Identifying the Morphological and Molecular Features of a Cell-Based Orthotopic Pancreatic Cancer Mouse Model during Growth over Time.

Pancreatic ductal adenocarcinoma (PDAC), characterized by hypovascularity, hypoxia, and desmoplastic stroma is one of the deadliest malignancies in humans, with a 5-year survival rate of only 7%. The anatomical location of the pancreas and lack of symptoms in patients with early onset of disease accounts for late diagnosis. Consequently, 85% of patients present with non-resectable, locally advanced, or advanced metastatic disease at diagnosis and rely on alternative therapies such as chemotherapy, immunotherapy, and others. The response to these therapies highly depends on the stage of disease at the start of therapy. It is, therefore, vital to consider the stages of PDAC models in preclinical studies when testing new therapeutics and treatment modalities. We report a standardized induction of cell-based orthotopic pancreatic cancer models in mice and the identification of vital features of their progression by ultrasound imaging and histological analysis of the level of pancreatic stellate cells, mature fibroblasts, and collagen. The results highlight that early-stage primary tumors are secluded in the pancreas and advance towards infiltrating the omentum at week 5-7 post implantation of the BxPC-3 and Panc-1 models investigated. Late stages show extensive growth, the infiltration of the omentum and/or stomach wall, metastases, augmented fibroblasts, and collagen levels. The findings can serve as suggestions for defining growth parameter-based stages of orthotopic pancreatic cancer models for the preclinical testing of drug efficacy in the future.

Deep-tissue localization of magnetic field hyperthermia using pulse sequencing.

OBJECTIVE: Deep-tissue localization of thermal doses is a long-standing challenge in magnetic field hyperthermia (MFH), and remains a limitation of the clinical application of MFH to date. Here, we show that pulse sequencing of MFH leads to a more persistent inhibition of tumor growth and less systemic impact than continuous MFH, even when delivering the same thermal dose. METHODS: We used an in vivo orthotopic murine model of pancreatic PANC-1 cancer, which was designed with a view to the forthcoming 'NoCanTher' clinical study, and featured MFH alongside systemic chemotherapy (SyC: gemcitabine and nab-paclitaxel). In parallel, in silico thermal modelling was implemented. RESULTS: Tumor volumes 27 days after the start of MFH/SyC treatment were 53% (of the initial volume) in the pulse MFH group, compared to 136% in the continuous MFH group, and 337% in the non-treated controls. Systemically, pulse MFH led to ca. 50% less core-temperature increase in the mice for a given injected dose of magnetic heating agent, and inflicted lower levels of the stress marker, as seen in the blood-borne neutrophil-to-lymphocyte ratio (1.7, compared to 3.2 for continuous MFH + SyC, and 1.2 for controls). CONCLUSION: Our data provided insights into the influence of pulse sequencing on the observed biological outcomes, and validated the nature of the improved thermal dose localization, alongside significant lowering of the overall energy expenditure entailed in the treatment.

MNP-Enhanced Microwave Medical Imaging by Means of Pseudo-Noise Sensing.

