Acacetin is a natural source of flavonoids with anti-inflammatory and antioxidant effects.This study determines acacetin’s protective effect and mechanism on myocardial ischaemia/reperfusion (I/R) injury.Sprague-Dawley rats were divided into sham and I/R injury and treatment with acacetin. Acacetin (10 mg/kg) was subcutaneously injected for 7 days. ECG and echocardiography were conducted to determine arrhythmia and heart function. The pathological characters of the heart were determined with triphenyl tetrazolium chloride staining, Haematoxylin & Eosin staining, and Masson staining. Expression of proteins in infarct tissues was examined with western blots.Administrated with acacetin in I/R rats significantly reduced the arrhythmia score from 4.90 to 2.50 and the reperfusion arrhythmia score from 3.79 to 1.82 in the vehicle or the acacetin group, respectively. LVEF was improved from 33.5% in the I/R group to 43.7% in the acacetin group, LVFS was increased from 16.4% to 24.5%, LVIDs was decreased from 6.5 to 5.3 mm. The inflammatory cell infiltration, myocardial fibrosis, and collagen 1 and 3 were reduced by acacetin. Acacetin promoted SOD and decreased MDA. In myocardial tissues, the expression level of TLR4 and IL-6 were restrained, and IL-10 was promoted. Apoptotic protein Bax was suppressed, and anti-apoptotic protein Bcl-2 was promoted in the acacetin group. Interestingly, the transcription factor Nrf-2/HO-1 pathway was also reversed by acacetin.Our findings indicated that acacetin has a potential therapeutic effect in clinical application on treating I/R-induced heart injury. Ischaemic heart disease is the deadliest disease globally  injury of H9C2 cardiomyoblasts and primary cultured neonatal rats cardiomyocytes via the AMPK/Nrf2 signalling pathway , sham rats treated with acacetin group (n = 6), vehicle group , and I/R rats treated with acacetin group  to detect cardiac function. Four parameters, left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular internal diameter end-diastole (LVIDd), and end-systole (LVIDd), were measured at least three consecutive cardiac cycles  were employed to detect the serum level of SOD and MDA following the manufacturer’s instructions.The serum level of IL-6 was conducted using an IL-6 ELISA kit  following the manufacturer’s instructions.After minced the rat heart samples in RIPA lysis buffer, the samples were supplemented with loading buffer and denatured at 95 °C for 5–10 min. The protein was separated with SDS-PAGE gel and transferred to the PVDF membrane. After being blocked with 5% skim milk for 1 h, the specific primary antibody was incubated overnight at 4 °C, and the secondary antibodies were incubated for an additional 1 h. Then the membranes were developed in the FluorChem E chemiluminescence imaging system. Image J image processing software performed band analysis. The primary antibodies used in the experiment were partly bought from Santa Cruz Biotechnology, including anti-IL-10 (sc-365858), anti-Bcl-2 (sc-7382), anti-Bax (sc-493), anti-TLR-4 (sc-293072), anti-GAPDH, or from Wuhan Boster Biological Technology, including anti-Nrf2 (PB0327), anti-HO-1 (PB0212), and anti-SOD1 (BA1401). The primary antibodies, such as anti-collagen I (67288), anti-collagen III (22734), and the secondary antibodies, such as peroxidase affiniPure goat anti-mouse IgG (H + L), peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L), were from Proteintech Technology.post hoc test. The incidence of arrhythmia Fisher’s test was used for statistics, and P < 0.05 was considered statistically significant.GraphPad Prism 8 statistical software was used for data analysis and graphing. Measurement data were expressed as Mean ± SEM. Differences between multiple groups were calculated by one-way ANOVA followed by Tukey’s In this study, the ECG of the rat was detected during ischaemia. The results in TTC staining was performed to detect rat myocardial infarcted area 7 days after reperfusion. The results showed treatment with acacetin did not exhibit any difference compared to sham rats. However, the infarct area of the I/R rats treated with vehicle reached about 30%. The I/R rats administrated with acacetin reduced the infarcted size to less than 20%.Echocardiography was employed to determine the left ventricular function of rats. As shown in H&E staining and Masson staining were applied to evaluate the structural damage and fibrosis of the rat heart  and malondialdehyde (MDA) reflect the level of oxidative stress , interleukin-6 (IL-6), and interleukin-10 (IL-10), play vital roles in the inflammatory reaction of cardiomyocytes. The expression levels of TLR4 in the vehicle group were significantly increased in the cardiac tissue, and the serum level of IL-6 and acacetin could dramatically inhibit the surge of TLR4  and IL-6Cell apoptosis occurs after inflammation and oxidative stress in cardiac I/R. The results showed that the proapoptotic protein was increased, and anti-apoptotic protein Bcl-2 was decreased in the I/R group. At the same time, acacetin significantly inhibited the decrease of Bcl-2 expression  and the The Nrf-2/HO-1 pathway is an essential regulator in the process of I/R. The results showed that the vehicle group significantly reduced the expression of Nrf-2  and HO-1O-methylated flavone extracted from the traditional Chinese medicine Xuelianhua or many other plants as a bioactive compound (Chen et al. Acacetin is an in vivo. Acacetin is a flavonoid compound, which has the antioxidant capability. Our study also verified the effect of acacetin on myocardial I/R; that is, acacetin can significantly reduce the level of MDA and restore the antioxidant capacity of SOD. Therefore, acacetin can protect against myocardial I/R by inhibiting oxidative stress.When myocardial I/R occurs, abnormal activation of the xanthine oxidase system and neutrophils and abnormal mitochondrial respiratory function produce excessive reactive oxygen species, which can cause oxidative stress and cell apoptosis, and damage to myocardial function (Monji et al. The inflammatory response is activated at the stage of myocardial ischaemia and is significantly increased during reperfusion (de Haan et al. Acacetin can reduce the inflammatory response of myocardial tissue by inhibiting the levels of TLR4 and IL-6 and increasing the levels of IL-10. Apoptosis in myocardial tissue results from multiple factors, such as myocardial I/R-induced oxidative stress and inflammatory (Wang et al. Nrf2 is an important transcription factor regulating cell protection mechanisms such as antioxidant and anti-inflammatory (Liu et al. Our present study demonstrated a novel protective effect of acacetin against myocardial I/R injury by reducing arrhythmia, myocardial infarction area and fibrosis, and restoring functional cardiac damage and structural damage. The protective function of acacetin on the I/R heart is mainly through regulating the Nrf2/HO-1 signalling pathway, thereby reducing oxidative stress and inflammation and inhibiting cell apoptosis.


IrO2 was prepared by a simple hydrolysis method and coated with a thin layer of mesoporous silica (MSN) to facilitate the physical adsorption of Chlorin e6 (Ce6). The PDA coating and IrO2 NPs of the nanoplatform demonstrated an improved photothermal conversion efficiency of 29.8% under NIR irradiation. Further, the Ce6 loading imparts materials with the ability to produce reactive oxygen species (ROS) under 660 nm NIR laser irradiation. It was also proved that the IrO2 NPs could catalyze the hydrogen peroxide (H2O2) in the tumor microenvironment (TME) to generate endogenous oxygen (O2), thereby enhancing the efficiency of PDT. The in vitro and in vivo experiments indicated that the nanocomposite was highly biocompatible and could produce a satisfactory tumor therapeutic effect. Thus, the findings of the present study demonstrate the viability of using theranostic nanoenzymes for translational medicine.The combination of photothermal therapy (PTT) and photodynamic therapy (PDT) has attracted attention due to its enhanced tumor therapy effect. This study proposes a novel nanoenzyme-based theranostic nanoplatform, IrOThe online version contains supplementary material available at 10.1186/s12951-021-00948-8. Cancer is currently one of the major obstacles preventing the reduction of the global mortality rate , 2. Trad2) in the cancer cells. This leads to the production of a type of highly toxic reactive oxygen species  that cause irreparable damage to the cancer cells [2 directly affects the efficiency of PDT [2 levels in solid tumors are inadequate, thereby reducing the therapeutic efficiency of PDT [2 concentration in tumors [2 concentration in tumors for PDT [2O2 level in tumor microenvironment (TME) is relatively high compared to the normal tissues, the amount of endogenous H2O2 is still insufficient to kill the cancer cells due to the cancer cells characteristically have a high antioxidant capacity [2O2 in TME can be utilized for the catalytic generation of endogenous O2 and facilitate tumor PDT [PDT involves the absorption of photon energy by a photosensitizing agent (PSA), resulting in the transfer of its electrons to the oxygen molecules  to raise the temperature of the surrounding environment and trigger the ablation and apoptosis of cancer cells , 21. The2) has recently drawn attention due to its high biocompatibility and photothermal conversion efficiency [2 has catalase (CAT)-like activity that enables it to catalyze H2O2 in the TME to generate endogenous O2, thereby enhancing the efficiency of PDT [2 nanomaterial-based nanoplatform systems for the combined PTT and PDT treatment of tumors. An IrO2@MSN@PDA-BSA nanocomposite was synthesized in this study for the PTT and PDT dual-mode therapeutic treatment of tumors. IrO2 was prepared by a simple hydrolysis method and coated with a thin layer of mesoporous silica (MSN) to facilitate the physical adsorption of Chlorin e6 (Ce6). The MSN layer can not only protect the oxidation of IrO2 but also provide abundant functional groups to support the further surface modification of the nanocomposite. Subsequently, PDA was coated on the surface of IrO2@MSN, followed by the grafting of bovine serum albumin (BSA) on the surface of IrO2@MSN@PDA as a stabilizer. The installation of IrO2@MSN@PDA-BSA (Ce6) serves several purposes. The PDA coating and IrO2 NPs demonstrate significant photothermal conversion under NIR irradiation. IrO2@MSN@PDA-BSA(Ce6) can produce ROS to kill cancer cells under photon activation. IrO2 can also catalyze the decomposition of H2O2 to enhance the production of O2 in the TME, thereby enhancing the therapeutic effect of PDT. In vitro and in vivo experiments have proved that IrO2@MSN@PDA-BSA(Ce6) is biocompatible and can passively target tumors