Hemocompatibility and Biomedical Potential of Poly(Gallic Acid) Coated Iron Oxide Nanoparticles for Theranostic Use

Polyacid covered core-shell iron oxide nanoparticles were designed for potential use in biomedicine with special attention to theranostics - magnetic resonance imaging (MRI), magnetic hyperthermia and magnetic drug targeting. The magnetite nanoparticles coated with a gallic acid shell polymerized in situ on the nanoparticle surface (PGA@MNPs) were tested for hemocompatibility in blood, sedimentation rate, blood smear and blood cell viability experiments and for antioxidant capacity in Jurkat cells in the presence of H2O2 as reactive oxygen species. No signs of interaction of the nanoparticles with whole blood cells were found. In addition, the PGA@MNPs reduced significantly the oxidative stress mediated by H2O2 supporting earlier findings of MTT tests, namely, the improvement of cell viability in their presence. The in vitro tests revealed that PGA@MNPs are not only biocompatible but also bioactive. Preliminary experiments revealed that the nanoparticles are especially efficient MRI and magnetic hyperthermia agents. The r2 relaxivity was found to be one of the highest among published values (387 mM-1s-1) and they possess a relatively significant specific absorption rate (SAR) value of 11 W/g magnetite

Different storage conditions influence biocompatibility and physicochemical properties of iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted increasing attention in many biomedical fields. In magnetic drug targeting SPIONs are injected into a tumour supplying artery and accumulated inside the tumour with a magnet. The effectiveness of this therapy is thus dependent on magnetic properties, stability and biocompatibility of the particles. A good knowledge of the effect of storage conditions on those parameters is of utmost importance for the translation of the therapy concept into the clinic and for reproducibility in preclinical studies. Here, core shell SPIONs with a hybrid coating consisting of lauric acid and albumin were stored at different temperatures from 4 to 45 °C over twelve weeks and periodically tested for their physicochemical properties over time. Surprisingly, even at the highest storage temperature we did not observe denaturation of the protein or colloidal instability. However, the saturation magnetisation decreased by maximally 28.8% with clear correlation to time and storage temperature. Furthermore, the biocompatibility was clearly affected, as cellular uptake of the SPIONs into human T-lymphoma cells was crucially dependent on the storage conditions. Taken together, the results show that the particle properties undergo significant changes over time depending on the way they are stored

Functionalized Superparamagnetic Iron Oxide Nanoparticles (SPIONs) as Platform for the Targeted Multimodal Tumor Therapy

Standard cancer treatments involve surgery, radiotherapy, chemotherapy, and immunotherapy. In clinical practice, the respective drugs are applied orally or intravenously leading to their systemic circulation in the whole organism. For chemotherapeutics or immune modulatory agents, severe side effects such as immune depression or autoimmunity can occur. At the same time the intratumoral drug doses are often too low for effective cancer therapy. Since monotherapies frequently cannot cure cancer, due to their synergistic effects multimodal therapy concepts are applied to enhance treatment efficacy. The targeted delivery of drugs to the tumor by employment of functionalized nanoparticles might be a promising solution to overcome these challenges. For multimodal therapy concepts and individualized patient care nanoparticle platforms can be functionalized with compounds from various therapeutic classes (e.g. radiosensitizers, phototoxic drugs, chemotherapeutics, immune modulators). Superparamagnetic iron oxide nanoparticles (SPIONs) as drug transporters can add further functionalities, such as guidance or heating by external magnetic fields (Magnetic Drug Targeting or Magnetic Hyperthermia), and imaging-controlled therapy (Magnetic Resonance Imaging)

Inert Coats of Magnetic Nanoparticles Prevent Formation of Occlusive Intravascular Co-aggregates With Neutrophil Extracellular Traps

If foreign particles enter the human body, the immune system offers several mechanisms of response. Neutrophils forming the first line of the immune defense either remove pathogens by phagocytosis, inactivate them by degranulation or release of reactive oxygen species or immobilize them by the release of chromatin decorated with the granular proteins from cytoplasm as neutrophil extracellular traps (NETs). Besides viable microbes like fungi, bacteria or viruses, also several sterile inorganic particles including nanoparticles reportedly activate NET formation. The physicochemical nanoparticle characteristics fostering NET formation are still elusive. Here we show that agglomerations of non-stabilized superparamagnetic iron oxide nanoparticles (SPIONs) induce NET formation by isolated human neutrophils, in whole blood experiments under static and dynamic conditions as well as in vivo. Stabilization of nanoparticles with biocompatible layers of either human serum albumin or dextran reduced agglomeration and NET formation by neutrophils. Importantly, this passivation of the SPIONs prevented vascular occlusions in vivo even when magnetically accumulated. We conclude that higher order structures formed during nanoparticle agglomeration primarily trigger NET formation and the formation of SPION-aggregated NET-co-aggregates, whereas colloid-disperse nanoparticles behave inert and are alternatively cleared by phagocytosis