Magnetic nanoparticles have been investigated for microwave imaging over the last decade. The use of functionalized magnetic nanoparticles, which are able to accumulate selectively within tumorous tissue, can increase the diagnostic reliability. This paper deals with the detecting and imaging of magnetic nanoparticles by means of ultra-wideband microwave sensing via pseudo-noise technology. The investigations were based on phantom measurements. In the first experiment, we analyzed the detectability of magnetic nanoparticles depending on the magnetic field intensity of the polarizing magnetic field, as well as the viscosity of the target and the surrounding medium in which the particles were embedded, respectively. The results show a nonlinear behavior of the magnetic nanoparticle response depending on the magnetic field intensity for magnetic nanoparticles diluted in distilled water and for magnetic nanoparticles embedded in a solid medium. Furthermore, the maximum amplitude of the magnetic nanoparticles responses varies for the different surrounding materials of the magnetic nanoparticles. In the second experiment, we investigated the influence of the target position on the three-dimensional imaging of the magnetic nanoparticles in a realistic measurement setup for breast cancer imaging. The results show that the magnetic nanoparticles can be detected successfully. However, the intensity of the particles in the image depends on its position due to the path-dependent attenuation, the inhomogeneous microwave illumination of the breast, and the inhomogeneity of the magnetic field. Regarding the last point, we present an approach to compensate for the inhomogeneity of the magnetic field by computing a position-dependent correction factor based on the measured magnetic field intensity and the magnetic susceptibility of the magnetic particles. Moreover, the results indicate an influence of the polarizing magnetic field on the measured ultra-wideband signals even without magnetic nanoparticles. Such a disturbing influence of the polarizing magnetic field on the measurements should be reduced for a robust magnetic nanoparticles detection. Therefore, we analyzed the two-state (ON/OFF) and the sinusoidal modulation of the external magnetic field concerning the detectability of the magnetic nanoparticles with respect to these spurious effects, as well as their practical application.

Hyperthermia affects collagen fiber architecture and induces apoptosis in pancreatic and fibroblast tumor hetero-spheroids in vitro.

Desmoplasia, an aberrant production of extracellular matrix (ECM), is considered as one predictive marker of malignancy of pancreatic cancer. In this paper, we study the effect of mild hyperthermia on fibrillary collagen architecture in murine Achilles tendons and in a pancreatic cancer model, in vitro, i.e. 3D hetero-type tumor spheroids, consisting of pancreatic cancer (Panc-1) cells and fibroblasts (WI-38), producing collagen fibers. We clearly demonstrate that i) mild hyperthermia (40 °C, 42 °C) damages the collagen architecture in murine Achilles tendons. ii) Mild extrinsic (hot air) and iron oxide nanoparticle based magnetic hyperthermia reduce the level of collagen fiber architecture in the generated hetero-type tumor spheroids. iii) Mild magnetic hyperthermia reduces cell vitality mainly through apoptotic and necrotic processes in the generated tumor spheroids. In conclusion, hetero-type 3D tumor spheroids are suitable for studying the effect of hyperthermia on collagen fibers, in vitro.

New generation CPPs show distinct selectivity for cancer and noncancer cells.

In the last three decades, many new cell-penetrating peptides (CPPs) were developed that exhibited enhanced cell selectivity. Thus, we aimed to validate the tumor cell selectivity of peptides from this new generation, namely fragments mini-crotamine and mini-maurocalcine. Both of these peptides are derived from venoms. Furthermore, we studied an analog of the classical CPP HIV-TAT(47-57) with alternating chirality of Arg residues. To allow covalent coupling of cargoes or fluorophores, a cysteine residue was introduced to the N-terminus of the synthesized peptides. The therapeutic antibody trastuzumab conjugated to different fluorescent dyes was used for internalization studies. Comparison of uptake efficiencies revealed that CPPs of the new generation are in contrast to MPG-peptides, nearly unable to internalize the noncovalently formed complexes with trastuzumab. Interestingly, the fluorescent derivative of the crotamine fragment was mainly observed in a subpopulation of breast cancer cells, whereas it was homogenously distributed in fibrosarcoma, colon cancer, and noncancerous endothelia cells. Thus, the fluorescent crotamine fragment reported herein is a potent theranostic tool for image-guided applications. This peptide can be used to pinpoint the level of heterogeneity present within tumors and aid in the generation of therapeutics that target heterogenic subpopulations.

The phenotype of target pancreatic cancer cells influences cell death by magnetic hyperthermia with nanoparticles carrying gemicitabine and the pseudo-peptide NucAnt.