through the enhanced permeability and retention (EPR) effect, thereby harnessing the clinical and combined effects of PTT and PDT for the tumors. The design of IrO2@MSN@PDA-BSA(Ce6) provides a framework for the design of multifunctional nanocomposite that can accurately treat cancer.The application of iridium oxide  at room temperature. Then, the solution pH was adjusted to 12 using NaOH solution (2.0 M), and the mixture was allowed to react at 80° C for 12 h with stirring. Finally, the mixture was further separated by centrifugation , and then rinsed three times with ethanol and distilled water. The sample in this experiment was freeze-dried and collected for the following usages.For the synthesis of IrO2@MSN nanoparticles, the as-prepared IrO2-PVP NPs were dissolved in 65 mL ethanol upon ultrasonication for 30 min. Then, 2.8 mL NH3·H2O (28 %) was added dropwise and the mixture was stirred at room temperature for 30 min. Subsequently, 0.1 mL TEOS was dissolved in 6.5 mL ethanol and added dropwise to the mixture at a rate of 0.5 mL/min under vigorous stirring. After stirring at room temperature for 6 h, the obtained products were centrifuged and washed twice with ethanol and once with distilled water. To further remove the surfactant template of CTAB, the product was stirred with saturated ammonium nitrate ethanol solution for 12 h. The resulting product was centrifuged, and washed it with double-distilled water extensively.For the synthesis of IrO2@MSN NPs. After stirring it at room temperature for 4 h, the color of the solution changed into dark brown because of the oxidation, and the formed PDA coated IrO2@MSN (IrO2@MSN@PDA) NPs were collected by centrifugation . The product was washed three times with ethanol and water to remove any possible remnants.In this step, we dissolved 0.2 g dopamine in Tris buffer solution , and then mixed it with the as-prepared IrO2@MSN@PDA-BSA was synthesized by the amidation reaction between BSA and PDA. Specifically, the as-prepared IrO2@MSN@PDA and 0.25 g BSA were added to 15 mL phosphate buffer  under 4 h of ultrasounding at room temperature. Finally, the product was conventionally centrifuged  and washed it three times with distilled water.IrO2@MSN@PDA-BSA(Ce6), Ce6 was dissolved in DMSO at a concentration of 1000 µg/mL. Afterwards, the prepared Ce6 stock solution was diluted into different concentrations and mixed with IrO2@MSN@PDA-BSA . Then, the mixture was magnetically stirred overnight at room temperature. Thereafter, the reaction products were centrifuged at 12,000 rpm for 8 min to obtain IrO2@MSN@PDA-BSA(Ce6) NPs. The supernatant containing excess Ce6 was carefully collected for the loading amount calculation of Ce6. In brief, we used UV–vis–NIR spectroscopy to read the absorbance at 404 nm of the supernatant to reckon the concentration of Ce6 as per the absorbance-concentration standard curve. The loading efficiency of Ce6 was calculated by Wt/W t and W stand for the weight of loading Ce6 and total Ce6, respectively.), and the loading percentage of Ce6 was calculated by Wt/WSt and Ws stand for the weight of loading Ce6 and the weight of IrO2@MSN@PDA-BSA, respectively.). The amount of released Ce6 was measured by using a UV–vis spectrometer. In detail, four groups of IrO2@MSN@PDA-BSA(Ce6)  were placed in a dialysis bag with a molecular weight cutoff of 14,000. Then, they were dialyzed in 4 mL of PBS solution (pH = 7.4) in a constant temperature oscillator at 37 °C) for 24 h. At each predetermined time point, 2 mL of buffer solution that contained the released Ce6 was taken out and 2 mL of the corresponding fresh buffer solution was added. The concentration of the released Ce6 was determined with UV–vis–NIR spectroscopy at 404 nm.For the synthesis of IrO2@MSN@PDA-BSA at different temperature (37 and 80 °C) was evaluated by mixing IrO2@MSN@PDA-BSA  with various concentration of H2O2 . After the addition of H2O2, we used an oxygen probe  to monitor the dissolved oxygen content, and use a digital camera to record the bubble releasing of the reaction. At the same condition, the IrO2@MSN@PDA without the addition of H2O2 was set as a control. The catalytic efficiency of IrO2@MSN@PDA-BSA under simulated TME  conditions was investigated. In details, IrO2@MSN@PDA-BSA (200 µg/mL) NPs were incubated in PBS solutions at different pH values (6.0 and 7.4) containing 500 µM H2O2. At predetermined time points, the generated dissolved O2 was measured by an oxygen probe .In this step, the catalase-like catalytic efficiency of IrO2@MSN@PDA-BSA(Ce6) in dimethyl sulfoxide was mixed with 50 µL DPBF in dimethyl sulfoxide. The terminal concentration of IrO2@MSN@PDA-BSA(Ce6) and DPBF was 20 µg/mL and 10 µM, respectively. Then, the mixture was added with H2O2 , while the mixture added with H2O (45 µL) was set as the control group. We exposed the above solutions to a 660 nm NIR laser (0.3 W/cm2) in a dark environment for 20 min, and used the UV–vis–NIR spectrophotometer  to record the absorption intensity at designed time intervals.1,3-diphenylisobenzofuran (DPBF), a classical probe for the measurement of singlet oxygen, was employed for the detection of the singlet oxygen. Briefly, 2.95 mL IrO2@MSN@PDA-BSA(Ce6) was prepared in different concentrations . Then, 100 µL of the above samples were added in each well of a 96-well plate and exposed to the 808 nm NIR laser . To investigate the power density-dependent thermal characteristics, IrO2@MSN@PDA-BSA(Ce6) with the concentration of 200 µg/mL was select to be irradiated with 808 nm NIR laser at different power  for 5 min. The photothermal stability of IrO2@MSN@PDA-BSA(Ce6) was also validated via the 808 nm NIR laser irradiation (1 W/cm2) for 5 on/off cycles (laser on for 10 min and laser off for 10 min in each cycle). We use FLIR™ E60 camera  to record the temperature increment (ΔT) of the above experiment for specific analysis.In order to explore the photothermal conversion performance, the as-prepared IrO2@MSN@PDA-BSA in vitro. On the one hand, the CCK8 kit was performed to detect the cell viability after cultured with IrO2@MSN@PDA-BSA(Ce6). In brief, L929 cells were seeded into 96-well plate (8000 cells/well) and maintained at 37 °C in a humidified atmosphere with 5% CO2. After 24 h, the medium was replaced by various concentrations  of IrO2@MSN@PDA-BSA(Ce6). Another 24 h later, 100 µL fresh RMPI1640 with 10 µL CCK8 test solution was added in each tested well to replace the old solution and then incubated for another 2 h. Using a microplate reader to read the absorbency of each well at 450 nm to calculate cell viability. On the other hand, calcein-AM/PI Live/Dead kit was used to further determine the cytocompatibility of IrO2@MSN@PDA-BSA(Ce6). L929 cells were cultured and disposed of with the same as the CCK8 assay but lastly stained with calcein-AM/PI (100 µL) based on the manufacturer for 30 min at 37 °C. The counterstained cellular morphology was recorded using a fluorescence microscope (Olympus BX53).In this section, L929 cells were chosen to assess the potential cellular cytotoxicity of IrO2@MSN@PDA-BSA(Ce6) in vitro, HT29 cells were seeded into 96-well plates at a density of 8 × 103 cells/well and maintained in a humidified cell-incubator with 5% CO2 at 37 °C overnight for cell attachment. Then, each well was filled with 100 µL IrO2@MSN@PDA-BSA(Ce6)  and continue culturing for 12 h. Then, we divided cells into four groups (n = 6). Cells in group 2 were irradiated with 808 nm laser  to assess the effect of PTT. Cells in group 3 were exposed to 660 nm laser  and cells in group 4 were treated with the medium containing H2O2 (100 µmol/L) and the pH was adjusted to 6.0 with HCl. Cells in group 5 were exposed to a combined NIR irradiation, including 808 nm (1 W/cm2) for 5 min and 660 nm (0.3 W/cm2) for 3 min. Cells treated with DMEM without other intervention were set as the control (group 1). Finally, we used CCK-8 and the calcein-AM/PI Live/Dead staining to evaluate the relative viabilities of cells.To study the cytotoxic effects of IrO2@MSN@PDA-BSA(Ce6) was investigated by the typical in vitro hemolysis assay as below: fresh complete blood samples (1 mL) were collected from healthy KM mice and centrifuged  to obtain the mouse red blood cells (mRBCs). After that, the harvested mRBCs were washed with PBS three times and reposited in PBS for further experiments. Then, the above mRBCs dispersions (0.3 mL) was mixed with 1 mL of different concentrations  of IrO2@MSN@PDA-BSA and cultured at 37 °C. Meanwhile, mRBCs dispersions (0.3 mL) were incubated with 1 mL of PBS (negative control) or 1 mL of DI water (positive control). After incubating for 4 h, the supernatant of the above mixture was gathered via centrifugation . Subsequently, we recorded the absorbance of the supernatants at 580 nm to compute the hemolysis percentage (HP) according to literature.The hemocompatibility of IrO2@MSN@PDA-BSA(Ce6) in vivo, the healthy KM female mice (SPF grade) were randomly sent into two groups (n = 24 per group). All animal experiments were conducted under the protocols approved by the Laboratory Animal Center of Changhai Hospital of Second Military Medical University, the policies of National Ministry of Hkiuealth. One group was intravenously (I.V.) injected with saline as a control, another group was injected with IrO2@MSN@PDA-BSA(Ce6) solution . The body weight of all experimental mice was weighed and recorded per 2 days. Mice were heart punctured for collect blood samples after anesthesia on 1, 7, 28 days and then were euthanized for organs collection. The blood samples were used for blood routine analysis and blood biochemical index testing in virtue of a Sysmex XS-800i automated hematology analyzer and a DxC 800 automatic biochemical analyzer. These crucial organs  were partly sectioned and immediately dipped in 4% polyformaldehyde for hematoxylin and eosin (H&E) staining. Besides, the remaining part of the organs was used for quantitative detection of the Silicon (Si) ion biodistribution through the Agilent 700 Series ICP-OES.To evaluate the biocompatibility and biodistribution of IrO7 cells per mouse, in 150 µL of serum-free DMEM) were subcutaneously implanted into the right back of the Balb/c nude mice . Three weeks after the cell-injection, a tumor nodule about 0.15–0.20 cm3 in each mouse was established. Then, the tumor mice were randomly allocated to five groups with six mice in each group as follows: mice in group 1 were I.V. injected with saline and served as the control group. Mice in group 2 were given with IrO2@MSN@PDA-BSA(Ce6) solution  for PTT. Mice in group 3 were treated with IrO2@MSN@PDA-BSA(Ce6) solution  for PDT. With regard to the dual-modal tumor therapy, 200 µL (I.V.) and 20 µL (I.T.) IrO2@MSN@PDA-BSA(Ce6) were separately injected in group 4 and group 