Human T cells loaded with superparamagnetic iron oxide nanoparticles retain antigen-specific TCR functionality

BackgroundImmunotherapy of cancer is an emerging field with the potential to improve long-term survival. Thus far, adoptive transfer of tumor-specific T cells represents an effective treatment option for tumors of the hematological system such as lymphoma, leukemia or myeloma. However, in solid tumors, treatment efficacy is low owing to the immunosuppressive microenvironment, on-target/off-tumor toxicity, limited extravasation out of the blood vessel, or ineffective trafficking of T cells into the tumor region. Superparamagnetic iron oxide nanoparticles (SPIONs) can make cells magnetically controllable for the site-specific enrichment. MethodsIn this study, we investigated the influence of SPION-loading on primary human T cells for the magnetically targeted adoptive T cell therapy. For this, we analyzed cellular mechanics and the T cell response after stimulation via an exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65 and compared them to T cells that had not received SPIONs.ResultsSPION-loading of human T cells showed no influence on cellular mechanics, therefore retaining their ability to deform to external pressure. Additionally, SPION-loading did not impair the T cell proliferation, expression of activation markers, cytokine secretion, and tumor cell killing after antigen-specific activation mediated by the TCR. ConclusionIn summary, we demonstrated that SPION-loading of T cells did not affect cellular mechanics or the functionality of the endogenous or an exogenous TCR, which allows future approaches using SPIONs for the magnetically enrichment of T cells in solid tumors

Magnetic Drug Targeting Reduces the Chemotherapeutic Burden on Circulating Leukocytes

Magnetic drug targeting (MDT) improves the integrity of healthy tissues and cells during treatment with cytotoxic drugs. An anticancer drug is bound to superparamagnetic iron oxide nanoparticles (SPION), injected into the vascular supply of the tumor and directed into the tumor by means of an external magnetic field. In this study, we investigated the impact of SPION, mitoxantrone (MTO) and SPIONMTO on cell viability in vitro and the nonspecific uptake of MTO into circulating leukocytes in vivo. MDT was compared with conventional chemotherapy. MTO uptake and the impact on cell viability were assessed by flow cytometry in a Jurkat cell culture. In order to analyze MTO loading of circulating leukocytes in vivo, we treated tumor-bearing rabbits with MDT and conventional chemotherapy. In vitro experiments showed a dose-dependent MTO uptake and reduction in the viability and proliferation of Jurkat cells. MTO and SPIONMTO showed similar cytotoxic activity. Non-loaded SPION did not have any effect on cell viability in the concentrations tested. Compared with systemic administration in vivo, MDT employing SPIONMTO significantly decreased the chemotherapeutic load in circulating leukocytes. We demonstrated that MDT spares the immune system in comparison with conventional chemotherapy

