Jonas Rosager Henriksen

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK(a) to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome-lysosome pathway.

Vesicle fluctuation analysis of the effects of sterols on membrane bending rigidity

Sterols are regulators of both biological function and structure. The role of cholesterol in promoting the structural and mechanical stability of membranes is widely recognized. Knowledge of how the related sterols, lanosterol and ergosterol, affect membrane mechanical properties is sparse. This paper presents a comprehensive comparison of the effects of cholesterol, lanosterol, and ergosterol upon the bending elastic properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles. Measurements are made using vesicle fluctuation analysis, a nonintrusive technique that we have recently improved for determining membrane bending rigidity. Giving a detailed account of the vesicle fluctuation analysis technique, we describe how the gravitational stabilization of the vesicles enhances image contrast, vesicle yield, and the quality of data. Implications of gravity on vesicle behaviour are also discussed. These recent modifications render vesicle fluctuation analysis an efficient and accurate method for determining how cholesterol, lanosterol, and ergosterol increase membrane bending rigidity.

Understanding detergent effects on lipid membranes: a model study of lysolipids.

Lysolipids and fatty acids are the natural products formed by the hydrolysis of phospholipids. Lysolipids and fatty acids form micelles in solution and acts as detergents in the presence of lipid membranes. In this study, we investigate the detergent strength of a homologous series of lyso-phosphatidylcholine lipids (LPCs) on 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine (POPC) lipid membranes by use of isothermal titration calorimetry and vesicle fluctuation analysis. The membrane partition coefficient (K) and critical micelle concentration (cmc) are determined by isothermal titration calorimetry and found to obey an inverse proportionality relation (cmc.K approximately 0.05-0.3). The partition coefficient and critical micelle concentration are used for the analysis of the effect of LPCs on the membrane bending rigidity. The dependency of the bending rigidity on LPC membrane coverage has been analyzed in terms of a phenomenological model based on continuum elastic theory, which yields information about the curvature-inducing properties of the LPC molecule. The results reveal: 1), an increase in the partition coefficient with increasing LPC acyl-chain length; and 2), that the degree of acyl-chain mismatch between LPC and POPC determines the magnitude of the membrane mechanical perturbation per LPC molecule in the membrane. Finally, the three-stage model describing detergent membrane interaction has been extended by a parameter D(MCI), which governs the membrane curvature stability in the detergent concentration range below the cmc-value of the LPC molecule.

Positron emission tomography evaluation of somatostatin receptor targeted 64Cu-TATE-liposomes in a human neuroendocrine carcinoma mouse model

Targeted therapeutic and diagnostic nanocarriers functionalized with antibodies, peptides or other targeting ligands that recognize over-expressed receptors or antigens on tumor cells have potential in the diagnosis and therapy of cancer. Somatostatin receptors (SSTRs) are over-expressed in a variety of cancers, particularly neuroendocrine tumors (NETs) and can be targeted with somatostatin peptide analogs such as octreotate (TATE). In the present study we investigate liposomes that target SSTR in a NET xenograft mouse model (NCI-H727) by use of TATE. TATE was covalently attached to the distal end of DSPE-PEG(2000) on PEGylated liposomes with an encapsulated positron emitter (64)Cu that can be utilized for positron emission tomography (PET) imaging. The biodistribution and pharmacokinetics of the (64)Cu-loaded PEGylated liposomes with and without TATE was investigated and their ability to image NETs was evaluated using PET. Additionally, the liposome accumulation and imaging capability was compared with free radiolabelled TATE peptide administered as (64)Cu-DOTA-TATE. The presence of TATE on the liposomes resulted in a significantly faster initial blood clearance in comparison to control-liposomes without TATE. PEGylated liposomes with or without TATE accumulated at significantly higher quantities in NETs (5.1±0.3 and 5.8±0.2 %ID/g, respectively) than the free peptide (64)Cu-DOTA-TATE (1.4±0.3 %ID/g) 24 h post-injection. Importantly, (64)Cu-loaded PEGylated liposomes with TATE showed significantly higher tumor-to-muscle (T/M) ratio (12.7±1.0) than the control-liposomes without TATE (8.9±0.9) and the (64)Cu-DOTA-TATE free peptide (7.2±0.3). The higher T/M ratio of the PEGylated liposomes with TATE suggests some advantage of active targeting of NETs, although no absolute benefit in tumor accumulation over the non-targeted liposomes was observed. Collectively, these data showed that (64)Cu-loaded PEGylated liposomes with TATE conjugated to the surface could be promising new imaging agents for visualizing tumor tissue and especially NETs using PET.

Design, calibration and application of broad-range optical nanosensors for determining intracellular pH

Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1–7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2–3 weeks.

