Kristin Mohr

Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers

The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.

Aggregation behavior of cationic nanohydrogel particles in human blood serum.

For systemic siRNA delivery applications, well-defined drug carriers are required that guarantee stability for both carrier and cargo. Among various concepts progressing in market or final development, cationic nanohydrogel particles may serve as novel transport media especially designed for siRNA-in vivo experiments. In this work, the interaction of nanohydrogel particles with proteins and serum components was studied via dynamic light scattering in human blood serum as novel screening method prior to applications in vivo. The formation of larger aggregates mostly caused by charge interaction with albumin could be suppressed by nanogel loading with siRNA affording a neutral zeta potential for the complex. Preliminary in vivo studies confirmed the results inside the light-scattering cuvette. Although both carrier and cargo may have limited stability on their own under physiological relevant conditions, they can form safe and stable complexes at a charge neutralized ratio and thus making them applicable to systemic siRNA delivery.

Aggregation Behavior of Polystyrene-Nanoparticles in Human Blood Serum and its Impact on the in vivo Distribution in Mice

The interactions between nanoparticles (NPs) and proteins in complex biological application media such as blood serum are capable of inducing aggregate formation which can lead to subsequent changes in biological activity. Here, we correlate surface charge, aggregation-tendency, and surface serum protein adsorption with cellular uptake and biodistribution in mice. Polystyrene-based NPs (80 - 170 nm) with different surface functionalizations were synthesized and incubated with human serum. Interaction of NPs with serum proteins and aggregate formation were analyzed by mass spectrometryanalysis and dynamic light-scattering. Influence of surface functionalization on specific cellular uptake and organdistribution was characterized. Localization and organ targeting of intravenously applied NPs preferentially depended on their aggregationbehavior in the presence of serum. Whereas strongly aggregating particles mainly located to liver, non-aggregating particles distributed to all organs. Determination of aggregate formation of NPs in the presence of serum and further analysis of the protein corona allows for pre-selection of NPs for in vivo application.

Polycarbonate and polystyrene nanoplastic particles act as stressors to the innate immune system of fathead minnow (Pimephales promelas)

Water pollution with large-scale and small-scale plastic litter is an area of growing concern. Macro-plastic litter is a well-known threat to aquatic wildlife; however, the effects of micro-sized and nano-sized plastic particles on the health of organisms are not well understood. Small-scale plastic particles can easily be ingested by various aquatic organisms and potentially interfere with their immune system; therefore, the authors used a freshwater fish species as a model organism for nanoplastic exposure. Characterization of polystyrene (41.0 nm) and polycarbonate (158.7 nm) nanoplastic particles (PSNPs and PCNPs, respectively) in plasma was performed, and the effects of PSNPs and PCNPs on the innate immune system of fathead minnow were investigated. In vitro effects of PSNPs and PCNPs on neutrophil function were determined using a battery of neutrophil function assays. Exposure of neutrophils to PSNPs or PCNPs caused significant increases in degranulation of primary granules and neutrophil extracellular trap release compared to a nontreated control, whereas oxidative burst was less affected. The present study outlines the stress response of the cellular component of fish innate immune system to polystyrene and polycarbonate nanoparticles/aggregates and indicates their potential to interfere with disease resistance in fish populations. Environ Toxicol Chem 2016;35:3093-3100.

HPMA-LMA Copolymer Drug Carriers in Oncology: An in Vivo PET Study to Assess the Tumor Line-Specific Polymer Uptake and Body Distribution

Polymeric drug carriers aim to selectively target tumors in combination with protecting normal tissue. In this regard polymer structure and molecular weight are key factors considering organ distribution and tumor accumulation of the polymeric drug delivery system. Four different HPMA based copolymer structures (random as well as block copolymers with lauryl methacrylate as hydrophobic block) varying in molecular weight, size and resulting architecture were analyzed in two different tumor models (AT1 prostate carcinoma and Walker-256 mammary carcinoma) in vivo. Polymers were labeled with (18)F and organ/tumor uptake was followed by μPET imaging and ex vivo biodistribution. Vascular permeability was measured by dextran extravasation and vascular density by immunohistochemistry. Cellular polymer uptake was determined in vitro using fluorescence-labeled polymers. Most strikingly, the high molecular weight HPMA-LMA random copolymer demonstrated highest tumor uptake and blood pool concentration. The molecular structure (e.g., amphiphilicity) is holding a higher impact on desired in vivo properties than polymer size. The results also revealed pronounced differences between the tumor models although vascular permeability was almost comparable. Accumulation in Walker-256 carcinomas was much higher, presumably due to a better cellular uptake in this cell line and a denser vascular network in the tumors. These investigations clearly indicate that the properties of the individual tumor determine the suitability of polymeric drug carriers. The findings also illustrate the general necessity of a preclinical screening to analyze polymer uptake for each individual patient (e.g., by noninvasive PET imaging) in order to individualize polymer-based chemotherapy.

