Non-canonical interplay between glutamatergic NMDA and dopamine receptors shapes synaptogenesis.

Direct interactions between receptors at the neuronal surface have long been proposed to tune signaling cascades and neuronal communication in health and disease. Yet, the lack of direct investigation methods to measure, in live neurons, the interaction between different membrane receptors at the single molecule level has raised unanswered questions on the biophysical properties and biological roles of such receptor interactome. Using a multidimensional spectral single molecule-localization microscopy (MS-SMLM) approach, we monitored the interaction between two membrane receptors, i.e. glutamatergic NMDA (NMDAR) and G protein-coupled dopamine D1 (D1R) receptors. The transient interaction was randomly observed along the dendritic tree of hippocampal neurons. It was higher early in development, promoting the formation of NMDAR-D1R complexes in an mGluR5- and CK1-dependent manner, favoring NMDAR clusters and synaptogenesis in a dopamine receptor signaling-independent manner. Preventing the interaction in the neonate, and not adult, brain alters in vivo spontaneous neuronal network activity pattern in male mice. Thus, a weak and transient interaction between NMDAR and D1R plays a structural and functional role in the developing brain.

Electrical stimulation facilitates NADPH production in pentose phosphate pathway and exerts an anti-inflammatory effect in macrophages.

Macrophages play an important role as effector cells in innate immune system. Meanwhile, macrophages activated in a pro-inflammatory direction alter intracellular metabolism and damage intact tissues by increasing reactive oxygen species (ROS). Electrical stimulation (ES), a predominant physical agent to control metabolism in cells and tissues, has been reported to exert anti-inflammatory effect on immune cells. However, the mechanism underlying the anti-inflammatory effects by ES is unknown. This study aimed to investigate the effect of ES on metabolism in glycolytic-tricarboxylic acid cycle (TCA) cycle and inflammatory responses in macrophages. ES was performed on bone marrow-derived macrophages and followed by a stimulation with LPS. The inflammatory cytokine expression levels were analyzed by real-time polymerase chain reaction and ELISA. ROS production was analyzed by CellRox Green Reagent and metabolites by capillary electrophoresis-mass spectrometry. As a result, ES significantly reduced proinflammatory cytokine expression levels and ROS generation compared to the LPS group and increased glucose-1-phosphate, a metabolite of glycogen. ES also increased intermediate metabolites of the pentose phosphate pathway (PPP); ribulose-5-phosphate, rebose-5 phosphate, and nicotinamide adenine dinucleotide phosphate, a key factor of cellular antioxidation systems, as well as α-Ketoglutarate, an anti-oxidative metabolite in the TCA cycle. Our findings imply that ES enhanced NADPH production with enhancement of PPP, and also decreased oxidative stress and inflammatory responses in macrophages.

Skeletal myotube-derived extracellular vesicles enhance itaconate production and attenuate inflammatory responses of macrophages.

Introduction: Macrophages play an important role in the innate immunity. While macrophage inflammation is necessary for biological defense, it must be appropriately controlled. Extracellular vesicles (EVs) are small vesicles released from all types of cells and play a central role in intercellular communication. Skeletal muscle has been suggested to release anti-inflammatory factors, but the effect of myotube-derived EVs on macrophages is unknown. As an anti-inflammatory mechanism of macrophages, the immune responsive gene 1 (IRG1)-itaconate pathway is essential. In this study, we show that skeletal muscle-derived EVs suppress macrophage inflammatory responses, upregulating the IRG1-itaconate pathway. Methods: C2C12 myoblasts were differentiated into myotubes and EVs were extracted by ultracentrifugation. Skeletal myotube-derived EVs were administered to mouse bone marrow-derived macrophages, then lipopolysaccharide (LPS) stimulation was performed and inflammatory cytokine expression was measured by RT-qPCR. Metabolite abundance in macrophages after addition of EVs was measured by CE/MS, and IRG1 expression was measured by RT-PCR. Furthermore, RNA-seq analysis was performed on macrophages after EV treatment. Results: EVs attenuated the expression of LPS-induced pro-inflammatory factors in macrophages. Itaconate abundance and IRG1 expression were significantly increased in the EV-treated group. RNA-seq analysis revealed activation of the PI3K-Akt and JAK-STAT pathways in macrophages after EV treatment. The most abundant miRNA in myotube EVs was miR-206-3p, followed by miR-378a-3p, miR-30d-5p, and miR-21a-5p. Discussion: Skeletal myotube EVs are supposed to increase the production of itaconate via upregulation of IRG1 expression and exhibited an anti-inflammatory effect in macrophages. This anti-inflammatory effect was suggested to involve the PI3K-Akt and JAK-STAT pathways. The miRNA profiles within EVs implied that miR-206-3p, miR-378a-3p, miR-30d-5p, and miR-21a-5p may be responsible for the anti-inflammatory effects of the EVs. In summary, in this study we showed that myotube-derived EVs prevent macrophage inflammatory responses by activating the IRG1-itaconate pathway.