In this paper we show that conjugation of magnetic nanoparticles (MNPs) with Gemcitabine and/or NucAnt (N6L) fostered their internalization into pancreatic tumor cells and that the coupling procedure did not alter the cytotoxic potential of the drugs. By treating tumor cells (BxPC3 and PANC-1) with the conjugated MNPs and magnetic hyperthermia (43 °C, 60 min), cell death was observed. The two pancreatic tumor cell lines showed different reactions against the combined therapy according to their intrinsic sensitivity against Gemcitabine (cell death, ROS production, ability to activate ERK 1/2 and JNK). Finally, tumors (e.g. 3 mL) could be effectively treated by using almost 4.2 × 105 times lower Gemcitabine doses compared to conventional therapies. Our data show that this combinatorial therapy might well play an important role in certain cell phenotypes with low readiness of ROS production. This would be of great significance in distinctly optimizing local pancreatic tumor treatments.

Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes.

BACKGROUND: Endoglin (CD105) is overexpressed on tumor cells and tumor vasculatures, making it a potential target for diagnostic imaging and therapy of different neoplasms. Therefore, studies on nanocarrier systems designed for endoglin-directed diagnostic and drug delivery purposes would expose the feasibility of targeting endoglin with therapeutics. METHODS: Liposomes carrying high concentrations of a near-infrared fluorescent dye in the aqueous interior were prepared by the lipid film hydration and extrusion procedure, then conjugated to single chain antibody fragments either selective for murine endoglin (termed mEnd-IL) or directed towards human endoglin (termed hEnd-IL). A combination of Dynamic Light Scattering, electron microscopy, cell binding and uptake assays, confocal microscopy and in vivo fluorescence imaging of mice bearing xenografted human breast cancer and human fibrosarcoma models were implemented to elucidate the potentials of the liposomes. RESULTS: The mEnd-IL and hEnd-IL were highly selective for the respective murine- and human endoglin expressing cells in vitro and in vivo. Hence, the hEnd-IL bound distinctly to the tumor cells and enabled suitable fluorescence imaging of the tumors, whereas the mEnd-IL bound the tumor vasculature, but also to the liver, kidney and lung vasculature of mice. CONCLUSIONS: The work highlights key differences between targeting vascular (murine) and neoplastic (human) endoglin in animal studies, and suggests that the hEnd-IL can serve as a delivery system that targets human endoglin overexpressed in pathological conditions. GENERAL SIGNIFICANCE: The endoglin-targeting liposomes presented herewith represent strategic tools for the future implementation of endoglin-directed neoplastic and anti-angiogenic therapies.

A fast and effective determination of the biodistribution and subcellular localization of fluorescent immunoliposomes in freshly excised animal organs.

BACKGROUND: Preclinical research implementing fluorescence-based approaches is inevitable for drug discovery and technology. For example, a variety of contrast agents developed for biomedical imaging are usually evaluated in cell systems and animal models based on their conjugation to fluorescent dyes. Biodistribution studies of excised organs are often performed by macroscopic imaging, whereas the subcellular localization though vital, is often neglected or further validated by histological procedures. Available systems used to define the subcellular biodistribution of contrast agents such as intravital microscopes or ex vivo histological analysis are expensive and not affordable by the majority of researchers, or encompass tedious and time consuming steps that may modify the contrast agents and falsify the results. Thus, affordable and more reliable approaches to study the biodistribution of contrast agents are required. We developed fluorescent immunoliposomes specific for human fibroblast activation protein and murine endoglin, and used macroscopic fluorescence imaging and confocal microscopy to determine their biodistribution and subcellular localization in freshly excised mice organs at different time points post intravenous injection. RESULTS: Near infrared fluorescence macroscopic imaging revealed key differences in the biodistribution of the respective immunoliposomes at different time points post injection, which correlated to the first-pass effect as well as the binding of the probes to molecular targets within the mice organs. Thus, a higher accumulation and longer retention of the murine endoglin immunoliposomes was seen in the lungs, liver and kidneys than the FAP specific immunoliposomes. Confocal microscopy showed that tissue autofluorescence enables detection of organ morphology and cellular components within freshly excised, non-processed organs, and that fluorescent probes with absorption and emission maxima beyond the tissue autofluorescence range can be easily distinguished. Hence, the endoglin targeting immunoliposomes retained in some organs could be detected in the vascular endothelia cells of the organs. CONCLUSIONS: The underlying work represents a quick, effective and more reliable setup to validate the macroscopic and subcellular biodistribution of contrast agents in freshly excised animal organs. The approach will be highly beneficial to many researchers involved in nanodrug design or in fluorescence-based studies on disease pathogenesis.