5. Twelve hours after the injection, mice in group 1 and group 2 were irradiated with 808 nm NIR laser (1.0 W/cm2) for 5 min, while mice in group 3 were subjected to the 660 nm laser (0.3 W/cm2) for 5 min. As the combined treatment group, the mice in group 4 and group 5 were successively exposed to 808 nm NIR laser (1.0 W/cm2) and 660 nm laser (0.3 W/cm2) for 5 min. The FLIR E60 infrared camera was exploited to keep the record of ΔT measurement and thermal imaging during the treatments. Mice in all groups were then raised for 4 weeks, and we measured the tumor volume every 3 days. The efficiency of in vivo tumor treatment was assessed by the relative tumor volume.HT29 cells (10All the data acquest from the above experiments were reported as mean ± standard deviation (SD) and statistically analyzed with one-way ANOVA. P values < 0.05 was regard as statistically significant. Data indicated with (*) deputize for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001.2 NPs (IrO2-PVP) were prepared through facile hydrolysis of IrCl3, the as-prepared IrO2-PVP nanoparticles showed a diameter of approximately (10.12 ± 2.10) nm  and leads to poor lymphatic drainage, which is the structural basis of the enhanced permeability and retention (EPR) effect. The EPR effect of the nanomedicine with larger size was lower than the most suitable size but it also can accumulate in tumor through EPR effect. EDS  and large pore volume (0.2 cm3/g). The average pore size was 5.7 nm  and amide II (–NH–) of BSA, respectively. The vibration peaks of the PDA and BSA amide groups due to the stretching of the –NH– group are located at 3400 cm− 1 and 3000 cm− 1, respectively. The peaks corresponding to the –C=C– stretching vibration of PDA is located at 1500 cm− 1. These results collectively confirm that PDA and BSA were successfully decorated onto the IrO2@MSN surface. The yields of IrO2-PVP, IrO2@MSN, IrO2@MSN@PDA-BSA, IrO2@MSN@PDA-BSA were 89.3%, 78.6%, 73.2 and 68.4%, respectively.Colloidal-stable polyvinylpyrrolidone (PVP)-decorated IrO2@MSN@PDA-BSA NPs are likely to facilitate guest molecule loading. A classical PSA named Ce6 was chosen as the model drug to be physically adsorbed in the mesoporous pore of MSN. It is essential to evaluate the loading performance of IrO2@MSN@PDA-BSA because the tumor PDT is dose-dependent. The UV–vis–NIR spectra of IrO2@MSN@PDA-BSA before and after the loading of Ce6 are shown in Fig. 2@MSN@PDA-BSA. This proves that Ce6 was successfully loaded onto IrO2@MSN@PDA-BSA. The Ce6 loading of IrO2@MSN@PDA-BSA was then optimized by varying the concentration of Ce6. Ce6 concentrations of 50, 100, and 200 µg/mL resulted in Ce6 loading values of approximately 76.3%, 61.3%, and 43.0% on IrO2@MSN@PDA-BSA, respectively. The Ce6 loading ratio was calculated by dividing the weight of the loaded Ce6 with the weight of IrO2@MSN@PDA-BSA, which was equal to 3.8%, 6.1%, and 8.6% at Ce6 concentrations of 50, 100, and 200 µg/mL, respectively  was thoroughly evaluated. As shown in Additional file The conspicuous pore sizes of the IrO2@MSN@PDA-BSA can improve the PDT efficiency by catalyzing the decomposition of H2O2 to generate endogenous O2 due to the catalase-like activity of IrO2. The addition of H2O2 to the IrO2@MSN@PDA-BSA solution results in the immediate generation of several O2 bubbles, as shown in Fig. 2. Further, the catalytic efficiency of IrO2@MSN@PDA-BSA was concentration-dependent. An increase in the IrO2@MSN@PDA-BSA concentration led to an increase in the concentration of the dissolved O2  conditions. As shown in Additional file 2O2 at pH 6.0. The IrO2@MSN@PDA-BSA NPs possess the required thermal stability and are superior to the traditional catalase, which is vulnerable to hyperthermia . In addition, it was observed that the rate of reduction in the light absorption increased with the addition of H2O2 , respectively. An E60 thermal imaging camera  was used to record the concentration, irradiation, and laser-power-density-dependent temperature surges of IrO2@MSN@PDA-BSA  were equal to 29.8% and 175.7 s, both of which are comparable to the values obtained for graphene and black phosphorus nanosheets  was assessed using a CCK-8 cell viability assay and calcein-AM/PI double staining. The cell viabilities were maintained above 95% after 24 h of co-cultivation with varying concentrations of IrO2@MSN@PDA-BSA(Ce6), as shown in the CCK-8 results depicted in Fig. 2@MSN@PDA-BSA(Ce6) is satisfactory within experimental dosages. The hemocompatibility of IrO2@MSN@PDA-BSA(Ce6) was evaluated using a hemolysis assay. The HP of IrO2@MSN@PDA-BSA(Ce6) remained lower than 5%, even at a high material concentration of 500 µg/mL. The HP values were equal to 0.4 ± 0.14%, 1.2 ± 0.25%, and 2.16 ± 0.3% at IrO2@MSN@PDA-BSA(Ce6) concentrations of 100, 250, and 500 µg/mL, respectively  in animals was also studied. The body weights of mice were measured every 2 days after the I.V. materials injection. The body weights of the experimental and control groups did not differ significantly (p > 0.05), as shown in Fig. 2@MSN@PDA-BSA(Ce6) were not similar to those of the control group (p > 0.05). The potential toxicity of IrO2@MSN@PDA-BSA(Ce6) to vital organs, such as the heart, liver, spleen, lungs, and kidneys, was also evaluated. The H&E staining of these organs demonstrated that no obvious damage was caused during the preset feeding periods . This indicates that IrO2@MSN@PDA-BSA(Ce6) does not cause acute or chronic organ damage. The potential metabolic pathway of IrO2@MSN@PDA-BSA(Ce6) was then investigated by conducting an in vivo biodistribution of Si ions. The accumulation of Si in the liver and kidney 1 day after the injection was higher than that observed in the other organs due to the non-specific uptake of the reticuloendothelial system. The amount of Si in the major organs gradually decreased with time. As a result, the concentration of Si in the tested organs was lower than 5 µg/g 28 days after the injection.The biocompatibility of IrOy p > 0.0, as showy p > 0.0, as show2@MSN@PDA-BSA(Ce6) in vitro and in vivo. The HT-29 cells cultured with IrO2@MSN@PDA-BSA(Ce6) in the absence of light intervention did not report excessive cell death. However, the survival rate of the cells after being subjected to 808 nm NIR laser irradiation was reduced to 48.9% . In comparison with the control group, the viability of the HT-29 cells irradiated with a 660 nm laser decreased to 65.8% . The addition of H2O2 to the DMEM under conditions similar to those of the PDT group drastically reduced the viability of the HT-29 cells to 17.6% . The differences in the cell viability values indicate that the co-existence of H2O2 and IrO2@MSN@PDA-BSA(Ce6) could enhance the tumor PDT effect. This is because IrO2 catalyzed the decomposition of H2O2 to produce endogenous oxygen, which was further sensitized by the Ce6 while being irradiated by a 660 nm laser. Owing to the PTT and CAT-mimicking abilities of IrO2@MSN@PDA-BSA(Ce6), the HT-29 cells were almost completely dead after being irradiated by the 808 nm and 660 nm lasers successively . The results of the calcein-AM/PI Live/Dead staining were similar to those of the CCK-8 cell viability assay. The HT-29 cells in the control group were stained with a strong green fluorescence, whereas most HT-29 cells treated with PTT, PDT, enhanced PDT, and PDT/PTT were stained red .The efficiency of the combined tumor therapy in HT-29 cells and tumor-bearing nude mice was studied to realize the desirable biocompatibility of IrOPDA-BSACe in vitroPDA-BSACe in vitro2@MSN@PDA-BSA(Ce6) is suitable for multimodal tumor therapy. This was verified by the tests conducted on animals. The variations in the temperatures of the tumor-bearing nude mice that received the material injections were measured using a thermal imaging camera. The ΔT of the control group was only 4.3 °C after being subjected to NIR irradiation at 808 mm (5 min), whereas ΔT for the mice injected with the I.V. and I.T. materials were 10.3 and 20.8 °C, respectively. The variation in the temperatures of the mice injected with the I.V. material also proved that IrO2@MSN@PDA-BSA(Ce6) could passively accumulate at the tumor site through the EPR effect. The volume of the tumors of the mice belonging to the control group increased to 9.12 times their original values after 28 days of feeding. However, the tumor volumes of the PTT group increased to 2.56 times their original values . This indicates that PTT can kill a few cancer cells while the surviving ones continue to grow. Similarly, the tumor volumes of the PDT group increased to approximately 3.42 times their original values . There was no obvious difference between the tumor volumes of the PDT and PTT groups (p > 0.05). This suggests that a single dose of PTT or PDT produces a certain therapeutic effect. However, the tumors of the mice in the combined therapy group were completely eradicated, irrespective of the material injection method  NPs.The in vitro therapeutic effect investigation provides preliminary confirmation that IrOhod Fig. c, d. Thi2@MSN@PDA-BSA(Ce6) nanoenzymatic composite platform for the treatment of tumors through the combined effects of PTT and PDT was designed in this study. The MSN coating introduced abundant pores for Ce6 loading, and the conjugated BSA endowed the NPs with sufficient biocompatibility in vitro and in vivo. The loading of Ce6 facilitated the photo-triggered generation of single oxygen, resulting in photodynamic cell death. IrO2 functions as a CAT-mimic to catalyze the decomposition of H2O2 in the TME to generate endogenous oxygen and alleviate the hypoxia of solid tumors. This enhances the PDT of Ce6 by offering local oxygen and promoting the generation of 1O2. In addition, the PDA coating and IrO2 NPs exhibited remarkably high photothermal conversion efficiencies, resulting in the ablation of solid tumors through hyperthermia. The therapeutic effect of IrO2 @MSN@PDA-BSA(Ce6) for multimodal tumor therapy was proved both in vitro and in vivo. The results indicated that the tumors of the mice in the combined therapy group could be completely eradicated. The findings of the present study highlight the feasibility of using theranostic nanoenzymes for translational medicine.An IrOAdditional file 1. 1. Materials. 2. Characterization of IrO2@MSN@PDA-BSA NPs. Fig. S1. (a) The TEM images of IrO2@MSN@PDA-BSA NPs; (b) the corresponding size distributions of IrO2-PVP nanoparticles. Fig. S2. EDS spectra of IrO2@MSN@PDA-BSA NPs. Fig. S3. FTIR spectra of PVP, PDA, and IrO2@MSN@PDA-BSA NPs. Fig. S4. Release of Ce6 from IrO2@MSN@PDA-BSA(Ce6) NPs. Fig. S5. Variation of the dissolved oxygen (DO) content at different pH values (pH 6.0 and 7.4). Fig. S6. Routine blood test of IrO2@MSN@PDA-BSA(Ce6) treated KM mice fed for different days.