Fluorophore-Decorated Mie Resonant Silicon Nanosphere for Scattering/Fluorescence Dual-Mode Imaging.

Inorganic nanoparticles with multiple functions have been attracting attention as multimodal nanoprobes in bioimaging, biomolecule detection, and medical diagnosis and treatment. A drawback of conventional metallic nanoparticle-based nanoprobes is the Ohmic losses that lead to fluorescence quenching of attached molecules and local heating under light irradiation. Here, metal-free nanoprobes capable of scattering/fluorescence dual-mode imaging are developed. The nanoprobes are composed of a silicon nanosphere core having efficient Mie scattering in the visible to near infrared range and a fluorophore doped silica shell. The dark-field scattering and photoluminescence images/spectra for nanoprobes made from different size silicon nanospheres and different kinds of fluorophores are studied by single particle spectroscopy. The fluorescence spectra are strongly modified by the Mie modes of a silicon nanosphere core. By comparing scattering and fluorescence spectra and calculated Purcell factors, the fluorescence enhancement factor is quantitatively discussed. In vitro scattering/fluorescence imaging studies on human cancer cells demonstrate that the developed nanoparticles work as scattering/fluorescence dual-mode imaging nanoprobes.

Synthesis of Chiral α-Substituted ß-Aminophosphine Derivatives through Asymmetric Hydrophosphinylation Utilizing Secondary Phosphine Sulfides.

The organocatalytic enantioselective hydrophosphinylation of various secondary phosphine sulfides with aromatic and aliphatic nitroalkenes is presented in this study. The reaction produced chiral ß-nitrophosphine sulfides with excellent yields and enantioselectivities (up to 99% yield and 99% ee). Furthermore, the chiral ß-nitrophosphine sulfides can be easily converted into α-substituted ß-aminophosphine, which is a family of useful P, N-ligands and phosphine catalysts.

In vivo stealthified molecularly imprinted polymer nanogels incorporated with gold nanoparticles for radiation therapy.

Radiation therapy is a representative therapeutic approach for cancer treatment, wherein the development of efficient radiation sensitizers with low side effects is critical. In this study, a novel stealth radiation sensitizer based on Au-embedded molecularly imprinted polymer nanogels (Au MIP-NGs) was developed for low-dose X-ray radiation therapy. Surface plasmon resonance measurements reveal the good affinity and selectivity of the obtained Au MIP-NGs toward the target dysopsonic protein, human serum albumin. The protein recognition capability of the nanogels led to the formation of the albumin-rich protein corona in the plasma. The Au MIP-NGs acquire stealth capability in vivo through protein corona regulation using the intrinsic dysopsonic proteins. The injection of Au MIP-NGs improved the efficiency of the radiation therapy in mouse models of pancreatic cancer. The growth of the pancreatic tumor was inhibited even at low X-ray doses (2 Gy). The novel strategy reported in this study for the synthesis of stealth nanomaterials based on nanomaterial-protein interaction control shows significant potential for application even in other approaches for cancer treatment, diagnostics, and theranostics. This strategy paves a way for the development of a wide range of effective nanomedicines for cancer therapy.