Internalization of Near-Infrared Fluorescently Labeled Activatable Cell-Penetrating Peptide and of Proteins into Human Fibrosarcoma Cell Line HT-1080.

The internalization of near-infrared fluorescently labeled cargos into living cells and tissues allows a highly sensitive detection without interference from skin, porphins or other fluorescent cell and tissue compounds. In this study, the uptake of labeled bovine serum albumin and an antibody, into fibrosarcoma (HT-1080) cells was triggered by the formation of non-covalent complexes with different cell-penetrating peptides; uptake efficiency and intracellular localization were determined. To improve selectivity of internalization into tumor cells, a fluorescent activatable cell-penetrating peptide (ACPP) was synthesized and functionally characterized. This 25-mer peptide was designed to be activatable by Matrix-Metallo-Proteases (MMPs). Its uptake selectivity was estimated using cells with different MMP activities.

Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery.

INTRODUCTION: Tumor cells can effectively be killed by heat, e.g. by using magnetic hyperthermia. The main challenge in the field, however, is the generation of therapeutic temperatures selectively in the whole tumor region. We aimed to improve magnetic hyperthermia of breast cancer by using innovative nanoparticles which display a high heating potential and are functionalized with a cell internalization and a chemotherapeutic agent to increase cell death. METHODS: The superparamagnetic iron oxide nanoparticles (MF66) were electrostatically functionalized with either Nucant multivalent pseudopeptide (N6L; MF66-N6L), doxorubicin (DOX; MF66-DOX) or both (MF66-N6LDOX). Their cytotoxic potential was assessed in a breast adenocarcinoma cell line MDA-MB-231. Therapeutic efficacy was analyzed on subcutaneous MDA-MB-231 tumor bearing female athymic nude mice. RESULTS: All nanoparticle variants showed an excellent heating potential around 500 W/g Fe in the alternating magnetic field (AMF, conditions: H=15.4 kA/m, f=435 kHz). We could show a gradual inter- and intracellular release of the ligands, and nanoparticle uptake in cells was increased by the N6L functionalization. MF66-DOX and MF66-N6LDOX in combination with hyperthermia were more cytotoxic to breast cancer cells than the respective free ligands. We observed a substantial tumor growth inhibition (to 40% of the initial tumor volume, complete tumor regression in many cases) after intratumoral injection of the nanoparticles in vivo. The proliferative activity of the remaining tumor tissue was distinctly reduced. CONCLUSION: The therapeutic effects of breast cancer magnetic hyperthermia could be strongly enhanced by the combination of MF66 functionalized with N6L and DOX and magnetic hyperthermia. Our approach combines two ways of tumor cell killing (magnetic hyperthermia and chemotherapy) and represents a straightforward strategy for translation into the clinical practice when injecting nanoparticles intratumorally.

Tangential Flow Ultrafiltration Allows Purification and Concentration of Lauric Acid-/Albumin-Coated Particles for Improved Magnetic Treatment.