Intrathecal drug delivery has a significant role in pain management and central nervous system (CNS) disease therapeutics. A fluid-physics based tool to assist clinicians in choosing specific drug doses to the spine or brain may help improve treatment schedules.max), and maximum concentration (Cmax), for each case.This study applied computational fluid dynamics (CFD) and in vitro model verification to assess intrathecal drug delivery in an anatomically idealized model of the human CSF system with key anatomic features of the CNS. Key parameters analyzed included the role of (a) injection location including lumbar puncture (LP), cisterna magna (CM) and intracerebroventricular (ICV), (b) LP injection rate, injection volume, and flush volume, (c) physiologic factors including cardiac-induced and deep respiration-induced CSF stroke volume increase. Simulations were conducted for 3-h post-injection and used to quantify spatial–temporal tracer concentration, regional area under the curve (AUC), time to maximum concentration  did not improve tracer exposure to the brain. An increase in cardiac and respiratory CSF movement improved tracer spread to the brain, basal cistern, and cerebellum up to ~ 2 logs compared to the baseline LP injection.The computational modeling approach provides ability to conduct in silico trials representative of CSF injection protocols. Taken together, the findings indicate a strong potential for delivery protocols to be optimized to reach a target region(s) of the spine and/or brain with a needed therapeutic dose. Parametric modification of bolus rate/volume and flush volume was found to have impact on tracer distribution; albeit to a smaller degree than injection location, with CM and ICV injections resulting in greater therapeutic dose to brain regions compared to LP. CSF stroke volume and frequency both played an important role and may potentially have a greater impact than the modest changes in LP injection protocols analyzed such as bolus rate, volume, and flush.The online version contains supplementary material available at 10.1186/s12987-022-00304-4. Cerebrospinal fluid (CSF) has a complex and dynamic movement that allows solutes to disperse around the brain and within the ventricles. CSF has a viscosity and density similar to water at body temperature and resides in the subarachnoid space (SAS) and ventricles of the brain with a total volume ranging from 250 to 400 mL in healthy adult humans , 2. CSF The combination of close proximity and naturally occurring solute transport along the neuro-axis make CSF an intriguing pathway for delivery and removal of solutes to and from the central nervous system (CNS). One advantage of bypassing the blood–brain-barrier through CSF drug delivery is potential for reduction of systemic toxicity because of increased dose efficiency to the CNS –14. CSF While CSF drug delivery is of growing interest, we have relatively little knowledge about the factors impacting CSF drug dispersion. These factors include: (1) catheter placement location, (2) drug injection protocol including flow rate, bolus volume and flush volume, (3) CSF physiology including cardiac-related stroke volume and respiratory fluctuations, (4) patient disease-specific anatomy, (5) drug injection formulation, baricity and chemical properties. After dispersion of a drug in the CSF, it is absorbed into the CNS tissue and other outflow pathways. This is also a complex process that involves movement of solutes across the pia-arachnoid complex membranes, along peri- and/or paravascular spaces, and into the interstitial spaces and ultimately into neurons. Understanding these factors is needed to minimize therapy risk, complications, and costs while maximizing benefits , 14. In Computational fluid dynamics (CFD) modeling is a powerful tool for conducting in silico trials that can be applied to help understand the above-mentioned factors impacting CSF drug dispersion. Several numerical modeling studies have investigated intrathecal drug delivery solute transport Table . KuttlerDespite the outstanding contributions available in literature, to the best of our knowledge, a 3D anatomically detailed model of the entire CSF system has not been used to investigate the possibility and a range of effects of injection parameters, injection locations, and CSF physiology on CSF drug delivery. The present study presents a numerical model of drug injection based on a patient-specific 3D geometry of the entire SAS with idealized anatomical structures.Our overall approach was to build a subject-specific multiphase CFD simulation to investigate the impact of the following parameters on tracer distribution within the CSF: (a) catheter position, (b) injection parameters  and (c) physiological parameters (deep respiration and stroke volume) Table . Each siA subject-specific 23-year-old female CSF system geometry  includinAn unstructured tetrahedral computational mesh was generated using ANSYS ICEM CFD . The computational mesh was further refined with a prism layer near boundaries and near the needle tip. To allow computation of CSF velocity profile at the needle termination, the mesh also included the internal needle lumen. The final computational mesh covering the needle, cranial, and spinal SAS, and ventricles had 18.7 million cells.3 and viscosity of 0.89 mpa-s.The model was oriented with the subject lying on the side to represent a typical patient position for LP injection. To represent CSF oscillations around the brain and spinal cord, an oscillatory velocity inlet boundary condition was defined at the caudal opening of the model by applying a user defined function. A representative CSF flow rate waveform was applied with a 1.0 mL stroke volume at the C2-C3 vertebral level  was used to solve the oscillatory CSF flow field using a laminar viscous model. The convergence criterion was set to 1E-06. A total computation time of two weeks was needed to simulate three hours of real-world time running in parallel mode with 126 GB RAM and 78 processors. Numerical sensitivity studies for time-step size and mesh resolution were carried out for axial distribution of cross-sectional average tracer concentration and tracer concentration across a rake of points located at the basal cistern, thoracic spine and lumbar spine at 3 h. The possible inaccuracy between two phases based on the numerical diffusion was addressed by refining the mesh near the injection location. A time-step size of 0.01 s and maximum mesh size of 0.5 mm was used for each simulation. The PISO Scheme (pressure-implicit with splitting of operators) with PRESTO! (pressure staggering options) pressure discretization method was used to solve the flow equations. Second-order upwind and first-order upwind was used for discretization of momentum and volume fraction. Under relaxation factors were set to default values. The implicit formulation was used for volume fraction parameters and a dispersed model was used for phases interface modeling. More details about the flow and phase equations is provided in our previous study .A summary of injection and physiological parameters utilized for each simulation is indicated in Table Our focus was to analyze the impact of injection parameters and physiological changes only under LP injection scenario. The effect of flush (5 mL-Flush) was investigated by injecting 5 mL of water in 1 min immediately after the end of bolus injection. To study the effect of injection bolus volume (3X-Bolus volume), the injection rate considered to be constant (5 mL/min) and the injection bolus volume increased 3X (15 mL). To keep the tracer dosage constant, the injection volume fraction was set to 0.33 for this larger bolus. To study the effect of injection bolus rate (5X-Bolus rate), the bolus volume was considered to be constant (5 mL) and the injection rate was set to 25 mL/min.Two more simulations were performed to study the effect of physiological parameters of stroke volume and respiration. The first simulation (2X-Stroke volume) was performed by increasing the CSF stroke volume, defined as the one half of the integrated flow for the entire CSF oscillation cycle, from 1 to 2 mL. This degree of change was selected to be representative of potential alterations in CSF stroke volume across subjects. For this simulation, only CSF flow magnitude was increased while cardiac cycle frequency was held constant. The second simulation studied the impact of deep respiration by adding the deep respiration flow waveform measured by Yildiz et al.  to the cmax) and time at which the maximum concentration occurred (Tmax) for each axial location. Tracer exposure, or integrated value of tracer concentration over time (3 h in this study), is defined by the area under the curve (AUC).Details about the multiphase simulation model is provided in our previous study . In brieTo help verify the numerical model results, an in vitro CSF model was constructed with an identical fluid domain geometry Fig. . The sheA continuous flow syringe pump was used for CSF production originating from the ventricles. Pump flow rates were verified by bucket-and-stopwatch before each experiment. An optical imaging system was used to quantify spatial–temporal tracer concentration as previously described by Khani et al. . ReliabiTo calibrate the imaging system, a group of data sets were needed as a standard for the actual fluorescein concentration with respect to the luminescence captured in the digital images. Thus, the system was filled with a uniform fluid of multiple fluorescein concentrations and images were captured with each concentration. Collection of each calibration image-set was replicated three times to improve the robustness of the measurement. These image sets were then used to interpolate raw luminescent intensity values into known molar concentrations throughout the model.A detailed comparison of numerical and in vitro results was performed by the following correlation analysis similar to that previously conducted by our group . The in In particular, Fig. The in vitro model supports our assumption for modeling CSF steady streaming as the main driving forces of solute dispersion in slow continuous CSF drug administration. Numerical simulations predicted slightly faster distribution rate compared to in