Reactive oxygen species-inducing titanium peroxide nanoparticles as promising radiosensitizers for eliminating pancreatic cancer stem cells.

BACKGROUND: Despite recent advances in radiotherapy, radioresistance in patients with pancreatic cancer remains a crucial dilemma for clinical treatment. Cancer stem cells (CSCs) represent a major factor in radioresistance. Developing a potent radiosensitizer may be a novel candidate for the eradication of pancreatic CSCs. METHODS: CSCs were isolated from MIA PaCa-2 and PANC1 human pancreatic cancer cell lines. Titanium peroxide nanoparticles (TiOxNPs) were synthesized from titanium dioxide nanoparticles (TiO2NPs) and utilized as radiosensitizers when added one hour prior to radiation exposure. The antitumor activity of this novel therapeutic strategy was evaluated against well-established pancreatic CSCs model both in vitro and in vivo. RESULTS: It is shown that TiOxNPs combined with ionizing radiation exhibit anti-cancer effects on radioresistant CSCs both in vitro and in vivo. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Notably, radiosensitizing TiOxNPs suppressed the growth of pancreatic xenografts following primary or dissociating sphere MIA PaCa-2 cell implantation. It is inferred that synergy is formed by generating intolerable levels of reactive oxygen species (ROS) and inactivating the AKT signaling pathway. CONCLUSIONS: Our data suggested the use of TiOxNPs in combination with radiation may be considered an attractive therapeutic strategy to eliminate pancreatic CSCs.

Pulsed-Ultrasound Irradiation Induces the Production of Itaconate and Attenuates Inflammatory Responses in Macrophages.

Background: Itaconate is a key metabolite in the innate immune system and exerts strong anti-inflammatory effects in macrophages. For the production of itaconate in macrophages, immune-responsive gene 1 (IRG1) is an imperative enzyme, and activating the IRG1-itaconate pathway is reported to alleviate inflammatory diseases by upregulating nuclear factor-erythroid 2-related factor 2 (NRF2). However, there are very few reports on strategies to increase itaconate production. Ultrasound therapy is a widely used intervention for anti-inflammatory and soft-tissue regeneration purposes. Here we show the effect of ultrasound irradiation on the production of itaconate in macrophages. Methods: Murine bone marrow-derived macrophages (BMDMs) were exposed to pulsed ultrasound (3.0 W/cm2) for 5 minutes. Three hours after irradiation, the intracellular levels of metabolites and mRNA expression levels of Irg1 and Nrf2 were measured using CE/MS and qPCR, respectively. To evaluate macrophage inflammation status, 3 h after irradiation, the cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 1.5 h and the mRNA expression levels of pro-inflammatory factors (Il-1ß, Il-6, and Tnf-α) were measured. Student's t-test, one-way ANOVA and Tukey's multiple comparison test were used for statistical processing, and the significance level was set to less than 5%. Results: Ultrasound irradiation significantly increased the intracellular itaconate level and the expression levels of Irg1 and Nrf2 in BMDMs. Upregulation of Il-1ß, Il-6, and Tnf-α by LPS was significantly suppressed in BMDMs treated with ultrasound. Ultrasound irradiation did not affect cell viability and apoptosis. Conclusion: Ultrasound irradiation induces the production of itaconate by upregulating Irg1 expression and attenuates inflammatory responses in macrophages via Nrf2. These results suggest that ultrasound is a potentially useful method to increase itaconate production in macrophages.

Biocompatible polymer-modified gold nanocomposites of different shapes as radiation sensitizers.

Radiation therapy is a powerful approach for cancer treatment due to its low invasiveness. The development of radiation sensitizers is of great importance as they assist in providing radiation therapy at a low dose. In this study, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-modified gold nanocomposites of different shapes were created using the grafting-to approach to serve as a novel radiation sensitizer with high cellular uptake. The effect of the shape of the nanocomposite on cellular uptake by the breast cancer cell line MCF-7 was also investigated. The PMPC-modified gold nanostars showed the highest cellular uptake compared to the other gold nanocomposites (spheres and rods), whereas cell cytotoxicity was negligible among all candidates. Furthermore, the therapeutic effect of radiation of PMPC-modified nanostars was the highest among all the gold nanocomposites. These results clearly indicate that the shape of the gold nanocomposite is an important parameter for cellular uptake and radiation sensitizing effects in breast cancer cells.

