precise phototherapies on brain tumor was developed in this work. Methods: The UCNP-based phototherapy delivery system was acquired by assembly of oleic acid-coated UCNPs with angiopep-2/cholesterol-conjugated poly(ethylene glycol) and the hydrophobic photosensitizers. The hybrid nanoparticles (ANG-IMNPs) were characterized by DLS, TEM, UV/vis and fluorescence spectrophotometer. Cellular uptake was examined by laser scanning confocal microscopy and flow cytometry. The PDT/PTT effect of ANG-IMNPs was evaluated using MTT assay. Tumor accumulation of NPs was determined by a non-invasive in vivo imaging system (IVIS). The in vivo anti-glioma effect of ANG-IMNPs was evaluated by immunohistochemical (IHC) examination of tumor tissues and Kaplan-Meier survival analysis. Results: In vitro data demonstrated enhanced uptake of ANG-IMNPs by murine astrocytoma cells (ALTS1C1) and pronounced cytotoxicity by combined NIR-triggered PDT and PTT. In consistence with the increased penetration of ANG-IMNPs through endothelial monolayer in vitro, the NPs have also shown significantly enhanced accumulation at brain tumor by IVIS. The IHC tissue examination confirmed prominent apoptotic and necrotic effects on tumor cells in mice receiving targeted dual photo-based therapies, which also led to enhanced median survival (24 days) as compared to the NP treatment without angiopep-2 (14 days). Conclusion: In vitro and in vivo data strongly indicate that the ANG-IMNPs were capable of selectively delivering dual photosensitizers to brain astrocytoma tumors for effective PDT/PTT in conjugation with a substantially improved median survival. The therapeutic efficacy of ANG-IMNPs demonstrated in this study suggests their potential in overcoming BBB and establishing an effective treatment against GBM.

Six-axis orthodontic force and moment sensing system for dentist technique training

The purpose of this study is to develop a sensing system device that measures three-axis orthodontic forces and three-axis orthodontic moments for dentist training. The developed sensing system is composed of six-axis force sensors, action sticks, sliders, and tooth models. The developed system also simulates various types of tooth row shape patterns in orthodontic operations, and measures a 14 × 6 axis orthodontic force and moment from tooth models simultaneously. The average force and moment error per loaded axis were 2.06 % and 2.00 %, respectively.

Rapid clearing of biological organs by using phosphoric acid, a hydrophilic solution with high refractive index

Tissue clearing is a fundamental challenge in biology and medicine to achieve high-resolution optical imaging of tissues deep inside intact organs. The clearing methods, reported up to now, require long incubation time or physical/electrical pressure to achieve tissue clearing, which is done by matching the refractive indices of the whole sample and medium to that of the lipid layer. Here we show that phosphoric acid increases the refractive index of the medium and can increase the transparency of formalin-fixed tissue samples rapidly. Immersion of fixed tissues of mice in phosphoric acid solutions increased their transparency within 60 min in the case of 3-mm-thick fixed tissue specimens. While phosphoric acid suppresses bright signals on the boundary of cells in their phase-contrast images, it does not damage the morphology of cell membrane with phospholipid bilayer. The protocol presented herein may contribute to develop better and faster soaking methods for tissue clearing than previously reported protocols. Highlights ▪ Phosphoric acid can reduce light scattering by tissue samples. ▪ Tissue clearing effect of phosphoric acid is fast and needs only 60-min incubation. ▪ Cell membrane was preserved during incubation using phosphoric acid.

Application of Ceramic Nanoparticles for Near Infrared Bioimaging

Bioimaging is an inevitable technique for biological studies and medical diagnosis. As for the fluorescence bioimaging, only wavelength up to 1000 nm has been used. However, by extend it to be over-1000-nm near infrared, the fluorescence bioimaging with ten times deeper, several centimeters, observation depth can be achieved in comparison with that with a currently used wavelength. The authors have developed both materials and system for the over-1000 nm near infrared bioimaging. The paper will review the development by using rare-earth doped ceramic nanoparticles.

PEGylated polymer micelles for anticancer drug delivery carrier

Abstract In this chapter, we provide an overview of poly(ethylene glycol) (PEG)-tethered (PEGylated) polymer micelles and their application for anticancer drug delivery carrier. An amphiphilic block copolymer forms a core–shell type of polymeric micelle in aqueous media that can be utilised as a carrier for the delivery of hydrophobic drugs. The incorporation of anticancer drugs into the core of PEGylated polymer micelles increases the solubility, stability and circulation of the drugs. Recently, new types of polymeric micelles have been proposed, including micelles characterised by pH-triggered disintegration and formation of a polyion complex of hydrophilic block polymers. For example, pH-sensitive PEGylated polymer micelles can control drug release and have been shown to be an effective chemotherapy with enhanced therapeutic efficacy against cancer cells. Therefore, PEGylated polymer micelles should perform well as a carrier for anticancer drug delivery.