Magnetic hyperthermia can cause localized thermal eradication of several solid cancers. However, a localized and homogenous deposition of high concentrations of magnetic nanomaterials into the tumor ...stroma and tumor cells is mostly required. Poorly responsive cancers such as the pancreatic adenocarcinomas are hallmarked by a rigid stroma and poor perfusion to therapeutics and nanomaterials. Hence, approaches that enhance the infiltration of magnetic nanofluids into the tumor stroma convey potentials to improve thermal tumor therapy. We studied the influence of the matrix-modulating enzymes hyaluronidase and collagenase on the uptake of magnetic nanoparticles by pancreatic cancer cells and 3D spheroids thereof, and the overall impact on magnetic heating and cell death. Furthermore, we validated the effect of hyaluronidase on magnetic hyperthermia treatment of heterotopic pancreatic cancer models in mice. Treatment of cultured cells with the enzymes caused higher uptake of magnetic nanoparticles (MNP) as compared to nontreated cells. For example, hyaluronidase caused a 28% increase in iron deposits per cell. Consequently, the thermal doses (cumulative equivalent minutes at 43 °C, CEM43) increased by 15-23% as compared to heat dose achieved for cells treated with magnetic hyperthermia without using enzymes. Likewise, heat-induced cell death increased. In in vivo studies, hyaluronidase-enhanced infiltration and distribution of the nanoparticles in the tumors resulted in moderate heating levels (CEM43 of 128 min as compared to 479 min) and a slower, but persistent decrease in tumor volumes over time after treatment, as compared to comparable treatment without hyaluronidase. The results indicate that hyaluronidase, in particular, improves the infiltration of magnetic nanoparticles into pancreatic cancer models, impacts their thermal treatment and cell depletion, and hence, will contribute immensely in the fight against pancreatic and many other adenocarcinomas.
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.
Liposomes encapsulated with high concentrations of a near IR fluorescent dye reveal high fluorescence quenching. These non‐targeted PEGylated fluorescence‐activatable liposomes lead to the release of the dye and an increase in fluorescence. In zymosan‐induced edema models, the liposomes are taken up by monocytes and macrophages, which migrate to the sites of inflammation and, upon activation, enhance prolonged and reliable in vivo near IR fluorescence imaging.
Due to their high X-ray attenuation, gold nanoparticles (GNPs) emerged as preclinical contrast agents by giving high vasculature contrast. For this reason, GNPs are regularly applied for computed ...tomography (CT) imaging of tumors but not for the visualization of inflammation. The aim of this study was to evaluate the biocompatibility and applicability of preclinical GNPs (AuroVist™) of two different sizes (1.9 nm and 15 nm) for the detection of inflammation-associated phagocytes in early-stage edema. Both GNP variants were stable under in vitro conditions and achieved high micro-CT (mCT) contrast after embedment into agarose. Fifteen-nanometer GNPs were detected after uptake into macrophages via mCT imaging exhibiting higher X-ray contrast than cells treated with 1.9 nm GNPs and untreated ones. Both 1.9 nm and 15 nm GNPs exhibited good biocompatibility on murine macrophages according to ATP and cellular dehydrogenase levels. Reactive oxygen species levels produced by phagocytic cells decreased significantly (P≤0.05) after co-incubation with GNPs regardless of the size of the nanoparticle (NP) in comparison to untreated control cells. In vivo mCT studies of inflammation imaging revealed that GNPs with a diameter of 15 nm accumulated within subcutaneous edema 2 hours after injection with a maximum signaling 8 hours postinjection and could be detected up to 48 hours within the edema region. In contrast, 1.9 nm GNPs were not shown to accumulate at the site of the inflammation region and were mostly excreted via the renal system 2-4 hours after injection. In conclusion, our study demonstrated that both GNP variants (1.9 nm and 15 nm) were stable and biocompatible under in vitro conditions. However, only 15 nm NPs have the potential as contrast agent for phagocyte labeling and applications in CT imaging of inflammation on a cellular level.