Seizure onset detection based on a Uni- or Multi-modal Intelligent Seizure Acquisition (UISA/MISA) system

An automatic Uni- or Multi-modal Intelligent Seizure Acquisition (UISA/MISA) system is highly applicable for onset detection of epileptic seizures based on motion data. The modalities used are surface electromyography (sEMG), acceleration (ACC) and angular velocity (ANG). The new proposed automatic algorithm on motion data is extracting features as “log-sum” measures of discrete wavelet components. Classification into the two groups “seizure” versus “non-seizure” is made based on the support vector machine (SVM) algorithm. The algorithm performs with a sensitivity of 91–100%, a median latency of 1 second and a specificity of 100% on multi-modal data from five healthy subjects simulating seizures. The uni-modal algorithm based on sEMG data from the subjects and patients performs satisfactorily in some cases. As expected, our results clearly show superiority of the multi-modal approach, as compared with the uni-modal one.

Selective Acylation Enhances Membrane Charge Sensitivity of the Antimicrobial Peptide Mastoparan-X

The partitioning of the wasp venom peptide mastoparan-X (MPX) into neutral and negatively charged lipid membranes has been compared with two new synthetic analogs of MPX where the N(α)-terminal of MPX was acylated with propanoic acid (PA) and octanoic acid (OA). The acylation caused a considerable change in the membrane partitioning properties of MPX and it was found that the shorter acylation with PA gave improved affinity and selectivity toward negatively charged membranes, whereas OA decreased the selectivity. Based on these findings, we hypothesize that minor differences in the embedding and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptides selectivity between neutral and negatively charged lipid membranes. This finding is unique compared to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge-a finding that should be investigated for other, more potent antimicrobial peptides in future studies.

Thermodynamic Profiling of Peptide Membrane Interactions by Isothermal Titration Calorimetry: A Search for Pores and Micelles

Antimicrobial peptides are known to interact strongly with negatively charged lipid membranes, initially by peripheral insertion of the peptide into the bilayer, which for some antimicrobial peptides will be followed by pore formation, and successive solubilization of the membranes resulting in mixed peptide-lipid micelles. We have investigated the mode of action of the antimicrobial peptide mastoparan-X using isothermal titration calorimetry (ITC) and cryo-transmission electron microscopy (cryo-TEM). The results show that mastoparan-X induces a range of structural transitions of POPC/POPG (3:1) lipid membranes at different peptide/lipid ratios. It has been established that ITC can be used as a fast method for localizing membrane transitions and when combined with DLS and cryo-TEM can elucidate structural changes, including the threshold for pore formation and micellation. Cryo-TEM was employed to confirm the structural changes associated with the thermodynamic transitions found by ITC. The pore-formation process has furthermore been investigated in detail and the thermodynamic parameters of pore formation have been characterized using a system-specific temperature where the enthalpy of peptide partitioning becomes zero (T(zero)). This allows for an exclusive study of the pore-formation process. The use of ITC to find T(zero) allows for characterization of the thermodynamic parameters of secondary processes on lipid membranes.

Adsorption of cationic peptides to solid surfaces of glass and plastic.

Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant experimental implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use analytical HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical experimental peptide concentrations, 90% or more of the peptides might be lost from solution due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting experiments on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the experimental impact of these adsorption effects.

Side Chain Hydrophobicity Modulates Therapeutic Activity and Membrane Selectivity of Antimicrobial Peptide Mastoparan-X

The discovery of new anti-infective compounds is stagnating and multi-resistant bacteria continue to emerge, threatening to end the “antibiotic era”. Antimicrobial peptides (AMPs) and lipo-peptides such as daptomycin offer themselves as a new potential class of antibiotics; however, further optimization is needed if AMPs are to find broad use as antibiotics. In the present work, eight analogues of mastoparan-X (MPX) were investigated, having side chain modifications in position 1, 8 and 14 to modulate peptide hydrophobicity. The self-association properties of the peptides were characterized, and the peptide-membrane interactions in model membranes were compared with the bactericidal and haemolytic properties. Alanine substitution at position 1 and 14 resulted in higher target selectivity (red blood cells versus bacteria), but also decreased bactericidal potency. For these analogues, the gain in target selectivity correlated to biophysical parameters showing an increased effective charge and reduction in the partitioning coefficient for membrane insertion. Introduction of an unnatural amino acid, with an octyl side chain by amino acid substitution, at positions 1, 8 and 14 resulted in increased bactericidal potency at the expense of radically reduced membrane target selectivity. Overall, optimized membrane selectivity or bactericidal potency was achieved by changes in side chain hydrophobicity of MPX. However, enhanced potency was achieved at the expense of selectivity and vice versa in all cases.