Mitoxantrone Loaded Superparamagnetic Nanoparticles for Drug Targeting: A Versatile and Sensitive Method for Quantification of Drug Enrichment in Rabbit Tissues Using HPLC-UV

In medicine, superparamagnetic nanoparticles bound to chemotherapeutics are currently investigated for their feasibility in local tumor therapy. After intraarterial application, these particles can be accumulated in the targeted area by an external magnetic field to increase the drug concentration in the region of interest (Magnetic-Drug-Targeting). We here present an analytical method (HPLC-UV), to detect pure or ferrofluid-bound mitoxantrone in a complex matrix even in trace amounts in order to perform biodistribution studies. Mitoxantrone could be extracted in high yields from different tissues. Recovery of mitoxantrone in liver tissue (5000 ng/g) was 76 ± 2%. The limit of quantification of mitoxantrone standard was 10 ng/mL ±12%. Validation criteria such as linearity, precision, and stability were evaluated in ranges achieving the FDA requirements. As shown for pilot samples, biodistribution studies can easily be performed after application of pure or ferrofluid-bound mitoxantrone

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)

Hydroxyapatite-coated SPIONs and their influence on cytokine release

Hydroxyapatite- or calcium phosphate-coated iron oxide nanoparticles have a high potential for use in many biomedical applications. In this study, a co-precipitation method for the synthesis of hydroxyapatite-coated nanoparticles (SPIONHAp), was used. The produced nanoparticles have been characterized by dynamic light scattering, X-ray diffraction, vibrating sample magnetometry, Fourier transform infrared spectrometry, atomic emission spectroscopy, scanning electron microscopy, transmission electron microscopy, selected area diffraction, and energy-dispersive X-ray spectroscopy. The results showed a successful synthesis of 190 nm sized particles and their stable coating, resulting in SPIONHAp. Potential cytotoxic effects of SPIONHAp on EL4, THP-1, and Jurkat cells were tested, showing only a minor effect on cell viability at the highest tested concentration (400 [my]g Fe/mL). The results further showed that hydroxyapatite-coated SPIONs can induce minor TNF-α and IL-6 release by murine macrophages at a concentration of 100 [my]g Fe/mL. To investigate if and how such particles interact with other substances that modulate the immune response, SPIONHAp-treated macrophages were incubated with LPS (lipopolysaccharides) and dexamethasone. We found that cytokine release in response to these potent pro- and anti-inflammatory agents was modulated in the presence of SPIONHAp. Knowledge of this behavior is important for the management of inflammatory processes following in vivo applications of this type of SPIONs

Functionalization Of T Lymphocytes With Citrate-Coated Superparamagnetic Iron Oxide Nanoparticles For Magnetically Controlled Immune Therapy

Purpose: Immune activation with T cell tumor infiltration is beneficial for the prognosis of patients suffering from solid cancer. Depending on their immune status, solid tumors can be immunologically classified into three groups: “hot” tumors are infiltrated with T lymphocytes, “cold” tumors are not infiltrated and “immune excluded” tumors are only infiltrated in the peripheral tumor tissue. Checkpoint inhibitors provide new therapeutic options for “hot” tumors by triggering the immune response of T cells. In order to enable this for cold tumors as well, T cells must be enriched in the tumor. Therefore, we use the principle of magnetic targeting to guide T cells loaded with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONCitrate) to the tumor by an externally applied magnetic field. Methods: SPIONCitrate were produced by alkaline coprecipitation of iron(II) and iron(III) chloride and in situ coating with sodium citrate. The concentration-dependent cytocompatibility of the particles was determined by flow cytometry and blood stability assays. Atomic emission spectroscopy was used for the quantification of the particle uptake into T lymphocytes. The attractability of the loaded cells was observed by live-cell imaging in the presence of an externally applied magnetic field. Results: SPIONCitrate displayed good cytocompatibility to T cells and did not show any sign of aggregation in blood. Finally, SPIONCitrate-loaded T cells were strongly attracted by a small external magnet. Conclusion: T cells can be “magnetized” by incorporation of SPIONCitrate for magnetic targeting. The production of the particle-cell hybrid system is straightforward, as the loading process only requires basic laboratory devices and the loading efficiency is sufficient for cells being magnetically controllable. For these reasons, SPIONCitrate are potential suitable candidates for magnetic T cell targeting

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)

Diverse Applications of Nanomedicine

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic. \ua9 2017 American Chemical Society