All over the world, different types of nanomaterials with a diversified spectrum of applications are designed and developed, especially in the field of nanomedicine. The great variety of ...nanoparticles (NPs), in vitro test systems and cell lines led to a vast amount of publications with conflicting data. To identify the decisive principles of these variabilities, we conducted an intercomparison study of collaborating laboratories within the German DFG Priority Program SPP1313, using well-defined experimental parameters and well-characterized NPs. The participants analyzed the in vitro biocompatibility of silica and polymer NPs on human hepatoma HepG2 cells. Nanoparticle mediated effects on cell metabolism, internalization, and inflammation were measured. All laboratories showed that both nanoparticle formulations were internalized and had a low cytotoxicity profile. Interestingly, small variations in nanoparticle preparation, cell handling and the type of culture slide influenced the nanoparticle stability and the outcomes of cell assays. The round robin test demonstrated the importance of the use of clearly defined and characterized NPs and parameters for reproducible results across laboratories. Comparative analyses of in vitro screening methods performed in multiple laboratories are absolutely essential to establish robust standard operation procedure as a prerequisite for sound hazard assessment of nanomaterials.
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.
Zusammenfassung
Die optische Bildgebung spielte immer schon eine zentrale Rolle bei der Aufklärung von biologischen und physiologischen Mechanismen in der modernen Biologie und Medizin. Ausgehend von ...den guten Erfahrungen in der Lichtmikroskopie, hat man in jüngster Zeit anspruchsvolle meso- und makroskopische optische Bildgebungssysteme geschaffen. Alle optischen Bildgebungsverfahren zeichnen sich durch hohe Benutzerfreundlichkeit und Empfindlichkeit aus, sie sind mit verhältnismäßig geringen Kosten verbunden und kommen ohne Radioaktivität aus. Ihre klinische Leistungsfähigkeit wird in der intraoperativen bildgebenden Darstellung des zu entfernenden Gewebeareals und in der Radiotracer-freien Diagnostik von Erkrankungen in Körperarealen mit guter Zugänglichkeit für Licht gesehen. Aus pathomorphologischer Sicht hat man sich dabei insbesondere auf die Darstellung von Tumoren und Entzündungen fokussiert. Im Sinne eines guten Signal-Hintergrund-Verhältnisses und eines verbesserten Informationsgewinns aus größeren Gewebetiefen ist die bildgebende Detektion von Fluoreszenzfarbstoffen mit Emissionsmerkmalen im nahen Infrarotbereich des Spektrums günstig. Eine große Herausforderung sind allerdings die vielseitigen Photoneninteraktionen mit dem Gewebe. Die bisherigen Forschungs- und Entwicklungsarbeiten haben verschiedene optische In-vivo-Bildgebungsverfahren hervorgebracht, die teilweise noch im experimentellen Stadium sind (z. B. fluoreszenzvermittelte Tomographie, multispektrale In-vivo-Bildgebung, Biolumineszenz, Raman-Spektroskopie etc.), während andere schon den Einzug in die klinische Situation vollzogen haben (z. B. Fluoreszenz-Reflexionsbildgebung, optoakustische Bildgebung). Die wichtigsten optischen Verfahren werden in diesem Übersichtsartikel vorgestellt.
Fluorochrome-labelled iron oxide magnetic nanoparticles (MNP) have been of great help in elucidating biological processes. Here, we used dually-fluorochrome-labelled MNP and studied to what extent ...fluorescence detection could reflect their fate in living animals. One day after application in mice (200 µmol Fe/kg body weight), the fluorescence of the dye attached to the core (DY-730) was very prominent and in agreement with the increase of iron in the liver and spleen of mice, but inconspicuous at time points thereafter. We attribute this fluorescence behavior to early degradation processes of the MNP´s core in the cellular lysosomal compartment. In contrast, the fluorescence of the dye DY-555 stuck to the PEG coating was not detectable in vivo. In summary, labelling of MNP with dyes at their metallic core could be of help when detecting first incidences of MNP biodegradation in vivo, as opposed to dyes attached to the MNP coating.
