Marina Pöttler

Pharmaceutical formulation of HSA hybrid coated iron oxide nanoparticles for magnetic drug targeting

In this work we present a new formulation of superparamagnetic iron oxide nanoparticles (SPIONs) for magnetic drug targeting. The particles were reproducibly synthesized from current good manufacturing practice (cGMP) - grade substances. They were surface coated using fatty acids as anchoring molecules for human serum albumin. We comprehensively characterized the physicochemical core-shell structure of the particles using sophisticated methods. We investigated biocompatibility and cellular uptake of the particles using an established flow cytometric method in combination with microwave-plasma assisted atomic emission spectroscopy (MP-AES). The cytotoxic drug mitoxantrone was adsorbed on the protein shell and we showed that even in complex media it is slowly released with a close to zero order kinetics. We also describe an in vitro proof-of-concept assay in which we clearly showed that local enrichment of this SPION-drug conjugate with a magnet allows site-specific therapeutic effects.

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 SEONLA-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 SEONLA-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 SEONLA-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.

Genotoxicity of Superparamagnetic Iron Oxide Nanoparticles in Granulosa Cells

Nanoparticles that are aimed at targeting cancer cells, but sparing healthy tissue provide an attractive platform of implementation for hyperthermia or as carriers of chemotherapeutics. According to the literature, diverse effects of nanoparticles relating to mammalian reproductive tissue are described. To address the impact of nanoparticles on cyto- and genotoxicity concerning the reproductive system, we examined the effect of superparamagnetic iron oxide nanoparticles (SPIONs) on granulosa cells, which are very important for ovarian function and female fertility. Human granulosa cells (HLG-5) were treated with SPIONs, either coated with lauric acid (SEONLA) only, or additionally with a protein corona of bovine serum albumin (BSA; SEONLA-BSA), or with dextran (SEONDEX). Both micronuclei testing and the detection of γH2A.X revealed no genotoxic effects of SEONLA-BSA, SEONDEX or SEONLA. Thus, it was demonstrated that different coatings of SPIONs improve biocompatibility, especially in terms of genotoxicity towards cells of the reproductive system.

Analysis of Hypericin-Mediated Effects and Implications for Targeted Photodynamic Therapy

The phototoxic effect of hypericin can be utilized for Photodynamic Therapy (PDT) of cancer. After intravenous application and systemic distribution of the drug in the patient’s body, the tumor site is exposed to light. Subsequently, toxic reactive oxygen species (ROS) are generated, inducing tumor cell death. To prevent unwanted activation of the drug in other regions of the body, patients have to avoid light during and after the treatment cycles, consequently impairing quality of life. Here, we characterize toxicity and hypericin-mediated effects on cancer cells in vitro and confirm that its effect clearly depends on concentration and illumination time. To reduce side effects and to increase therapy success, selective accumulation of hypericin in the tumor region is a promising solution. Loading hypericin on superparamagnetic iron oxide nanoparticles (SPIONs) and guiding them to the desired place using an external magnetic field might accomplish this task (referred to as Magnetic Drug Targeting (MDT)). Thus, using a double targeting strategy, namely magnetic accumulation and laser induced photoactivation, might improve treatment effectivity as well as specificity and reduce toxic side effects in future clinical applications.

Novel nanoparticulate drug delivery systems

Multifunctional GM1–modified lipoprotein-like nanoparticles for the therapy of Alzheimer’s Disease Evaluation of: Huang M, Hu M, Song Q et al. GM1–modified lipoproteinlike nanoparticle: multifunctional nanoplatform for the combination therapy of Alzheimer’s disease. ACS Nano. 9(11), 10801–10816 (2015). Alzheimer’s disease (AD) is the most common neurodegenerative disorder, affecting about 11% of people aged 65 years and older in the USA alone. According to the estimates, this number may nearly triple by 2050 [1] resulting in an increased health and economic burden, which urgently calls for more effective therapies. The molecular basis for the formation of the extracellular amyloid plaques found in the brain of AD patients involves the strong neurotoxic effects of soluble amyloid-β (Aβ) [2]. In their recent paper, Huang et al. have developed a flexible multifunctional noninvasive drug delivery system for accelerated Aβ clearance, protection of neurons and inhibition of other pathological cascades induced by Aβ accumulation. Reconstituted high-density lipoprotein (rHDL) and monosialotetrahexosylganglioside (GM1)-rHDL nanoparticles were prepared by self-assembly from lipid-free apoE3 and 1,2–dimyristoyl-sn-glycero–3–phosphocholine (DMPC) liposomes or GM1–DMPC liposomes, respectively [3]. Surface plasmon resonance analysis showed a very high affinity of GM1–rHDL to monomeric and oligomeric Aβ 1–42 , comparable with anti-Aβ antibody binding. Following the extensive physicochemical analyses, in vivo experiments demonstrated a high level of GM1–rHDL in the brain after intranasal application in mice, confirming the feasibility of noninvasive administration of these nanoparticles. Subsequent in vitro and in vivo experiments revealed a strong improvement of different Aβ clearing pathways in the presence of GM1– rHDL as compared with rHDL. In primary microglia, GM1–rHDL facilitated the cellular uptake and intracellular degradation of Aβ 1–42 . Increased Aβ degradation and an accelerated Aβ brain-to-blood efflux after injection of I-labeled Aβ 1–42 oligomers into the unilateral hippocampus was also achieved in vivo with intranasally administered GM1–rHDL. Beside increased Aβ levels, AD is characterized by loss of neurons and synapses leading to memory loss, cognitive deficits and ultimately to death. Consequently, a successful therapy should combine strategies for Aβ clearance and neuroprotection. To verify the drugdelivery capacity of GM1–rHDL, the authors selected a neuroprotective octapeptide, NAP, as a model drug for AD. The resulting αNAP-GM1–rHDL particles increased the viability of Aβ 1–42 oligomer-treated primary neurons and reversed neuronal dysfunction by increasing the neurite length and branch point counts. Additionally, αNAP-GM1–rHDL Novel nanoparticulate drug delivery systems