Developement of 18F labelled, subtype selective radioligands for the dopamine D4 receptor for the application within positron emission tomography

Ziele der vorliegenden Arbeit waren: 1. Synthese hochaffiner und Subtyp-selektiver Radioliganden für den Dopamin D4-Rezeptor sowie geeigneter Vorläuferverbindungen für eine entsprechende Radiomarkierung mit 18F. 2. Entwicklung und Optimierung von 18F-Markierungssynthesen zur Darstellung 18F-markierter Dopamin D4-Rezeptorliganden. 3. In-Vitro und In-Vivo-Charakterisierung ausgewählter 18F- markierter Kandidaten. D4 Subtyp-selektive Radioliganden wurden als Derivate bereits vom Lehrstuhl für Pharmazeutische Chemie der Friedrich-Alexander-Universität Erlangen-Nürnberg charakterisierter Leitstrukturen entwickelt. Aus der Leitstruktur FAUC 316 (Ki(D4)= 1,0 nM; (Ki(D2/D4)= 19000) wurden die 5-Cyano-Indol Verbindungen 16a (Ki(D4)= 2,1 nM; (Ki(D2//D4)= 5,7; Ki(D3//D4)= 32) und 16b (Ki(D4)= 9,6 nM; (Ki(D2/D4)= 410; Ki(D3//D4)= 250) entwickelt. Die entsprechenden Markierungsvorläufer 16c(Ki(D4)= 1,6 nM; (Ki(D2/D4)= 460; Ki(D3//D4)= 180) und 16d (Ki(D4)= 3,1 nM; (Ki(D2/D4)= 110; Ki(D3//D4)= 1500) sind Edukte für eine zweistufigen 18F Markierungssynthese. Die Rezeptorbindungsdaten von Methoxy-ethoxy substituierten Benzylderivaten motivierten zur Entwicklung einer Serie an Fluorethoxysubstituierten Benzylderivaten welche zu vielversprechenden potentiellen D4-Rezeptorliganden führten. Verbindung 19h hat neben sehr hoher D4-Affinität (Ki(D4)= 1,7 nM)die höchste D4-Subtypselektivität (Ki(D2/D4)= 2058; Ki(D3//D4)= 223) aller charakterisierten fluorethoxysubstituierten Benzylderivate. Die im Rahmen dieser Arbeit dargestellten Verbindungen mit Pyrazolo[1,5-a]pyridin-Grundkörper, weisen eine zum Teil sehr hohe D4-Rezeptoraffinität (Ki(D4)= 1,3-28 nM) bei gleichzeitig niedriger Affinität den anderen Dopaminrezeptorsubtypen gegenüber auf. Der Ligand 31b wurde mit herausragender Subtyp-selektivität (Ki(D2/D4)=2143; Ki(D3//D4)= 1154) bei gleichzeitig hoher D4-Affinität (Ki(D4)= 13 nM) charakterisiert. Mit 31b konnte mit Hilfe eines [35S]GTPS-Bindungsassays, erstmals ein inverser D4-Agonist gefunden werden. Es ergeben sich damit neue Zusammenhänge für das Bindungsverhalten des Liganden, denn inverse Agonisten binden bevorzugt am inaktiven G-Protein-gekoppelten Rezeptor. Der entsprechende PET Ligand macht die inaktive Konformation des G-Protein-gekoppelten Rezeptors sichtbar und könnte somit einen Beitrag zur Aufklärung der funktionellen Rolle des Dopamin D4-Rezeptors leisten. Durch eine zweistufige Markierungssynthese mit [18F]Fluorethyltosylat konnte über die jeweiligen Precursoren nach Optimierung der Reaktionsparameter, [18F]16a und [18F]16b mit zerfallskorrigierten radiochemischen Ausbeuten von 80% (T=120°C, t=5 min) respektive 47% (T=140°C, t=5 min) bezogen auf [18F]Fluorethyltosylat dargestellt werden. Das Benzaldehyd [18F]19h war ebenfalls in einer zweistufigen Markierungssynthese via [18F]Fluorethyltosylat und dem Markierungsvorläufer 20 darstellbar. Die maximale radiochemische Ausbeute betrug 95% (T=110°C, t=3 min). Die einstufige 18F-für-Brom-Substitutionsreaktion lieferte die Pyridinyl-Derivate [18F]27a (T=180°C, RCA= 69%, t= 45 min) und [18F]28a (T=180°C, RCA= 80%, t= 60 min). Eine nicht weiter optimierte Radiosynthese erzeugte ausgehend von den Tosylat-Precursoren 34b und 34a die Radioliganden [18F]31b und [18F]30b in radiochemischen Ausbeuten von 88% bzw. 26% (T=85°C, t=10 min). Beide Verbindungen konnten erfolgreich mittels präparativer HPLC isoliert werden. Sämtliche mittels präparativer HPLC isolierbaren Tracer ([18F]19h, [18F]27a, [18F]28a, [18F]31b, [18F]30b), metabolisierten nicht in humanem Serum und waren darin mindestens 90 min bei 37°C stabil (Stabilitätsnachweis per Radio-DC > 96%). Die Lipophilie, ermittelt als dekadischer Logarithmus des Verteilungskoeffizienten eines Oktanol/PBS-Puffer Zweiphasensystems (LogD7,4), rangierte bei den genannten Radiotracern zwischen 1,78 und 2,77. Die durch die Software ClogP (Biobyte) berechneten Werte lagen zwischen 1,9 und 2,9 und damit etwa in einem Bereich, der als ideal für die Permeabilität der Blut-Hirnschranke gilt. [18F]31b ist besonders für umfassende in-vitro Studien am Rattenhirn geeignet. Nach intravenöser Applikation kam es hauptsächlich zu Anreicherung des Tracers in der Leber (3-7 % ID/g, 5-90 min p.i.) und im Hirn (0,8 %ID/g, 5 min p.i.). Die in-vitro Autoradiographie der Rattenhirnschnitte zeigte spezifische Bindungen von [18F]31b im Hippocampus (Gyrus Dentatus), Hypothalamus, den Kernen der Habenulae, im medialen Thalamus (Nucleus centralis), Septum, sowie dem Cortex und konnte durch den nicht selektiven D4-Antagonisten Eticloprid zu 65%-80% bzw dem selektiven D4-Antagonisten FAUC 213 zu 73%-93% blockiert werden. Dieses Bindungsmuster wurde durch ex-vivo Autoradiographiemessunge 90 min nach Injektion des Tracers bestätigt. Mit dem inversen D4-Agonisten [18F]31b konnte somit erfolgreich ein Subtyp-selektiver, PET-fähiger D4-Radioligand entwickelt werden, der in ausführlicheren tierexperimentellen Studien genauer charakterisiert werden soll.Aims of this thesis: 1. Synthesis of highly selective and highly affine radioligands for the dopamine D4-receptor plus appropriate precursors for 18F-radiofluorination. 