Nanocarrier for Oral Peptide Delivery Produced by Polyelectrolyte Complexation in Nanoconfinement

The hydrophilic peptide YY (PYY) is a promising hormone-based antiobesity drug. We present a new concept for the delivery of PYY from pH-responsive chitosan-based nanocarriers. To overcome the drawbacks while retaining the merits of the polyelectrolyte complex (PEC) method, we propose a one-pot approach for the encapsulation of a hydrophilic peptide drug in cross-linked PEC nanocarriers. First, the hydrophilic peptide is encapsulated via polyelectrolyte complexation within water-in-oil miniemulsion droplets. In a second step, the PEC surface is reinforced by controlled interfacial cross-linking. PYY is efficiently encapsulated and released upon pH change. Such nanocarriers are promising candidates for the fight against obesity and, in general, for the oral delivery of protein drugs.

Interaction of pHPMA-pLMA copolymers with human blood serum and its components.

Immediately after administration, polymer therapeutics are exposed to complex biological media like blood which may influence and alter their physicochemical properties due to interactions with proteins or serum components. Among such interactions those leading to larger sized aggregates can be sensitively detected by dynamic light scattering (DLS) as a pre in vivo screening method. Random copolymers from N-(2-hydroxypropyl)methacrylamide and lauryl methacrylate p(HPMA-co-LMA) and copolymers loaded with the model drug domperidone were characterized by DLS in isotonic salt solution and in blood serum. The bare amphiphilic copolymer micelles (Rh=30 nm in isotonic salt solution) formed large aggregates in serum of over 100 nm radius which were shown to originate from interactions with very low density lipoproteins (VLDLs). Encapsulation of the hydrophobic drug domperidone resulted, at first, in drug-copolymer formulations with larger hydrodynamic radii (39 nm<Rh<49 nm) which, however, did not induce aggregate formation in human serum. Since p(HPMA-co-LMA) copolymers were demonstrated to have a high potential for drug delivery into the brain, the knowledge of serum-copolymer interactions provides a better understanding of their function in the biological context.

Biodegradable Protein Nanocontainers

The application of synthetic polymers for drug delivery often requires tremendous efforts to ensure biocompatibility and -degradation. To use the bodys own substances can help to overcome these problems. Herein, we present the first synthesis of nanocontainers entirely composed of albumin proteins. These protein nanocontainers (PNCs) were loaded with hydrophilic compounds and release of the payload is triggered through natural lysis in vitro in human monocyte-derived dendritic cells (moDCs). No aggregation of PNCs in human blood plasma was observed, indicating stability for blood circulation. As the PNCs were readily taken up by moDCs, they are considered as a promising delivery platform for vaccination strategies and could minimize the risk of side effects caused by foreign carrier substances.

Pentafluorophenyl Ester-based Polymersomes as Nanosized Drug-Delivery Vehicles

In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate-based precursor polymers, an N-(2-hydroxypropyl) methacrylamide (HPMA)-analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cytotoxicity and no protein adsorption-induced aggregation in human blood serum occurs, making the vesicles interesting candidates as nanosized drug carriers.

Fluorescence labels may significantly affect the protein adsorption on hydrophilic nanomaterials

Fluorescently labelled proteins are often used to study processes in vitro, e.g. the binding of proteins to cell surfaces or the adsorption of plasma proteins on drug nanocarriers. However, the fact that the fluorescent labelling may affect the protein properties is frequently neglected. On the example of a simple model system, we reiterate the importance of this issue by showing that even a single label may perturb interactions between hydrophilic starch-based nanocapsules and serum albumin and thus prevent binding.