Concurrent recordings of hippocampal neuronal spikes and prefrontal synaptic inputs from an awake rat.

A major challenge in neuroscience is linking synapses to cognition and behavior. Here, we developed an experimental technique to concurrently conduct a whole-cell recording of a prefrontal neuron and a multiunit recording of hippocampal neurons from an awake rat. This protocol includes surgical steps to establish a cranial window and 3D printer-based devices to hold the rat. The data sets allow us to directly compare how subthreshold synaptic transmission is associated with suprathreshold spike patterns of neuronal ensembles. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2021).

Prefrontal synaptic activation during hippocampal memory reactivation.

Cooperative reactivation of hippocampal and prefrontal neurons is considered crucial for mnemonic processes. To directly record synaptic substances supporting the interregional interactions, we develop concurrent spike recordings of hippocampal neuronal ensembles and whole-cell patch-clamp recordings of medial prefrontal neurons in awake rats. We find that medial prefrontal neurons depolarize when hippocampal neurons synchronize. The depolarization in medial prefrontal neurons is larger when hippocampal place cells that encoded overlapping place fields and place cells that encoded a novel environment are synchronously reactivated. Our results suggest a functional circuit-synapse association that enables prefrontal neurons to read out specific memory traces from the hippocampus.

Titanium oxide nano-radiosensitizers for hydrogen peroxide delivery into cancer cells.

Polyacrylic acid-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers that enhance the therapeutic effect of X-ray irradiation after local injection into tumors. However, the mechanism for this reaction has remained unclear with the exception of the involvement of hydrogen peroxide (H2O2), which is released by PAA-TiOx NPs to a liquid phase during dispersion. In the present study, a clonogenic assay was used to compare PAA-TiOx NPs with free H2O2 molecules to investigate the effect exerted on the radiosensitivity of cancer cells in vitro. A cell-free dialysis method revealed that a portion of the H2O2 adsorbed onto the PAA-TiOx NPs during synthesis could be released during a treatment regimen. The H2O2 release lasted for 7 h, which was sufficient for one radiation treatment procedure. For in vitro experiments, cultured human pancreatic cancer cells took up PAA-TiOx NPs in 10 min after administration. Interestingly, when the cells were washed with a buffer after treatment with either a PAA-TiOx NP or H2O2 solution, the intracellular H2O2 levels remained higher with PAA-TiOx NP treatment compared with the H2O2 solution treatment. Furthermore, the effects of subsequent X-ray irradiation corresponded to the intracellular H2O2 levels. These results indicate that PAA-TiOx NPs are efficient carriers of H2O2 into cancer cells and thus enhance the radiosensitivity.

Urethane anesthesia suppresses hippocampal subthreshold activity and neuronal synchronization.

Urethane, an anesthetic utilized for animal experiments, induces neocortical slow oscillations in which a large number of neurons emit rhythmic synchronized activity. However, it remains unclear how urethane affects neuronal activity in the hippocampus. In this study, we obtained in vivo patch-clamp recordings from dorsal hippocampal CA1 neurons in mice and found a reduction in the fluctuation of subthreshold membrane potentials during urethane anesthesia, implying reduced synaptic activity in the hippocampus. We then performed spike unit recordings from dorsal hippocampal CA1 neuronal ensembles in rats and found prominent reductions in the spike rates of the majority of hippocampal units, especially spatially selective units, during urethane anesthesia, whereas a subset of nonspatial units exhibited increased spike rates. The overall reductions in neuronal spike rates induced by urethane led to prominent decreases in spike synchronization across neuronal units. Consistently, the magnitude of hippocampal sharp wave ripples was also reduced by urethane. The suppression of hippocampal neuronal synchronization by urethane may lead to the disruption of offline memory reactivation mechanisms.