2. Development and optimization of 18F-radiolabeling methods for the preparation of 18F marked dopamine D4-receptor ligands. 3. In-vitro characterization of the 18F-labelled candidates. D4-subtype selective radioligands were derived from formerly characterized lead compounds, which were developed at the Department of Chemistry and Pharmacy of the Friedrich-Alexander-University Erlangen-Nürnberg. The 5-cyanoindoles derived from the lead compound FAUC 316 (Ki(D4)= 1.0 nM (Ki(D2/D4)= 19000) 16a (Ki(D4)= 2.1 nM (Ki(D2//D4)= 5.7; Ki(D3//D4)= 32) and 16b (Ki(D4)= 9.6 nM; (Ki(D2/D4)= 410; Ki(D3//D4)= 250) were developed. The corresponding precursors 16c (Ki(D4)= 1.6 nM (Ki(D2/D4)= 460; Ki(D3//D4)= 180) and 16d (Ki(D4)= 3.1 nM (Ki(D2/D4)= 110; Ki(D3//D4)= 1500) serve as educts of a two-step 18F- labelling synthesis. Data of the binding profile of methoxy-ethoxy substituted benzyl- derivatives motivated us to deduct a series of fluorethoxy-substituted benzyl-derivatives, which lead to promising D4 receptor ligand candidates. 2-Methoxyphenylpiperazines tend to higher D4-affinity (19a-d: Ki(D4)= 1.3-3.7 nM) than the according 4-chlorophenylpiperazines (19e-h) except for 19h (Ki(D4)= 1.7 nM), which shows a very high D4-affinity plus superior D4 subtype selectivity (Ki(D2/D4)= 2058; Ki(D3//D4)= 223). Compounds with pyrazolo[1,5-a]pyridine moiety partially exhibit a very high D4R-affinity (Ki(D4)= 1.3-28 nM) and simultaneous low affinity for other dopamine receptor subtypes. 31b was characterized by both. Outstanding subtype selectivity (Ki(D2/D4)=2143; Ki(D3//D4)= 1154) and high D4R affinity (Ki(D4)= 13 nM). To our knowledge, 31b has been identified as the first inverse D4R-agonist using a [35S]GTPS functional assay, which was established and performed at the Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nürnberg. This implies new correlations of the ligand binding profile, because inverse agonists preferably bind to the inactive status of G-protein-coupled receptors. Therefor an analogous PET-Ligand possibly affords new cognitions for the functional role of the D4R. Starting from 16c and 16d, the radioligands [18F]16a and [18F]16b could be produced with radio chemical yields of 80% (120°C, 5 min) and 47% (140°C, 5 min) respectively, using a two-step radiosynthesis with [18F]fluoroethyltosylate. [18F]19h is also available by a two step labeling synthesis starting from precursor 20. The maximum radiochemical yield of 95 % was not aimed at in favor of high purity of the substance. Using a lower educt amount and separating just pure fractions of [18F]19h, the pure tracer was isolated with a radiochemical yield of 44%. The one-step bromo-for-18F substitution leads to [18F]27a (T=180°C, RCA= 69%, t= 45 min) and [18F]28a (180°C, 80%, 60 min). Starting from 34b and 34a a non optimized radiosynthesis generated [18F]31b and [18F]30b with radiochemical yields of 88% and 26%, respectively (85°C, 10 min). Both labeled compounds could be isolated via preparative HPLC. Due to the superior binding profile of the other groups, 5-cyanoindoles have not been used for further in-vitro characterization. All isolable tracers ([18F]19h, [18F]27a, [18F]28a, [18F]31b, [18F]30b) do not metabolize in human serum. The detectable stability is more than 96% after 90 min. Lipophilicity, expressed as the common logarithm of the n-octanol-water partition coefficient ranges between 1.78 and 2.77. LogP by Biobyte® software, is calculated between 1.9 and 2.9 thus fullfilling the criteria postulated for an ideal permeability of the blood-brain-barrier. [18F]31b is especially appropriate for extensive in-vitro investigations in rat brain. After i.v. application, the main accumulation is seen in liver (3-7 %ID/g, 5-90 min p.i.) and brain. In-vitro autoradiography of brain slices shows specific binding in the hippocampus (gyrus dentatus), hypothalamus, the habenular nuclei, in the medial thalamus (nucleus centralis), the septum and the cortex of the rat. [18F]31b could be displaced by the non selective D4 antagonist eticlopride (65%-80%) as well as the selective D4-antagonist FAUC 213 (73-93%). This binding pattern could be confirmed by ex-vivo autoradiography 90 min p.i. of [18F]31b and inhibition of the binding sites by co-injecting the animals with the D4 selective antagonists L-750667 and FAUC 213. In summary, [18F]31b is the first subtype-selective D4-Radioligand to be characterized in-vitro and in-vivo and appropriate for PET-Imaging