vitro Fig. a1. OveraDifferences between spatial–temporal cross-sectional average tracer concentration over 3-h obtained from in vitro and CFD were quantified using Bland–Altman plots. A relatively strong linear correlation was observed between the numerical and in vitro results for LP  within the spinal SAS compared to average of 0.11 and 0.05 μM · hr for CM and ICV, respectively  compared to 3-h simulated post injection Fig. a. Howeveses Fig. b.Based on our results, total AUC in the basal cistern and cerebellum was greater than left and right hemisphere for all simulations Fig. b1-3. HigIntrathecal drug administration has gained a significant role in pain management and drug delivery for CNS disease therapeutics. Despite this role, there are few in silico or in vitro trial platforms to study and optimize injection guidelines. A fluid-physics based tool to assist clinicians in choosing the parameters of bolus injection or drug pump settings to administer specific drug doses to the spine or the brain could improve treatment schedules.This study applied CFD to assess intrathecal drug delivery in an anatomically idealized model of the CSF system with in vitro verification. Overall, our findings suggest that intrathecal drug delivery is sensitive to many factors and that fluid-physics based computational modeling can offer insight into how individual factors may be tuned to produce a desired drug delivery profile. Our approach was to model the tracer dispersion within the flow field using multiphase model and parametrically assess the impact of the following parameters on the tracer spread over 3-h: (a) 5X-Bolus rate increase, (b) 3X-Bolus volume increase, (c) injection location comparing LP, CM and ICV Table .Studies show that wide variability exists in CFD modeling techniques, and the choice of numerical solvers and settings, are complex and can yield disparate results for biofluids simulations . At pres2 = 0.89, Fig. 2 = 0.6–0.7, Figs. Strong correlation of the CFD and corresponding in vitro simulation results were present, indicating robustness of the CFD model to capture the relevant multiphase fluid transport physics. Comparison of spatial–temporal cross-sectional average tracer concentration profiles revealed similar dispersion trends for both CFD and in vitro results under different injection location simulations Fig. . A stronmax, and Tmax values to the greatest degree . In contrast, for the LP injection, a high concentration tracer region was present for a period ~ 5 min after injection in both CFD  filled the basal intracranial cisterns and cerebellum within the first 15 min [A study by Wolf et al. showed that CM injection of two different contrast agents  due to challenges, including computational times required for this method and limited computational resources available. This is an important aspect since the physiology includes FSI and it could be addressed in future studies. Other limitations including physiologic interactions related to connected systems should be addressed in the future studies. For example, in vivo cardiac-related CSF dynamics are coupled to periodic volume changes within the cranial cavity subject to the Monro-Kellie doctrine . Also, tIn the present study of tracer transport, the effect of diffusion was neglected due to the relatively slow rate of molecular diffusion compared to streaming transport . This asThe present study did not include any in vivo validation of results as this required invasive procedures which were not possible for our research team to collect at the time of the study. Thus, the computational model was verified with controlled in vitro experiments. Future studies are needed for validation of CFD results with real-world imaging in human participants.Our model did not include tracer absorption due to microvasculature into the CNS tissue. This could have an impact on tracer concentration (particularly for LP) over long time periods. Our approach was focused on early pharmacokinetic transport in the CSF within a relatively short biologic timescale. In addition, the computational geometry did not include arachnoid trabeculae, structures that have been shown in the past to potentially alter the CSF flow field . The proOur 3D model had some limitations regarding the anatomy, mainly due to the highly complex nature of the CSF system. The CFD model did not distinguish compliance within the intracranial compartments and spinal cord tissue since the geometry of the spinal canal is extremely complex. This important aspect can be addressed in the future study.Both CM and ICV injections showed that the tracer spread earlier to the left hemisphere compared to the right hemisphere for all simulations conducted in this study Fig. . The reaThis study addressed intrathecal solute transport within the complete CSF system and the role of injection location , injection rate and volume, flush, as well as physiologic factors of cardiac-induced and deep respiration-induced CSF stroke volume on tracer distribution. For the injection scenarios analyzed in our study, CM injection was found to be the best way to improve tracer delivery to the brain; albeit CM injection can present significant risk to the patient. Injection location was found to affect the tracer distribution profile more than other injection parameters. LP injection generated a parabolic concentration profile within the spinal SAS. ICV injection enhanced tracer distribution inside the brain while reducing the peak concentration in the cerebellum compared to cervical injection. The effect of injection bolus volume and rate on maximum tracer exposure within the model was at most 2X greater compared to LP while higher stroke volume and deep respiration resulted in ~ 2 logs greater tracer exposure within the cranial SAS. The computational modeling approach provides detailed insight into how the tracer concentration is impacted within the CSF while in vitro modeling generated results faster. The threshold at which injection location would make a clinically significant difference is not yet known. We cannot extract general working guidelines based on these results; however, they suggest choosing an injection location in close proximity to the targeted therapeutic area. Also, that relatively modest modification of the injection protocols can have a noticeable impact for LP injections.Additional file 1. Spatial-temporal experimental (EXP) and computational fluid dynamics (CFD) results. EXP results provided only for verification studies conducted for the ICV, CM, and LP case (Fig. ase Fig. . CFD res


Background: Febrile neutropenia (FN) remains one of the most challenging problems in medical oncology and is a very severe side effect of chemotherapy. Its late consequences, when it is recurrent or of a severe grade, are dose reduction and therapy delays. Current guidelines allow the administration of granulocyte-colony-stimulating factors (G-CSF) for profound FN (except for the case when a pegylated form of G-CSF is administrated with prophylactic intention) in addition to antibiotics and supportive care. Methods: This is a prospective study that included 96 patients with confirmed malignancy, treated with chemotherapy, who developed FN during their oncological therapy, and were hospitalized. They received standard treatment plus a dose of G-CSF of 16 µg/Kg/day IV continuous infusion. Results: The gender distribution was almost symmetrical: Male patients made up 48.96% and 51.04% were female patients, with no significance on recovery from FN (p = 1.00). The patients who received prophylactic G-CSF made up 20.21%, but this was not a predictive or prognostic factor for the recovery time from aplasia (p = 0.34). The median chemotherapy line where patients with FN were included was two and the number of previous chemotherapy cycles before FN was three. The median serological number of neutrophils (PMN) was 450/mm3 and leucocytes (WBC) 1875/mm3 at the time of FN. Ten patients possess PMN less than 100/mm3. The median time to recovery was 25.5 h for 96 included patients, with one failure in which the patient possessed grade 5 FN. Predictive factors for shorter recovery time were lower levels of C reactive protein (p < 0.001) and procalcitonin (p = 0.002) upon hospital admission and higher WBC (p = 0.006) and PMN (p < 0.001) at the time of the provoking cycle of chemotherapy for FN. The best chance for a shorter duration of FN was a short history of chemotherapy regarding the number of cycles) (p < 0.0001). Conclusions: Continuous IV administration of G-CSF could be an alternative salvage treatment for patients with profound febrile neutropenia, with a very fast recovery time for neutrophiles. Cancer is a disease described from ancient times and many attempts to treat it have been conducted. From initial natural ointments, pastes, herbal solutions to actual synthetic treatments such as cytotoxic and hormonal agents, targeted therapy, and immunomodulatory therapies, many attempts were made to cure this disease. Whether the attempts were natural or synthetic, all of them have side effects.The last decades showed an increased survival rate of cancer patients due to extensive research performed in oncology; With the emergence of new and effective drugs, the added survival benefit approximated to be at least 21% compared to those with surgery performed alone [The burden of post-chemotherapy febrile neutropenia is represented by infections that could endanger the patients’ lives, increase treatment costs, decrease in the quality of life, and lower life expectancy.Managing febrile neutropenia can be a very difficult task depending on what the oncologist has at his disposal in terms of logistics—imaging department, bacteriological laboratory, pharmacy, intensive care unit, and access to a specialist in infectious diseases.The discovery of granulocyte colony-stimulating factor (G-CSF) represents a big breakthrough in changing the prognostic, natural history, and lifesaving of oncological patients with severe postchemotherapy toxicity, which could become a therapeutical emergency such as deep febrile neutropenia. The role of G-CSF in preventing aplasia for patients with systemic chemotherapy is well known and it is indicated in current guidelines . ConcernThe study design was submitted