Die Bedeutung des CRP für die Clearance sterbender Zellen

An efficient clearance by phagocytes of apoptotic cells is of major importance to avoid secondary necrosis. The leakage of intracellular autoantigens from improperly cleared cells may provoke inflammation and finally autoimmunity. Therefore, the clearance of dying and dead cells is secured by a plethora of soluble adaptor molecules and receptors that recognize apoptotic cell associated molecular patterns (ACAMP) on the surfaces of the target cells. A pattern recognition molecule of the acute phase reaction is C-reactive protein (CRP), whose serum levels rise massively in response to infection, inflammation or tissue damage. CRP binds phosphocholine-related lipids of certain bacteria and disturbed mammalian membranes as well as nuclear components and opsonizes its targets for complement activation and phagocytic clearance. Aim of this thesis was to analyse the binding to dying and dead cells of CRP and to investigate the role of CRP in clearance under healthy and autoimmune conditions. Here we show that during apoptosis the major CRP target lysophosphatidylcholine (LPC) is cleaved and released from the cells’ membranes as soluble CRP antagonist, molecularly identified by mass spectrometry as glycerophosphocholine (GPC). The binding of CRP is strongly reduced in post-apoptotic necrosis when compared with primary necrotic cells, since the former are deprived of their CRP targets. The release of GPC is executed by phospholipase-A2 (PLA2) type 6, which is activated by caspase-3-cleavage and has both PLA2 and lysophospholipase-A1 (LPLA1) activities. In clearance assays CRP augmented the uptake by macrophages of aged granulocytes. In addition, CRP increased binding of C1q to apoptotic cells and necrotic debris. Both activities were antagonized by soluble GPC. Treatment of necrotic cells with DNase-1 increased CRP binding significantly. Presumably, the loosening of the tight chromatin structure enables CRP to access DNA related target proteins more efficiently. However, neutrophil extracellular traps (NETs) relased from neutrophil granulocytes were not opsonized by CRP, thus questioning chromatin-associated targets. Usually, the opsonisation of dying and dead cells with CRP supports the anti-inflammatory clearance. However, in many patients with systemic lupus erythema-tosus (SLE) surface bound CRP is targeted by anti-CRP autoantibodies. We showed that complexes of secondary necrotic cell material, CRP and anti-CRP autoantibodies fostered the release of pro-inflammatory cytokines. We conclude that opsonisation by CRP and CRP-mediated complement activation serve as backup mechanism for cells which have escaped early clearance mechanisms. Post-apoptotic cells deprived of their CRP targets may accumulate as “unclearable” material. Low DNase-1 activity may further reduce accessibility for CRP of nuclear autoantigens and solubilisation of NETs by the recruitment of complement in patients with SLE. Problematically, remaining surface bound CRP is detected by anti-CRP autoantibodies shifting the clearance towards inflammation and further precipitating autoimmunity.Eine effiziente Entsorgung apoptotischer Zellen durch Phagozyten ist notwendig, um sekundäre Nekrose zu vermeiden. Das Auslaufen intrazellulärer Antigene aus nicht ordnungsgemäß entsorgten Zellen kann Entzündung und letztendlich Autoimmunität auslösen. Daher wird die Entsorgung sterbender und toter Zellen durch eine Fülle von löslichen Adaptermolekülen und Rezeptoren sichergestellt, die bestimmte Muster auf der Oberfläche apoptotischer Zielzellen erkennen (ACAMP). Ein Mustererkennungsmolekül der Akuten-Phase-Reaktion ist das C-reaktive Protein (CRP), dessen Serumkonzentration als Reaktion auf Infektion, Entzündung oder Gewebezerstörung massiv ansteigt. CRP bindet nukleäre Strukturen und Phosphocholin-Lipide verschiedener Bakterien und beschädigter Säugerzellen und markiert seine Zielzellen so für Komplementaktivierung und Aufnahme durch Phagozyten. Ziel dieser Arbeit war es, die CRP-Opsonisierung sterbender und bereits toter Zellen zu analysieren und die Rolle von CRP im Entsorgungsprozess unter physiologischen und autoimmunen Bedingungen zu untersuchen. Im Rahmen dieser Arbeit wurde gezeigt, dass während der Apoptose die CRP-Hauptbindungsstelle Lysophosphatidylcholin (LPC) gespalten und als löslicher CRP-Antagonist aus der Zellmembran gelöst wird. Der CRP-Antagonist wurde mit Hilfe der Massenspektrometrie als Glycerophosphocholin (GPC) identifiziert. Verglichen mit primär nekrotischen Zellen war die Bindung von CRP auf post-apoptotischen Zellen stark reduziert, da letztere ihre CRP-Bindungsstellen verloren haben. Die Phospholipase A2 (PLA2) ist für das Abspalten von GPC verantwortlich. PLA2 wird durch Caspase-Spaltung aktiviert und besitzt sowohl PLA2 als auch Lysophospholipase-A1 (LPLA1)-Aktivität. In Fressversuchen förderte CRP die Aufnahme gealterter Granulozyten durch Makrophagen. Zusätzlich verstärkte CRP die Bindung von C1q an apoptotische Zellen und nekrotische Zellreste. Lösliches GPC antagonisierte beide CRP-vermittelten Aktivitäten. Die Behandlung nekrotischer Zellen mit DNase-1 verstärkte die CRP-Bindung signifikant, da die Auflockerung der kompakten Chromatinstruktur DNA-assoziierte Proteine für CRP zugänglich machte. CRP opsonisierte jedoch nicht das Chromatin, das von neutrophilen Granulozyten als „Pathogenfalle“ in den extrazellulären Raum externalisiert wurde (NETs). Normalerweise unterstützt die Opsonisierung mit CRP die anti-inflammatorische Entsorgung sterbender und toter Zellen. Allerdings weisen viele Patienten mit systemischem Lupus erythematodes (SLE) Autoantikörper gegen gebundenes CRP auf. Komplexe aus sekundär nekrotischem Zellmaterial, CRP und anti-CRP Autoantikörpern förderten die Sekretion pro-inflammatorischer Zytokine. Wir folgern, dass die Opsonisierung mit CRP und die CRP-vermittelte Komplementaktivierung als Backup-Mechanismus für Zellen dienen, die den Entsorgungsmechanismen für früh-apoptotische Zellen entgangen sind. Post-apoptotische Zellen mit fehlender CRP-Zielstruktur akkumulieren daher möglicherweise als schwer entsorgbares Material. Niedrige DNase-1 Aktivität in Patienten mit SLE könnte weiterhin die Zugänglichkeit nukleärer Autoantigene für CRP und die Auflösung der NETs aufgrund fehlender Komplementrekrutierung reduzieren. Problematischerweise wird gebundenes CRP in Patienten mit SLE von anti-CRP Autoantikörpern gebunden. Diese treiben die Entsorgung sterbender Zellen in Richtung Inflammation und verschärfen so die pathologischen Konsequenzen der Autoimmunität