In Vitro Analysis of Superparamagnetic Iron Oxide Nanoparticles Coated with APTES as Possible Radiosensitizers for HNSCC Cells

Superparamagnetic iron oxide nanoparticles (SPION) are being investigated for many purposes, e.g., for the amplification of ionizing radiation and for the targeted application of therapeutics. Therefore, we investigated SPIONs coated with (3-Aminopropyle)-Triethoxysilane (SPION-APTES) for their influence on different head and neck squamous cell carcinoma (HNSCC) cell lines, as well as for their suitability as a radiosensitizer. We used 24-well microscopy and immunofluorescence microscopy for cell observation, growth curves to determine cytostatic effects, and colony formation assays to determine cytotoxicity. We found that the APTES-SPIONs were very well taken up by the HNSCC cells. They generally have a low cytotoxic effect, showing no significant difference in clonogenic survival between the control group and cells treated with 20 µg Fe/mL (p > 0.25) for all cell lines. They have a cytostatic effect on some cell lines cells (e.g., Cal33) that is visible across different radiation doses (1, 2, 8 Gy, p = 0.05). In Cal33, e.g., SPION-APTES raised the doubling time at 2 Gy from 24.53 h to 41.64 h. Importantly, these findings vary notably between the cell lines. However, they do not significantly alter the radiation effect: only one out of eight cell lines treated with SPION-APTES showed a significantly reduced clonogenic survival after ionizing radiation with 2 Gy, and only two showed significantly reduced doubling times. Thus, although the APTES-SPIONs do not qualify as a radiosensitizer, we were still able to vividly demonstrate and analyze the effect that the APTES-SPIONs have on various cell lines as a contribution to further functionalization