Liposomes are biocompatible nanocarriers with promising features for targeted delivery of contrast agents and drugs into the tumor microenvironment, for imaging and therapy purposes. Liposome-based ...simultaneous targeting of tumor associated fibroblast and the vasculature is promising, but the heterogeneity of tumors entails a thorough validation of suitable markers for targeted delivery. Thus, we elucidated the potential of bispecific liposomes targeting the fibroblast activation protein (FAP) on tumor stromal fibroblasts, together with endoglin which is overexpressed on tumor neovascular cells and some neoplastic cells. Fluorescence-quenched liposomes were prepared by hydrating a lipid film with a high concentration of the self-quenching near-infrared fluorescent dye, DY-676-COOH, to enable fluorescence detection exclusively upon liposomal degradation and subsequent activation. A non-quenched green fluorescent phospholipid was embedded in the liposomal surface to fluorescence-track intact liposomes. FAP- and murine endoglin-specific single chain antibody fragments were coupled to the liposomal surface, and the liposomal potentials validated in tumor cells and mice models. The bispecific liposomes revealed strong fluorescence quenching, activatability, and selectivity for target cells and delivered the encapsulated dye selectively into tumor vessels and tumor associated fibroblasts in xenografted mice models and enabled their fluorescence imaging. Furthermore, detection of swollen lymph nodes during intra-operative simulations was possible. Thus, the bispecific liposomes have potentials for targeted delivery into the tumor microenvironment and for image-guided surgery.
Liposomes represent suitable tools for the diagnosis and treatment of a variety of diseases, including cancers. To study the role of the human epidermal growth factor receptor 2 (HER2) as target in ...cancer imaging and image-guided deliveries, liposomes were encapsulated with an intrinsically quenched concentration of a near-infrared fluorescent dye in their aqueous interior. This resulted in quenched liposomes (termed LipQ), that were fluorescent exclusively upon degradation, dye release, and activation. The liposomes carried an always-on green fluorescent phospholipid in the lipid layer to enable tracking of intact liposomes. Additionally, they were functionalized with single-chain antibody fragments directed to fibroblast activation protein (FAP), a marker of stromal fibroblasts of most epithelial cancers, and to HER2, whose overexpression in 20–30% of all breast cancers and many other cancer types is associated with a poor treatment outcome and relapse. We show that both monospecific (HER2-IL) and bispecific (Bi-FAP/HER2-IL) formulations are quenched and undergo HER2-dependent rapid uptake and cargo release in cultured target cells and tumor models in mice. Thereby, tumor fluorescence was retained in whole-body NIRF imaging for 32–48 h post-injection. Opposed to cell culture studies, Bi-FAP/HER2-IL-based live confocal microscopy of a high HER2-expressing tumor revealed nuclear delivery of the encapsulated dye. Thus, the liposomes have potentials for image-guided nuclear delivery of therapeutics, and also for intraoperative delineation of tumors, metastasis, and tumor margins.
To assess relevant parameters for the minimally invasive elimination of breast tumors by using a selective application of magnetite and exposure of the breast to an alternating magnetic field.
The ...specific absorption rate (SAR) of different magnetite samples was determined calorimetrically. Temperature elevations based on magnetite mass (7-112 mg) and magnetic field amplitude (1.2-6.5 kA/m frequency, 400 kHz) were investigated by using human breast tissue. Parameter combinations (21 mg +/- 9 SD, 242-second magnetic field exposure, 6.5-kA/m amplitude) were tested in 10 immunodeficient mice bearing human adenocarcinomas (MX-1 cells). Histologic sections of heated tumor tissue were analyzed.
SAR data of different magnetite particle types ranged from 3 to 211 W/g. Temperature elevation (DeltaT) as a function of the magnetite mass increased linearly up to 28 mg; at higher masses, a saturation of DeltaT was observed at nearly 88 degrees C. The dependence of DeltaT on magnetic field amplitude (H) revealed a third-order power law: DeltaT = 0.26 degrees C/(kA/m)(3). H(3), with r(2) = 0.95. A mean temperature of 71 degrees C +/- 8 was recorded in the tumor region at the end of magnetic field exposure of the mice. Typical macroscopic findings included tumor shrinkage after heating. Histologically nuclear degenerations were observed in heated malignant cells.
Magnetic heating of breast tumors is a promising technique for future interventional radiologic treatments.
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