for approval to the institutional Ethics Committee; the approval must be obtained as part of the institutional procedure for the Oncology Institute of Cluj-Napoca, Romania (number: 42/8 December 2015). The study was performed and respected the principles and recommendations of the Declaration of Helsinki. Before any oncological treatment or blood sample prelevations, all patients signed and provided informed consent. All data  were anonymized before and during all steps of the study in accordance with the General Data Protection Regulations.Age ≥ 18 years;Patients presenting histologically with confirmed solid tumor or hemopathy;Patients being treated with chemotherapy (regardless of the cycle);Patients who were prescribed treatment with G-CSF as adjunctive therapy for neutropenia ;Fever is defined per institutional protocol as an oral or axillary temperature above 38 °C, with a presumed infectious etiology  in the absence of paraneoplastic or non-infectious causes, for example, blood transfusion;3) induced by curative or palliative chemotherapy regimens, without a cause of bone marrow failure;Neutropenia .The exclusion criteria:Patients with prior chronic or acute antibiotic therapy for a bacterial infection;Patients with shock (whatever the etiology) ;Patients subject to a bone marrow transplantation procedure;2 surface body);Patients with severe renal failure or impairment ;Patients with pregnancy positive test or breast-feeding;Patients allergic to antibiotics or any of the ingredients of G-CSF product;Patients presenting with a myelodysplastic syndrome;Patients not treated with chemotherapy;Patients that were included and excluded from a clinical trial less than 90 days from the actual study.The inclusion criteria:Study objectivesShortening the recovery time from febrile neutropenia.This is a prospective study; 96 patients were enrolled between 2015 and 2018. Following the institutional protocol, all patients were admitted to the hospital. Each patient signed informed consent before any procedures or inclusion in the study. During the aplasia period, daily blood samples were taken in accordance to the institutional protocol. From their medical records, we assessed some clinical and laboratory results which could influence the length of chemotherapy-induced neutropenia. These include demographic data, baseline lab tests , treatment intention, and data regarding treated neoplasia (type of the tumor and stage).The length of the aplasia was established by considering the date of the diagnostic and the date of the resolution. The length in minutes was calculated by taking into consideration the hour and minute of the blood sample registration to the internal lab informatics IT system.p-value of <0.05 was statistically significant. Descriptive statistics were assessed using counts and percentages for categorical and continuous data and means , medians, interquartile ranges, and ranges for quantitative data that did not follow the normal distribution. The bias-corrected and accelerated bootstrapped 95% confidence intervals were computed for the main variables of interest. Comparisons between two independent groups regarding non-normal quantitative data were performed with Wilcoxon rank-sum test. R.I. statistical analysis was performed using Excel 2010 and R version 3.5.1 Microsoft Windows version 7. A two-tailed 3) which was ameliorated to grade 3 following the study protocol as detailed in From the initial population of 96 included patients, 95 recovered from grade 4 aplasia, with only one death event in a patient with initially severe neutropenia  and 85.64% were resolved within less than 72 h. The exact numbers of patients with 1 day aplasia, 2 days, and so on are detailed in The median duration of FN episodes in the included population, expressed in days and hours, is stated in G-CSF is a recombinant stimulating factor for the growth and maturation of myeloid progenitor cells. For this action, it requires the presence of G-CSF receptors at different levels upon the differentiation in the neutrophilic lineage . In vivoGranulocytes are under the control of several cytokines, such as CXCL12  and CXCL2 (macrophage inflammatory protein), both of them having the role of increasing the chemotactic signal and consequently stimulating the neutrophils to move into the bloodstream . These cG-CSF accelerates the mitosis and maturation process, increases the motility and elution from the bone marrow of the neutrophils, and another possible mechanism (other than those mentioned above) could be performed through CXCR2 . Both foNeutrophil recovery represents the main issue for FN in oncological patients. Neutropenia could be responsible for chemotherapy regimen administration delay, unintended dose reduction, and secondary response rate failure. Since the overall incidence of cancer is continuously rising, the need and exposure to systemic treatment will continue to represent an important issue for oncological patients .To administrate or not the G-CSF in FN?Febrile neutropenia is considered an oncologic emergency that endangers patients’ lives. In order to diminish the risk of FN, the main guidelines recommend the use of G-CSF as secondary prophylaxis, especially for high and intermediate-risk chemotherapy regimens ,4. MorbiWith respect to primary or secondary prophylactic administration, the vast majority of the guidelines strongly support and recommend the use of G-CSF ,13,15,16In the management of FN, the data regarding the utility of G-CSF administration are scarce and usually related to a low number of patients. The objectives of these trials are not the same in terms of the investigated items, but the focuses are mainly the recovery time for neutrophils, length of hospitalization, and use of antibiotics. Shortening the recovery time by the administration of G-CSF was demonstrated by Yoshida et al. on 214 febrile neutropenic episodes; Ozkaynak on 67 pediatric oncological patients; Soda et al. on 33 patients; Carbonero et al. on 210 patients with FN; and Cochrane meta-analysis on 1335 patients ,19,20,213) or if the physician expects a prolonged period of aplasia (more than ten days) [Even today, using G-CSF as adjunctive therapy is not recommended by all oncological professional associations. ASCO does not recommend it for all patients and it is kept as an option mainly for those with profound aplasia (neutrophils below 100/mmen days) .What type of G-CSF to administrate in oncological patients with FN?For clinical use, the oncologists may prescribe either short or long-acting granulocyte colony-stimulating factors. Both types of myeloid stimulating agents are approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA) for administration during an FN episode to reduce the duration of neutropenia ,13,22,23Dosage and duration of IV continuous administration of G-CSF in F.N.In our study, we administrated a continuous IV perfusion of G-CSF at a dosage level of 16 microgram/kg body/day. The main objective of the study—diminishing the recovery time from aplasia—was achieved. The median time for recovery for the included patients was 25.5 h, which is much lower than indicated in other published works.3 [The standard dose for G-CSF is 5 micrograms/kg body/day or 250 micrograms/sqm/day for Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF) or 6 mg/day for pegylated G-CSF [3 .The median duration of post-chemotherapy aplasia is between 6–8 days . Reducti3, neither peg G-CSF nor subcutaneous G-CSF could quickly reverse the neutropenia. For the pegylated form of G-CSF, asymmetry between the serum number of neutrophils and G-CSF levels is approximated to be reached within 15 days and deep aplasia requires a higher stimulation which could be assured by IV continuous perfusion [Mathematical models showed that subcutaneous administration is not capable of maintaining optimal serological levels of G-CSF due to the rapid clearance of the drug; this suboptimal stimulation could be surpassed by a twice-daily injection or by peg G-CSF formulations . A higheerfusion . For thoerfusion .Regarding the dose of G-CSF, for healthy donors for bone marrow transplant (BMT) , FDA and EMA recommend a standard dose of G-CSF of 10 micrograms/kg body/day, with a safe increase in the dose up to 12 or even 24 micrograms/kg body/day in continuous subcutaneous perfusion if needed . MoreoveIn a published phase 1 study for patients who were treated with high-dose chemotherapy followed by an autologous BMT, a 14 day continuous intravenous infusion of G-CSF was compared with the 4 h infusion. The levels of the G-CSF dose were between 4 and 32 microgram/kg/day . G-CSF aThese differences could be explained by the molecular mechanism of neutrophil’s response to G-CSF stimulation, which is essentially antagonistically driven by CXCR4 or CXCR2-linked Rac regulators .Methods of administration for G-CSF?Both the short and long-acting forms of granulocyte-colony stimulating factors could be administrated intravenously or subcutaneously ,8.The pharmacokinetics of G-CSF is very complex since it is non-linear. It could be explained by the dependence of the absorption of G-CSF on the receptor-mediated endocytosis processes, renal clearance, and neutrophile serum levels . Researc3, 50–300/mm3, and less than 50/mm3 upon the dose of G-CSF, which are considered standard for short-acting at five microgram/kg or long-acting at 100 microgram/kg in the subcutaneous or continuous intravenous administration of 10 microgram/kg/day [On the other hand, in deep aplasia, the stimulation is not equal for both types of G-CSF and standard dose depending on the level of neutropenia. Mathematical modeling suggests that the magnitude of the effect of G-CSF could differ with respect to the grade of neutropenia 300–500/mmm/kg/day .A study published in 2014 included 120 patients with hematological malignancies who were randomized between standard subcutaneous dose administration and the same dose administered by IV bolus injection . The meaIn conclusion, our prospective study showed that continuous IV administration of G-CSF with a high dose (16 micrograms/kg/day) could represent an effective alternative for neutropenic patients, especially for those without any response within 24 h to standard treatment or profound neutropenia.