Superparamagnetic iron oxide nanoparticles (SPIONs) are frequently used for drug targeting, hyperthermia and other biomedical purposes. Recently, we have reported the synthesis of lauric acid-/albumin-coated iron oxide nanoparticles SEON(LA-BSA), which were synthesized using excess albumin. For optimization of magnetic treatment applications, SPION suspensions need to be purified of excess surfactant and concentrated. Conventional methods for the purification and concentration of such ferrofluids often involve high shear stress and low purification rates for macromolecules, like albumin. In this work, removal of albumin by low shear stress tangential ultrafiltration and its influence on SEON(LA-BSA) particles was studied. Hydrodynamic size, surface properties and, consequently, colloidal stability of the nanoparticles remained unchanged by filtration or concentration up to four-fold (v/v). Thereby, the saturation magnetization of the suspension can be increased from 446.5 A/m up to 1667.9 A/m. In vitro analysis revealed that cellular uptake of SEON(LA-BSA) changed only marginally. The specific absorption rate (SAR) was not greatly affected by concentration. In contrast, the maximum temperature Tmax in magnetic hyperthermia is greatly enhanced from 44.4 °C up to 64.9 °C by the concentration of the particles up to 16.9 mg/mL total iron. Taken together, tangential ultrafiltration is feasible for purifying and concentrating complex hybrid coated SPION suspensions without negatively influencing specific particle characteristics. This enhances their potential for magnetic treatment.

High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature dosages in the tumor area.

PURPOSE: Tumor cells can be effectively inactivated by heating mediated by magnetic nanoparticles. However, optimized nanomaterials to supply thermal stress inside the tumor remain to be identified. The present study investigates the therapeutic effects of magnetic hyperthermia induced by superparamagnetic iron oxide nanoparticles on breast (MDA-MB-231) and pancreatic cancer (BxPC-3) xenografts in mice in vivo. METHODS: Superparamagnetic iron oxide nanoparticles, synthesized either via an aqueous (MF66; average core size 12 nm) or an organic route (OD15; average core size 15 nm) are analyzed in terms of their specific absorption rate (SAR), cell uptake and their effectivity in in vivo hyperthermia treatment. RESULTS: Exceptionally high SAR values ranging from 658 ± 53 W*gFe (-1) for OD15 up to 900 ± 22 W*gFe (-1) for MF66 were determined in an alternating magnetic field (AMF, H = 15.4 kA*m(-1) (19 mT), f = 435 kHz). Conversion of SAR values into system-independent intrinsic loss power (ILP, 6.4 ± 0.5 nH*m(2)*kg(-1) (OD15) and 8.7 ± 0.2 nH*m(2)*kg(-1) (MF66)) confirmed the markedly high heating potential compared to recently published data. Magnetic hyperthermia after intratumoral nanoparticle injection results in dramatically reduced tumor volume in both cancer models, although the applied temperature dosages measured as CEM43T90 (cumulative equivalent minutes at 43°C) are only between 1 and 24 min. Histological analysis of magnetic hyperthermia treated tumor tissue exhibit alterations in cell viability (apoptosis and necrosis) and show a decreased cell proliferation. CONCLUSIONS: Concluding, the studied magnetic nanoparticles lead to extensive cell death in human tumor xenografts and are considered suitable platforms for future hyperthermic studies.

Identification of impactful imaging biomarker: Clinical applications for breast and prostate carcinoma. / Ermittlung aussagekräftiger Bildgebungsbiomarker: Klinische Anwendungen bei Mamma- und Prostatakarzinomen.