A Comparative Assessment of Mechanisms and Effectiveness of Radiosensitization by Titanium Peroxide and Gold Nanoparticles.

The development of potentially safe radiosensitizing agents is essential to enhance the treatment outcomes of radioresistant cancers. The titanium peroxide nanoparticle (TiOxNP) was originally produced using the titanium dioxide nanoparticle, and it showed excellent reactive oxygen species (ROS) generation in response to ionizing radiation. Surface coating the TiOxNPs with polyacrylic acid (PAA) showed low toxicity to the living body and excellent radiosensitizing effect on cancer cells. Herein, we evaluated the mechanism of radiosensitization by PAA-TiOxNPs in comparison with gold nanoparticles (AuNPs) which represent high-atomic-number nanoparticles that show a radiosensitizing effect through the emission of secondary electrons. The anticancer effects of both nanoparticles were compared by induction of apoptosis, colony-forming assay, and the inhibition of tumor growth. PAA-TiOxNPs showed a significantly more radiosensitizing effect than that of AuNPs. A comparison of the types and amounts of ROS generated showed that hydrogen peroxide generation by PAA-TiOxNPs was the major factor that contributed to the nanoparticle radiosensitization. Importantly, PAA-TiOxNPs were generally nontoxic to healthy mice and caused no histological abnormalities in the liver, kidney, lung, and heart tissues.

Dual real-time in vivo monitoring system of the brain-gut axis.

The brain-gut axis which is an interaction between recognition and emotion and the gut sensory system for food and microbiota is important for health. However, there is no real-time monitoring system of the brain and the gut simultaneously so far. We attempted to establish a dual real-time monitoring system for the brain-gut axis by a combination of intravital Ca2+ imaging of the gut and electroencephalogram. Using a conditional Yellow Cameleon 3.60 expression mouse line, we performed intravital imaging of the gut, electrophysiological recordings of the vagus nerve, and electroencephalogram recordings of the various cortical regions simultaneously upon capsaicin stimuli as a positive control. Upon capsaicin administration into the small intestinal lumen, a simultaneous response of Ca2+ signal in the enteric nervous system and cortical local field potentials (LFPs) was successfully observed. Both of them responded immediately upon capsaicin stimuli. Capsaicin triggered a significant increase in the frequency of vagus nerve spikes and a significant decrease in the slow-wave power of cortical LFPs. Furthermore, capsaicin induced delayed and sustained Ca2+ signal in intestinal epithelial cells and then suppressed intestinal motility. The dual real-time monitoring system of the brain and the gut enables to dissect the interaction between the brain and the gut over time with precision.

In Vivo Evaluation of the ZHER2-BNC/LP Carrier Encapsulating an Anticancer Drug and a Radiosensitizer.

Radiosensitizing therapy for cancer treatment that enhances the effect of existing radiation therapy and enables noninvasive therapy has attracted attention. In this study, to achieve target cell-specific noninvasive cancer treatment using a ZHER2-bionanocapsule/liposome (BNC/LP), a carrier that binds to human epidermal growth factor receptor 2 (HER2), we evaluated the delivery of anticancer drugs and radiosensitizers and treatment effects in vitro and in vivo in mice. Target cell-specific cytotoxic activity and antitumor effects were confirmed following delivery of doxorubicin-encapsulated particles. In addition, cell damage due to radiosensitizing effects was confirmed in combination with X-ray irradiation following delivery of particles containing polyacrylic acid-modified titanium peroxide nanoparticles as a radiosensitizer. Furthermore, even when the particles were injected via the tail vein of mice, they accumulated in the tumor and exhibited an antitumor effect because of radiosensitization. Therefore, ZHER2-BNC/LP is expected to be a carrier that releases small-molecule drugs into the target cell cytoplasm and delivers a radiosensitizer such as inorganic nanoparticles, enabling combination therapy with X-rays to the target tumor.

Sniffing behaviour-related changes in cardiac and cortical activity in rats.