The anti-human T-cell leukemia virus type 1 (HTLV-1) antibody assay in common use has changed from the particle agglutination (PA) method to chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA). These assays were validated in serum but not in cerebrospinal fluid (CSF). However, anti-HTLV-1 antibody positivity in CSF is a requisite for diagnosing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The anti-human T-cell leukemia virus type 1 (HTLV-1) antibody assay in common use has changed from the particle agglutination (PA) method to chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA). These assays were validated in serum but not in cerebrospinal fluid (CSF). However, anti-HTLV-1 antibody positivity in CSF is a requisite for diagnosing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We qualitatively compared the assays in CSF from 47 HAM/TSP patients diagnosed using PA, 15 HTLV-1 carriers (HCs), and 18 negative controls. In determining the positivity or negativity of CSF anti-HTLV-1 antibodies, we used serum cutoff points for CLIA and CLEIA because CSF cutoff points had not been decided. Truth table analysis revealed that the performance of CLIA was closer to that of PA and that CLEIA had low sensitivity. CSF antibodies from HAM/TSP patients were all positive by PA and CLIA but 83.0% positive by CLEIA. CSF antibodies from HCs were positive in 73.3%, 80.0%, and 6.7% by PA, CLIA, and CLEIA, respectively. Receiver operator characteristic curve analysis for CSF revealed that with the default cutoff point used for serum, CLIA and PA had comparable performances and CLEIA was less sensitive. The best performances of CLIA and CLEIA with adjusted cutoff points were 94.8% sensitivity and 95.5% specificity and 89.7% sensitivity and 95.5% specificity, respectively. We conclude that low-sensitivity CLEIA can underdiagnose HAM/TSP and that CLIA is a better alternative to PA in anti-HTLV-1 antibody assay for CSF with the current cutoff points. Human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus that causes adult T-cell leukemia 13, HTLV-14–A screening assay for anti-HTLV-1 Ab was established in 1983 . Therefo20–Ab titer by PA is determined by macroscopic findings in serial dilutions comparing sensitized and unsensitized reactions. Recent automated Ab assays used in diagnosing HTLV-1 infection include chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA). The former utilizes acridinium ester-labeled mouse antibody to human IgG, and the latter uses alkaline phosphatase-labeled mouse antibody to human IgG. These two assays detect antibodies to p19, p21, and p24 of HTLV-1 –29. Thes–Therefore, we compared anti-HTLV-1 Ab assays in CSF to validate PA, CLIA, and CLEIA anti-HTLV-1 Ab assays for accurate HAM/TSP diagnosis.We used sets of serum, CSF, and peripheral blood mononuclear cell (PBMC) samples from 47 subjects with HAM/TSP, 15 HCs, and 18 negative controls (NCs), from whom written informed consent was obtained. The sample sets from a subject were obtained within a week, and the patients were not prescribed any anti-inflammatory drugs. Subjects suspected of adult T-cell leukemia or other HTLV-1-related diseases were excluded. HAM/TSP diagnosis was made when seropositive subjects fulfilled the WHO diagnostic criteria  as previously described . The deth HTLV-1 . In thisWe measured anti-HTLV-1 Ab levels by PA, CLIA, and CLEIA. The cryopreserved serum or CSF in a tube was thawed, and the three assays were performed simultaneously. Serum anti-HTLV-1 Ab titer by PA  was measured according to the manufacturer’s protocol and interpretation criteria. Seropositivity (PA positivity in serum) was defined as a titer greater than 16× by PA , while aA true-positive anti-HTLV-1 Ab in serum  was defined as when both anti-HTLV-1 Ab in serum by PA and real-time PCR for HTLV-1 PVL were positive. On the other hand, PA positivity in CSF was expediently regarded as true when PA showed a titer of 4× or more without PCR because of the difficulty to collect enough DNA from a CSF sample.+). PA-negative samples were those from HCs with PA-negative CSF (HC-CSF−) and NCs. Additionally, we investigated CSF samples for the consistency of the Ab results by the three assays.Truth table analysis was performed to evaluate the similarity of performance (sensitivity and specificity) of Ab assays (CLIA or CLEIA) to that of PA when PA results were regarded as true. The performance of anti-HTLV-1 Ab assays in serum and CSF was calculated by 2 × 2 truth tables. For serum, PA-positive or PA-negative samples included those from HAM/TSP patients and HCs or those from NCs, respectively. For CSF, PA-positive samples included those from HAM/TSP patients and HCs with PA-positive CSF (HC-CSFReceiver operating characteristic (ROC) curve analysis was performed to predict the best potential performance of CLIA or CLEIA for serum and CSF from HAM/TSP patients, HCs, and NCs under the situation in which the cutoff points could be modified. We calculated the maximum Youden index (sensitivity + specificity − 1) for each ROC curve to determine the best cutoff points.The anti-HTLV-1 Ab levels were logarithmically transformed to bring their distribution closer to a normal distribution. Raw data for PA titer were transformed into a 2-based logarithm, and those data by CLIA (S/CO) or CLEIA (COI) were transformed into a 10-based logarithm. The base 2 of PA corresponds to the serial 2-fold dilution protocol. PA Ab titers below 16× in serum and those below 4× in CSF could not be measured because of the actual procedure, and those logarithmically transformed values were assigned as 0. Similarly, since the detection limits of CLIA and CLEIA were 0.1, the transformed values of Ab data below 0.1 from these assays were assigned as −1.Anti-HTLV-1 Ab levels were compared between serum and CSF and between HCs and HAM/TSP patients. A correlation analysis was performed to assess the consistency of anti-HTLV-1 Ab levels among assays.A correlation analysis was conducted between either serum or CSF anti-HTLV-1 Ab levels measured by the three assays and HTLV-1 PVL in PBMCs.P value of less than 0.05 was considered statistically significant. ROC curve analysis was performed using GraphPad Prism 6.0 . Statistics were performed using Statcel version 3.0  or GraphPad Prism 6.0.We compared the ages of sample groups by one-way analysis of variance (ANOVA). We performed a two-tailed Mann-Whitney U test with Bonferroni correction to compare Ab levels, Spearman correlation analysis, and simple linear regression analysis. A 4 PBMCs, and all seronegative subjects had negative PCR for HTLV-1 provirus. All assays matched the Ab results (positive or negative) in serum.The characteristics of subjects for each group, including diagnosis, sex, age, and PVL, are summarized in +) and 22 true-negative CSF samples (4 HC-CSF− and 18 NCs) . Under the assumption that CSF PA results were true, there were 58 true-positive CSF samples (47 HAM/TSP and 11 HC-CSF 18 NCs) . CLIA shCSF samples from all HAM/TSP patients showed anti-HTLV-1 Ab positivity at 100.0% (47/47) by PA and CLIA but 83.0% (39/47) by CLEIA. Those from all HCs were positive at 73.3% (11/15) by PA, 80.0% (12/15) by CLIA, and 6.7% (1/15) by CLEIA. CSF samples from NCs were negative by all assays. Next, we focused on the inconsistent results among the three assays as shown in In the table of inconsistency, CSF samples from 17.0% (8/47) of HAM/TSP patients and 86.7% (13/15) of HCs showed inconsistent Ab results among the three assays . The 13 + and those from HC-CSF− and NCs, respectively. The areas under the curves (AUCs) in serum by all assays were 1.0 (P < 0.05), indicating that CLIA performed more closely to PA  and PA-negative (true negative) categories for serum and CSF. For serum, PA-positive and PA-negative categories included assay results from HAM/TSP patients and HCs and those from NCs, respectively. For CSF, PA-positive and PA-negative categories included assay results from HAM/TSP patients and HC-CSFwere 1.0 . Meanwhily to PA . These dly to PA .Next, we determined the best cutoff points by maximum Youden index. CLIA showed the best potential performance in CSF, with 94.8% sensitivity and 95.5% specificity by the cutoff S/CO of 1.50 . IP < 0.01  to F).4 PBMCs, respectively (P < 0.01). We investigated the relationship between serum or CSF anti-HTLV-1 Ab levels and HTLV-1 PVL in PBMCs  copies/10ectively . The PVLin PBMCs . The serAnti-HTLV-1 Ab assays have been validated in serum but not in CSF because it was enough to examine the Ab in serum to know whether subjects were infected with HTLV-1. However, after the discovery of HAM/TSP, it became necessary to determine whether CSF Ab is positive or negative for the diagnosis. We qualitatively validated anti-HTLV-1 Ab assays in CSF samples from HAM/TSP patients, HCs, and NCs. When determining anti-HTLV-1 Ab CSF positivity or negativity using serum cutoff points, the truth table analysis revealed that CLIA in CSF had a well-balanced performance. The assay showed 96.6% sensitivity and 86.4% specificity; however, CLEIA had 69.0% sensitivity and 100.0% specificity . TherefoThe low sensitivity of CLEIA has a crucial impact on HAM/TSP diagnosis, given that anti-HTLV-1 Ab positivity in CSF is a requisite for diagnosis. In other words, assays with low sensitivity may exclude HAM/TSP cases with low Ab levels in CSF. The qualitative Ab assay results were all consistent in serum by all methods. However, there were inconsistent results in 21 CSF samples: all were negative by CLEIA . A totalAUCs in the ROC curve analysis supported the truth table analysis results, indicating that CLIA for CSF is better than CLEIA to determine anti-HTLV-1Ab positivity at the default cutoff point . On the Although PA titer was measured macroscopically, the values in CLIA and CLEIA were calculated as ratios of measured values of specimens to those of the standards. Therefore, the difference in diluents and dilution ratios of specimens among assays probably did not affect the comparison between the assays. As a quantitative analysis, we compared Ab levels measured by the three assays. We found that HAM/TSP patients had higher anti-HTLV-1 Ab levels in serum and CSF than HCs except for serum by CLIA . HoweverFor HAM/TSP patients, anti-HTLV-1 Ab levels in CSF were lower than those in serum . The fin4 PBMCs, and all seronegative subjects showed no provirus. Diagnosis of HTLV-1 infection needs both a serum anti-HTLV-1 Ab screening test  and a confirmatory test such as Western blotting or PCR. On the other hand, the diagnosis of HAM/TSP requires CSF anti-HTLV-1 Ab positivity in HTLV-1-infected individuals but not PCR positivity. Therefore, we detected CSF anti-HTLV-1 Ab PA titers over 4× but did not perform HTLV-1 PCR as a confirmatory test for anti-HTLV-1 Ab in CSF. Detection of HTLV-1 provirus in CSF may be difficult because HTLV-1 is usually detected in one of many cells, and PCR requires a large number of CSF cells. For example, even if we use a maximum of 10 ml of CSF for PCR, the total number of cells is limited to an order of magnitude of 104. When the number of HTLV-1 provirus is fewer than 10 copies in the CSF sample, detection of HTLV-1 provirus can fail by the quantitative PCR with a detection limit of 10 copies/104 cells. A study reported that PCR detected no PVL in CSF from subjects with serum Ab less than 64× (Anti-HTLV-1 Ab was regarded as showing true positivity in this study when both serum anti-HTLV-1 Ab by PA and PCR for HTLV-1 were positive, indicating that the subject was infected with the virus. All seropositive subjects showed PVLs of more than 10 copies/10than 64× . A methoWe randomly recruited HAM/TSP patients regardless of the severity of the disease. Although serum anti-HTLV-1 Ab levels are not different between HAM/TSP patients with either rapid or slow progression, patients with rapid progression have higher Ab levels in CSF than those with slow progression . As a reWe recruited 47 HAM/TSP patients, 15 HCs, and 18 NCs for this study, and the sample size of each group was small. Also, our samples did not shape Gaussian distribution, probably due to the small size of the samples. We selected HAM/TSP patients who were not treated at the sampling; therefore, the number of HAM/TSP patients almost reached the maximum in our cohort. Another problem is that the numbers of HCs and NCs were small compared to that of the HAM/TSP group. However, we analyzed both serum and CSF from the subjects, and it was difficult to recruit more HCs and NCs because lumbar puncture to obtain CSF is somewhat harmful. We could not recruit any individuals without neurological symptoms because conducting the examination on healthy subjects is an ethical problem. To increase the subject numbers, joint research with other facilities would be needed.We conclude that low-sensitivity CLEIA can underdiagnose HAM/TSP in some cases and that CLIA is a better anti-HTLV-1 Ab assay for CSF with the current cutoff point as a substitute for PA.