BACKGROUND: Imaging biomarkers are quantitative parameters from imaging modalities, which are collected noninvasively, allow conclusions about physiological and pathophysiological processes, and may consist of single (monoparametric) or multiple parameters (bi- or multiparametric). METHOD: This review aims to present the state of the art for the quantification of multimodal and multiparametric imaging biomarkers. Here, the use of biomarkers using artificial intelligence will be addressed and the clinical application of imaging biomarkers in breast and prostate cancers will be explained. For the preparation of the review article, an extensive literature search was performed based on Pubmed, Web of Science and Google Scholar. The results were evaluated and discussed for consistency and generality. RESULTS AND CONCLUSION: Different imaging biomarkers (multiparametric) are quantified based on the use of complementary imaging modalities (multimodal) from radiology, nuclear medicine, or hybrid imaging. From these techniques, parameters are determined at the morphological (e. g., size), functional (e. g., vascularization or diffusion), metabolic (e. g., glucose metabolism), or molecular (e. g., expression of prostate specific membrane antigen, PSMA) level. The integration and weighting of imaging biomarkers are increasingly being performed with artificial intelligence, using machine learning algorithms. In this way, the clinical application of imaging biomarkers is increasing, as illustrated by the diagnosis of breast and prostate cancers. KEY POINTS: · Imaging biomarkers are quantitative parameters to detect physiological and pathophysiological processes.. · Imaging biomarkers from multimodality and multiparametric imaging are integrated using artificial intelligence algorithms.. · Quantitative imaging parameters are a fundamental component of diagnostics for all tumor entities, such as for mammary and prostate carcinomas.. CITATION FORMAT: · Bäuerle T, Dietzel M, Pinker K et al. Identification of impactful imaging biomarker: Clinical applications for breast and prostate carcinoma. Fortschr Röntgenstr 2024; 196: 354 - 362.

Motivation and success factors in radiological research in Germany - results of a survey by the Methodology and Research Working Group of the German Radiological Society. / Motivation und Erfolgsfaktoren in der radiologischen Forschung in Deutschland ­ Ergebnisse einer Umfrage der AG Methodik und Forschung.

Investigation of motivation and identification of success factors in radiology research in Germany.Using a German online survey (54 questions, period: 3.5 months), demographic aspects, intrinsic and extrinsic success characteristics, as well as personal and organizational success factors were surveyed based on a career success model. The survey results were reported descriptively. The correlations between success factors and success characteristics were examined using linear, binary-logistic, and multinomial regression models.176 people (164 academically active, 10 not academically active) answered the survey. Most participants (80%, 139/174) worked at a university hospital. 32% had privatdozent or professor as their highest academic title (56/173). The researchers' main motivation was intrinsic interest in research (55%, 89/163), followed by a desire to increase their own career opportunities (25%, 41/163). The following were identified as factors for intrinsic success: i) support from department management (estimate=ß=0.26, p<0.001), ii) good work-life balance (ß=0.37, p<0.001), and iii) the willingness to pursue science even after reaching the career goal (ß=0.16, p<0.016). Relevant factors for extrinsic scientific success were mentoring, protected research time, and activities in professional societies.Researchers in German radiology are mainly intrinsically motivated. Factors known from the literature that determine intrinsic and extrinsic scientific success were confirmed in this study. Knowledge of these factors allows targeted systematic support and could thus increase scientific success in German radiology. · Main motivation for German radiology research is intrinsic interest, followed by career opportunities.. · Factors for intrinsic scientific success are good work-life balance and support by department management.. · Factors for extrinsic scientific success are mentoring, activities in professional societies, and protected research time.. · Wegner F, Heinrichs H, Stahlmann K et al. Motivation and success factors in radiological research in Germany - results of a survey by the Methodology and Research Working Group of the German Radiological Society. Fortschr Röntgenstr 2024; DOI 10.1055/a-2350-0023.

Liposomal encapsulation of a near-infrared fluorophore enhances fluorescence quenching and reliable whole body optical imaging upon activation in vivo.