KEY POINTS: High-frequency (HF) sniffing represents active odour sampling and an increase in the animal's motivation. We examined how HF sniffing affects the physiological activity of the brain-body system. During HF sniffing, heart rates and the ratio of theta to delta critical local field potential power were comparable to those observed during motion periods. Vagus nerve spike rates did not vary depending on HF sniffing. Our results suggest that physiological factors in the central nervous system and the periphery are not simply determined by locomotion but are crucially associated with HF sniffing. ABSTRACT: Sniffing is a fundamental behaviour for odour sampling, and high-frequency (HF) sniffing, generally at a sniff frequency of more than 6 Hz, is considered to represent an animal's increased motivation to explore external environments. Here, we examined how HF sniffing is associated with changes in physiological signals from the central and peripheral organs in rats. During HF sniffing while the rats were stationary, heart rates, the magnitude of dorsal neck muscle contraction, and the ratio of theta to delta local field potential power in the motor cortex were comparable to those observed during motion periods and were significantly higher than those observed during resting respiration periods. No pronounced changes in vagus nerve spike rates were detected in relation to HF sniffing. These results demonstrate that central and peripheral physiological factors are crucially associated with the emergence of HF sniffing, especially during quiescent periods. Behavioural data might be improved to more accurately evaluate an animal's internal psychological state if they are combined with a sniffing pattern as a physiological marker.

Vagus nerve spiking activity associated with locomotion and cortical arousal states in a freely moving rat.

The vagus nerve serves as a central pathway for communication between the central and peripheral organs. Despite traditional knowledge of vagus nerve functions, detailed neurophysiological dynamics of the vagus nerve in naïve behavior remain to be understood. In this study, we developed a new method to record spiking patterns from the cervical vagus nerve while simultaneously monitoring central and peripheral organ bioelectrical signals in a freely moving rat. When the rats transiently elevated locomotor activity, the frequency of vagus nerve spikes was correspondingly increased, and this activity was retained for several seconds after the increase in running speed terminated. Spike patterns of the vagus nerve were not robustly associated with which arms the animals entered on an elevated plus maze. During sniffing behavior, vagus nerve spikes were nearly absent. During stopping, the vagus nerve spike patterns differed considerably depending on external contexts and peripheral activity states associated with cortical arousal levels. Stimulation of the vagus nerve altered rat's running speed and cortical arousal states depending on running speed at the instant of stimulation. These observations are a new step for uncovering the physiological dynamics of the vagus nerve modulating the visceral organs such as cardiovascular, respiratory, and gastrointestinal systems.

Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae.

BACKGROUND: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels. RESULTS: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose. CONCLUSION: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.

In vivo tissue distribution and safety of polyacrylic acid-modified titanium peroxide nanoparticles as novel radiosensitizers.

Polyacrylic acid (PAA)-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers. PAA-TiOx NPs were synthesized from commercial TiO2 nanoparticles that were modified with PAA and functionalized by H2O2 treatment. To realize practical clinical uses for PAA-TiOx NPs, their tissue distribution and acute toxicity were evaluated using healthy mice and mice bearing tumors derived from xenografted MIAPaCa-2 human pancreatic cancer cells. Healthy mice were injected with PAA-TiOx NPs at 25 mg/kg body weight via the tail vein, and tumor-bearing mice were injected either into the tumor locally or via the tail vein. The concentration of PAA-TiOx NPs in major organs was determined over time using inductively coupled-plasma atomic emission spectrometry. After 1 h, 12% of the PAA-TiOx NP dose had accumulated in the tumor, and 2.8% of the dose remained after 1 week. Such high accumulation could be associated with enhanced permeability and retention effects of the tumor, as PAA-TiOx NPs are composed of inorganic particles and polymers, without tumor-targeting molecules. The liver accumulated the largest proportion of the injected nanoparticles, up to 42% in tumor-bearing mice. Blood biochemical parameters were also investigated after intravenous injection of PAA-TiOx NPs in healthy mice. PAA-TiOx NPs invoked a slight change in various liver-related biochemical parameters, but no liver injury was observed over the practical dose range. In the future, PAA-TiOx NPs should be modified to prevent accumulation in the liver and minimize risk to patients.