Targeting of drug-loaded nanoparticles to tumor sites increases cell death and release of danger signals

ABSTRACT Innovative strategies fighting cancer by inducing anti‐tumor responses from the immune system are urgently needed. Amongst others, chemotherapeutics from the class of the anthracyclines have been shown to induce death of cancer cells with immunogenic features and triggering immunogenic anti‐tumor responses. However, the patients immune system often becomes severely impaired by the unspecific action of these cytotoxic drugs in systemic chemotherapy, preventing any effective immune reactions. To reduce systemic side effects and to accumulate the drug exclusively in the tumor region, we developed an iron oxide nanoparticle‐based system for the magnetically targeted delivery of mitoxantrone to the tumor, which has previously proven its long‐term therapeutic efficacy in tumor bearing rabbits. Here, we show in vitro that superparamagnetic iron oxide nanoparticles (SPIONs), loaded with the chemotherapeutic drug mitoxantrone, are able to induce cell death with immunogenic features and concomitant maturation of dendritic cells, comparable to the free drug, whereas unloaded nanoparticles are very biocompatible. We conclude that the targeted delivery of mitoxantrone to the tumor region might be a promising possibility to selectively modulate the tumor microenvironment and to locally stimulate immune responses against the tumor. Thus, SPIONs can be used as a platform to specifically bring immunogenic cell death inducers (e.g. mitoxantrone, hypericin, doxorubicin) to the tumor region by sparing the immune system from their toxic effects.

Clinical Evaluation of Mucin-1 (MUC1) and P16 in Laryngeal Cancer

Background: Adapted from results in the field of cervical cancer, a direct connection between HPV infection and oropharyngeal carcinoma development could be established. Aim of this study was to evaluate p16 and TA-MUC1 in laryngeal cancer and their correlation to diagnostic, since TA-MUC1 is primarily restricted to malignancies. Methods: Paraffin-embedded laryngeal cancer specimens (n=129) and normal tissue (n=5) were analyzed for TA-MUC1 expression using hPankoMab-GEXTM antibody and evaluated according the immunoreactive score. Survival was assessed via log-rank test and Kaplan-Meier-survival analysis. Results: Significant correlation with tumor grading and staging was exhibited by TA-MUC1staining, while being negative in normal tissues. Expression of p16 significantly increased in T4 compared to T1 tumors. Significant differences in overall survival were found in correlation to TNM-classification, grading and relapse. TA-MUC1 showed a positive trend correlating to p16. Conclusion: Because of this positive trend, we suggest a HPV association in head and neck tumors. Most likely due to an insufficient quantity of HPV-positive patients, no statistical significance could be established. However, targeting TA-MUC1 would improve tumor therapy by linking hPankoMab-GEXTM to the overexpressed galectin. Systematic analysis of HPV-association should be performed generally in laryngeal cancer to gain further information about the interaction of HPV and malignancies.

Laryngeal dynamics and vocal fold tissue engineering in a rabbit model

Vocal fold surgery, especially due to cancer treatment, yields reduced voice quality and consequently reduced quality of life for patients. Hence, the development of vocal fold implants, to restore missing vocal fold tissue after surgery, is an urgent clinical need. To achieve this, a rabbit model is applied as a first step. Ex-vivo dynamic experiments were performed on twelve rabbit larynges providing normative phonatory data. The larynges were phonated at sustained phonation for different elongation levels at varying subglottal pressures. Laryngeal vibrations, airflow, and acoustics were recorded. Subsequently, for each larynx, a defined area of one vocal fold was resected, simulating the surgical intervention, and were phonated again with the same stimulations. The untreated larynges showed expected behavior regarding flow-pressure relation, acoustics and dynamics. In contrast, the phonatory quality of the resected larynges was significantly reduced showing, as expected, highly disturbed dynamics and a...