In the past decade, there has been significant progress in the development of water soluble near-infrared fluorochromes for use in a wide range of imaging applications. Fluorochromes with high photo and thermal stability, sensitivity, adequate pharmacological properties and absorption/emission maxima within the near infrared window (650-900 nm) are highly desired for in vivo imaging, since biological tissues show very low absorption and auto-fluorescence at this spectrum window. Taking these properties into consideration, a myriad of promising near infrared fluorescent probes has been developed recently. However, a hallmark of most of these probes is a rapid clearance in vivo, which hampers their application. It is hypothesized that encapsulation of the near infrared fluorescent dye DY-676-COOH, which undergoes fluorescence quenching at high concentrations, in the aqueous interior of liposomes will result in protection and fluorescence quenching, which upon degradation by phagocytes in vivo will lead to fluorescence activation and enable imaging of inflammation. Liposomes prepared with high concentrations of DY-676-COOH reveal strong fluorescence quenching. It is demonstrated that the non-targeted PEGylated fluorescence-activatable liposomes are taken up predominantly by phagocytosis and degraded in lysosomes. Furthermore, in zymosan-induced edema models in mice, the liposomes are taken up by monocytes and macrophages which migrate to the sites of inflammation. Opposed to free DY-676-COOH, prolonged stability and retention of liposomal-DY-676-COOH is reflected in a significant increase in fluorescence intensity of edema. Thus, protected delivery and fluorescence quenching make the DY-676-COOH-loaded liposomes a highly promising contrast agent for in vivo optical imaging of inflammatory diseases.

Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alzheimer's disease brains.

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC50=19nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72h post fertilization.

Hepatic excretory function in sepsis: implications from biophotonic analysis of transcellular xenobiotic transport in a rodent model.

INTRODUCTION: Hepatobiliary elimination of endo- and xenobiotics is affected by different variables including hepatic perfusion, hepatocellular energy state and functional integrity of transporter proteins, all of which are altered during sepsis. A particular impairment of hepatocellular transport at the canalicular pole resulting in an accumulation of potentially hepatotoxic compounds would have major implications for critical care pharmacology and diagnostics. METHODS: Hepatic transcellular transport, that is, uptake and hepatobiliary excretion, was studied in a rodent model of severe polymicrobial sepsis by two different biophotonic techniques to obtain insights into the handling of potentially toxic endo- and xenobiotics in sepsis. Direct and indirect in vivo imaging of the liver was performed by intravital multifluorescence microscopy and non-invasive whole-body near-infrared (NIRF) imaging after administration of two different, primarily hepatobiliary excreted xenobiotics, the organic anionic dyes indocyanine green (ICG) and DY635. Subsequent quantitative data analysis enabled assessment of hepatic uptake and fate of these model substrates under conditions of sepsis. RESULTS: Fifteen hours after sepsis induction, animals displayed clinical and laboratory signs of multiple organ dysfunction, including moderate liver injury, cholestasis and an impairment of sinusoidal perfusion. With respect to hepatocellular transport of both dyes, excretion into bile was significantly delayed for both dyes and resulted in net accumulation of potentially cytotoxic xenobiotics in the liver parenchyma (for example, specific dye fluorescence in liver at 30 minutes in sham versus sepsis: ICG: 75% versus 89%; DY635 20% versus 40% of maximum fluorescence; P<0.05). Transcutaneous assessment of ICG fluorescence by whole body NIRF imaging revealed a significant increase of ICG fluorescence from the 30th minute on in the bowel region of the abdomen in sham but not in septic animals, confirming a sepsis-associated failure of canalicular excretion. CONCLUSIONS: Hepatocytes accumulate organic anions under conditions of sepsis-associated organ dysfunction. These results have potential implications for monitoring liver function, critical care pharmacology and the understanding of drug-induced liver injury in the critically ill.

In vivo applications of magnetic nanoparticle hyperthermia.

Hyperthermia is considered to be a promising tool for the treatment of tumours. Intensive research activities reveal a distinct impact not only on the cellular level but also on tumour physiology which favours the combination with the classical oncologic modalities radio- and chemotherapy. Different techniques have been established so far. Among them, magnetic hyperthermia exploits the intrinsic magnetic properties of iron oxide nanoparticles (magnetite and maghemite) which induce heating during the exposure to an alternating magnetic field. Beyond the advantage that heating is generated within the tumour and not from outside the body, the amounts of magnetic material and their intratumoral distribution patterns are key factors determining the therapeutic outcome. They can be influenced by the use of different application routes, which will be discussed in this paper.