Our aim was to investigate the potential use of the PET radiotracer [18F]FLUDA to non-invasively determine the A2A-AR availability for diagnosis of the A2AR status. Therefore, we compared mice with cardiomyocyte-specific overexpression of the human A2A-AR (A2A-AR TG) with the respective wild type (WT). We determined: (1) the functional impact of the selective A2AR ligand FLUDA on the contractile function of atrial mouse samples, (2) the binding parameters (Bmax and KD) of [18F]FLUDA on mouse and human atrial tissue samples by autoradiographic studies, and (3) investigated the in vivo uptake of the radiotracer by dynamic PET imaging in A2A-AR TG and WT. After A2A-AR stimulation by the A2A-AR agonist CGS 21680 in isolated atrial preparations, antagonistic effects of FLUDA were found in A2A-AR-TG animals but not in WT. Radiolabelled [18F]FLUDA exhibited a KD of 5.9 ± 1.6 nM and a Bmax of 455 ± 78 fmol/mg protein in cardiac samples of A2A-AR TG, whereas in WT, as well as in human atrial preparations, only low specific binding was found. Dynamic PET studies revealed a significantly higher initial uptake of [18F]FLUDA into the myocardium of A2A-AR TG compared to WT. The hA2A-AR-specific binding of [18F]FLUDA in vivo was verified by pre-administration of the highly affine A2AAR-specific antagonist istradefylline. Conclusion: [18F]FLUDA is a promising PET probe for the non-invasive assessment of the A2A-AR as a marker for pathologies linked to an increased A2A-AR density in the heart, as shown in patients with heart failure.A An anti-inotropic effect towards the human A2A-AR was observed after the subsequent adding of 1 µM FLUDA. The FOC was decreased by 15.0 ± 2.4% in the A2A-AR TG atria. Hence, these results revealed an antagonistic effect of FLUDA towards a stimulated A2A-AR.The yet unknown agonistic or antagonistic action of FLUDA towards the human A2A-AR TG . As show2A-AR specificity of [18F]FLUDA towards the heart tissue of WT and A2A-AR TG   , which i18F]FLUDA  and a higher TAC peak value in the myocardium of A2A-AR TG (+10.4%) compared to WT. Hence, the integrated activity concentration over time was higher in both compartments. In the initial phase, a 2.3 times higher AUC value  was found in the myocardium, while it was 1.6 times higher  in the blood, as a result of the functional overexpression of the hA2A-AR. To normalise for unspecific physiological effects, AUC1–10 ratios (SUVr) of the myocardium to blood  of FLUDA into the myocardium (p = 0.017), whereas the TAC peak value in the muscle was slightly increased  compared to baseline WT. However, we found no significant changes in the AUC1–10 and AUC0–60 for these and other investigated tissues in comparison to the untreated WT. In A2A-AR TG, pre-injection of istradefylline abolished the increased uptake of [18F]FLUDA into the myocardium, as shown by the significant reduction of the AUC1–10, about 0.6 times (p = 0.032 vs. WT). These results were validated by the normalisation of the AUC1–10 of the tissues to the blood compartment, as well as to muscle. It was apparent that tissue normalisation to the blood compartment was less prone to fluctuation compared to the muscle, as shown by the SEM values  FLUDA is a functional antagonist towards the human A2A-AR and (2) [18F]FLUDA binds specifically and with high binding affinity to the human A2A-AR in a mouse model with transgenic overexpression of the receptor in the cardiac tissue and also to A2A-AR in human cardiac tissue in vitro. Furthermore, we found an increased uptake in the initial phase of the in vivo biodistribution of [18F]FLUDA in the A2A-AR overexpressing mouse model by dynamic PET imaging.The present work demonstrates the usefulness of the new radiotracer [2A-AR in a human subject by PET imaging using the methylxanthine derivative of KF17837 [11C]TMSX [11C]TMSX was later used in subsequent studies to compare the A2A-AR density in hearts between endurance athletes and untrained men [18F]FESCH was one of the first 18F-labelled A2A-AR PET-imaging probes [18F]FLUDA was recently developed and evaluated in vitro as well as in vivo by our group [2A-AR by a functional assay.Ishiwata and colleagues provided evidence of the successful non-invasive assessment of A11C]TMSX . [11C]TMined men ,23. The g probes ,25, and ined men FFESCH wa18F]FLUDA, we determined a KD value of 4.30 ± 0.73 nM and a Bmax value of 556 ± 143 fmol/mg wet weight in the striatum of healthy CD-1 mice and a KD value of 0.68 nM, as well as a Bmax value of 218 fmol/mg wet weight in the striatum of piglets FLUDA in cardiac cryosections of A2A-AR TG (KD of 5.9 ± 1.6 nM and a Bmax of 455 ± 78 fmol/mg protein) as in the striatum of healthy male CD-1 mice, although reliable binding kinetic parameters for cardiac cryosections of female FVB/N mice (WT) could not be determined , which was accompanied with a decreased ligand binding affinity (KD = 2.4 ± 0.1 nmol/L) when compared to control  [2A-AR-specific binding with [18F]FLUDA in the human atrial samples of patients with no diagnosed heart failure  . As an i failure , which i2A-AR agonists are often used in the clinic to dilate the coronary arteries in patients and to assess the severity and functional consequences of impaired vasodilation in angina pectoris [18F]FLUDA is not expected to be useful to image the coronary A2A-AR density, specifically, as the amount of smooth muscle cells and endothelial cells is much lower compared to the number and volume of cardiomyocytes. Thus, we would expect a binding of [18F]FLUDA mainly to the A2A-AR in cardiomyocytes. The in vivo studies in mice revealed increased uptake of [18F]FLUDA under baseline conditions into the myocardium of A2A-AR TG compared to WT. However, this was restricted to the initial phase after administration of the radioligand. In a single ex vivo autoradiography, we could confirm increased A2A-AR-specific uptake into the myocardia of A2A-AR TG even after 15 min p.i. (18F]FLUDA into the cardiac tissue of mice could not be observed as it was shown in other studies for [11C]TMSX [11C]TMSX was displaceable by just 40% with carrier and 8-(3-chlorostyryl)caffeine (each in A2A-AR-saturable concentrations), which may be caused by a higher amount of unspecific binding of this tracer, as it was shown for the binding in brain regions with low A2A-AR expression [11C]TMSX was very stable in human plasma (> 90% intact radioligand), whereas in mice, only 54% in plasma and 76% in heart tissue were measured [18F]FLUDA was non-metabolised in plasma samples of healthy mice 15 min post-injection [18F]FLUDA-derived metabolites in the cardiac tissue over time.Apectoris . Howevermin p.i. . An accu11C]TMSX . Interespression ,30. Addimeasured . In a repectoris FFLUDA is18F]FLUDA PET imaging could be useful to assess the receptor occupancy in the heart by A2A-AR-targeting drugs. This might be useful when treating Parkinsonian patients with A2A antagonists to avoid adverse side effects. Another potential application could be the non-invasive determination of an elevated A2AAR density in patients with fibrillation as a diagnostic marker. Hence, it would be possible to establish a cause-effect relationship between A2A-AR density and atrial fibrillation in a non-invasive manner in long-term follow-up. The next step will be to test the eligibility of [18F]FLUDA in patients and to determine the uptake of [18F]FLUDA in the heart.Clinical outlook: FLUDA are described elsewhere -FLUDA  or with addition of 10 µM ZM241385 (non-specific binding). The homologous competition assays were performed in the presence of FLUDA in the concertation range 10−6 to 10−12. The sections were then washed twice with ice-cold 50 mM TRIS-HCl (pH 7.4), dipped in ice-cold deionised water, dried in a stream of cold air, and exposed overnight on an imaging plate. The plates were scanned using an image plate scanner . The digitized autoradiograms were analyzed with AIDA 2.31 software . The Bmax and KD values were calculated from the homologous competition of [18F]FLUDA with FLUDA using the Cheng-Prusoff equation (KD = IC50 − [[18F]FLUDA] and Bmax = Top - Bottom (KD + [[18F]FLUDA])/[[18F]FLUDA]) FLUDA]) . InhibitEx vivo autoradiography of the murine hearts was performed immediately after euthanising the animals at 15 min p.i. of the radioligand. The hearts were isolated, frozen by immersion in isopentane at −20 °C, cryosectioned , and the dried sections were exposed to a phosphor imaging plate for 120 min and processed as described above.2A-TG and WT) received an i.v. injection of [18F]FLUDA . Control animals  received a vehicle solution containing DMSO/Kolliphor/NaCl, 1:2:7 (v/v/v) 10 min prior to [18F]FLUDA i.v. injection. For the determination of the A2AAR-specific uptake, animals received an i.v. injection of 2.5 mg/kg bodyweight tozadenant (WT and A2A-TG: n = 4) or 1mg/kg istradyfelline (WT and A2A-TG: n = 3) 10 min prior the radiotracer administration. The data were collected in list mode . Subsequently, after the PET scan, a T1-weighted whole-body MR scan  was performed for anatomical orientation and attenuation correction at the reconstruction step . The reconstructed images were analysed with PMOD (Version 3.802). For analysing the [18F]FLUDA uptake into the myocardium and the activity concentration in the left ventricle (blood compartment), VOIs were delineated in an averaged image of the first 10 min of the PET imaging after injection of [18F]FLUDA, and co-registered T1 images from MR. Non-parametrical analyses of achieved time-activity curves (TACs) were performed with Microsoft Excel to determine the time to peak, the TAC peak value, and the area-under-the-curve (AUC):c (radioactivity) is expressed as a standardised uptake value normalised to the bodyweight in g (SUV). GraphPad Prism 9  was used for graphical presentation.The animals were initially anaesthetised with 5% isoflurane and were positioned prone into a small-animal PET/MR  on a temperature-controlled bed system (37 °C) while respiration rate was continuously monitored. The anaesthesia  was maintained at 2.1–1.3% isoflurane in a 60% oxygen/40% air gas mixture  with 250 mL/min airflow. Prior to the 60 min PET scan, a scout image MR sequence was performed to outline the animal dimensions. Animals  with Bonferroni’s post-hoc test or Student’s t-test if applicable, were considered as significant.Values are represented as mean ± standard error of the mean (SEM). Statistical analyses were performed with Microsoft Excel and GraphPad Prism , with