Intravenous Injection of PEI-Decorated Iron Oxide Nanoparticles Impacts NF-kappaB Protein Expression in Immunologically Stressed Mice.

Nanoparticle-based formulations are considered valuable tools for diagnostic and treatment purposes. The surface decoration of nanoparticles with polyethyleneimine (PEI) is often used to enhance their targeting and functional properties. Here, we aimed at addressing the long-term fate in vivo and the potential "off-target" effects of PEI decorated iron oxide nanoparticles (PEI-MNPs) in individuals with low-grade and persistent systemic inflammation. For this purpose, we synthesized PEI-MNPs (core-shell method, PEI coating under high pressure homogenization). Further on, we induced a low-grade and persistent inflammation in mice through regular subcutaneous injection of pathogen-associated molecular patterns (PAMPs, from zymosan). PEI-MNPs were injected intravenously. Up to 7 weeks thereafter, the blood parameters were determined via automated fluorescence flow cytometry, animals were euthanized, and the organs analyzed for iron contents (atomic absorption spectrometry) and for expression of NF-κB associated proteins (p65, IκBα, p105/50, p100/52, COX-2, Bcl-2, SDS-PAGE and Western blotting). We observed that the PEI-MNPs had a diameter of 136 nm and a zeta-potential 56.9 mV. After injection in mice, the blood parameters were modified and the iron levels were increased in different organs. Moreover, the liver of animals showed an increased protein expression of canonical NF-κB signaling pathway members early after PEI-MNP application, whereas at the later post-observation time, members of the non-canonical signaling pathway were prominent. We conclude that the synergistic effect between PEI-MNPs and the low-grade and persistent inflammatory state is mainly due to the hepatocytes sensing infection (PAMPs), to immune responses resulting from the intracellular metabolism of the uptaken PEI-MNPs, or to hepatocyte and immune cell communications. Therefore, we suggest a careful assessment of the safety and toxicity of PEI-MNP-based carriers for gene therapy, chemotherapy, and other medical applications not only in healthy individuals but also in those suffering from chronic inflammation.

Hyperthermia Influences the Secretion Signature of Tumor Cells and Affects Endothelial Cell Sprouting.

Tumors are a highly heterogeneous mass of tissue showing distinct therapy responses. In particular, the therapeutic outcome of tumor hyperthermia treatments has been inconsistent, presumably due to tumor versus endothelial cell cross-talks related to the treatment temperature and the tumor tissue environment. Here, we investigated the impact of the average or strong hyperthermic treatment (43 °C or 47 °C for 1 h) of the human pancreatic adenocarcinoma cell line (PANC-1 and BxPC-3) on endothelial cells (HUVECs) under post-treatment normoxic or hypoxic conditions. Immediately after the hyperthermia treatment, the distinct repression of secreted pro-angiogenic factors (e.g., VEGF, PDGF-AA, PDGF-BB, M-CSF), intracellular HIF-1α and the enhanced phosphorylation of ERK1/2 in tumor cells were detectable (particularly for strong hyperthermia, 2D cell monolayers). Notably, there was a significant increase in endothelial sprouting when 3D self-organized pancreatic cancer cells were treated with strong hyperthermia and the post-treatment conditions were hypoxic. Interestingly, for the used treatment temperatures, the intracellular HIF-1α accumulation in tumor cells seems to play a role in MAPK/ERK activation and mediator secretion (e.g., VEGF, PDGF-AA, Angiopoietin-2), as shown by inhibition experiments. Taken together, the hyperthermia of pancreatic adenocarcinoma cells in vitro impacts endothelial cells under defined environmental conditions (cell-to-cell contact, oxygen status, treatment temperature), whereby HIF-1α and VEGF secretion play a role in a complex context. Our observations could be exploited for the hyperthermic treatment of pancreatic cancer in the future.