Publications

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

JTD Keywords: Antimalarial, Cell penetrating peptide, Chloroquine, Erythrocyte fluorescence, Flow cytometry, Hemolysis, Microscopy, Plasmodium, Primaquine, Red blood cell

The present investigation reports the development of PEG-modified liposomes for the delivery of naturally occurring resveratrol. PEG-modified liposomes were prepared by direct sonication of the phospholipid aqueous dispersion, in the presence of two PEG-surfactants. Small, spherical, unilamellar vesicles were produced, as demonstrated by light scattering, cryo-TEM, and SAXS. The aging of the vesicles was assessed by using the Turbiscan® technology, and their physical stability was evaluated in vitro in simulated body fluids, results showing that the key features of the liposomes were preserved. The biocompatibility of the formulations was demonstrated in an ex vivo model of hemolysis in human erythrocytes. Further, the incorporation of resveratrol in PEG-modified liposomes did not affect its intrinsic antioxidant activity, as DPPH radical was almost completely inhibited, and the vesicles were also able to ensure an optimal protection against oxidative stress in an ex vivo human erythrocytes-based model. Therefore, the proposed PEG-modified liposomes, which were prepared by a simple and reliable method, represent an interesting approach to safely deliver resveratrol, ensuring the preservation of the carrier structural integrity in the biological fluids, and the antioxidant efficacy of the polyphenol to be exploited against oxidative stress associated with cancer.

JTD Keywords: Resveratrol, Antioxidant, PEG-surfactants, PEG-modified liposomes, Human erythrocytes

JTD Keywords: antimalarial, heparin, magic bullet, malaria, nanomedicine, nanotechnology, nanovector, Plasmodium, polymers, targeted drug delivery, chloroquine, immunoliposome, liposome, nanoparticle, solid lipid nanoparticle, Anopheles, antimalarial activity, drug delivery system, drug efficacy, erythrocyte, human, IC50, malaria, malaria control, nanoencapsulation, nonhuman, pathophysiology, Plasmodium, Review

Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. Here we have explored if both properties could be joined into a drug delivery strategy where heparin would have a dual role as antimalarial and as a targeting element of drug-loaded nanoparticles. Confocal fluorescence and transmission electron microscopy data show that after 30. min of being added to living pRBCs fluorescein-labeled heparin colocalizes with the intracellular parasites. Heparin electrostatically adsorbed onto positively charged liposomes containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane and loaded with the antimalarial drug primaquine was capable of increasing three-fold the activity of encapsulated drug in Plasmodium falciparum cultures. At concentrations below those inducing anticoagulation of mouse blood in vivo, parasiticidal activity was found to be the additive result of the separate activities of free heparin as antimalarial and of liposome-bound heparin as targeting element for encapsulated primaquine. From the Clinical Editor: Malaria remains an enormous global public health concern. In this study, a novel functionalized heparin formulation used as drug delivery agent for primaquine was demonstrated to result in threefold increased drug activity in cell cultures, and in a murine model it was able to provide these benefits in concentrations below what would be required for anticoagulation. Further studies are needed determine if this approach is applicable in the human disease as well.

JTD Keywords: Heparin, Liposomes, Malaria, Plasmodium, Targeted drug delivery, Heparin, Malaria, Plasmodium, Red blood cell, Targeted drug delivery, Liposomes, 1,2 dioleoyl 3 trimethylammoniopropane, fluorescein, heparin, liposome, nanoparticle, primaquine, adsorption, animal experiment, anticoagulation, antimalarial activity, Article, binding affinity, confocal microscopy, controlled study, drug targeting, encapsulation, erythrocyte, female, fluorescence microscopy, human, human cell, in vivo study, liposomal delivery, mouse, nonhuman, Plasmodium falciparum, transmission electron microscopy

JTD Keywords: Antimalarial, Erythrocyte, Glycosaminoglycan, Heparin, Magic bullet, Malaria, Nanomedicine, Nanovector, Plasmodium, Targeted drug delivery

JTD Keywords: Antimalarial, Erythrocyte, Glycosaminoglycan, Heparin, Magic bullet, Malaria, Nanomedicine, Nanovector, Plasmodium, Targeted drug delivery

Cell migration and spreading involve the coordination of membrane trafficking, actomyosin contraction, and modifications to plasma membrane tension and area. The biochemical or biophysical basis for this coordination is however unknown. In this study, we show that during cell spreading, lamellipodia protrusion flattens plasma membrane folds and blebs and, once the plasma membrane area is depleted, there is a temporary increase in membrane tension by over twofold that is followed by activation of exocytosis and myosin contraction. Further, an artificial increase in plasma membrane tension stopped lamellipodia protrusion and activated an exocytotic burst. Subsequent decrease in tension restored spreading with activation of contraction. Conversely, blebbistatin inhibition of actomyosin contraction resulted in an even greater increase in plasma membrane tension and exocytosis activation. This spatio-temporal synchronization indicates that membrane tension is the signal that coordinates membrane trafficking, actomyosin contraction, and plasma membrane area change. We suggest that cells use plasma membrane tension as a global physical parameter to control cell motility.

JTD Keywords: Surface-area regulation, Cytoskeleton adhesion, Erythrocyte-membrane, Extensional flow, Elastic tether, Force

Summary During definitive erythropoiesis, erythroid precursors undergo differentiation through multiple nucleated states to an enucleated reticulocyte, which loses its residual RNA/organelles to become a mature erythrocyte. Over the course of these transformations, continuous changes in membrane proteins occur, including shifts in protein abundance, rates of expression, isoform prominence, states of phosphorylation, and stability. In an effort to understand when assembly of membrane proteins into an architecture characteristic of the mature erythrocyte occurs, we quantitated the lateral diffusion of the most abundant membrane protein, band 3 (AE1), during each stage of erythropoiesis using single particle tracking. Analysis of the lateral trajectories of individual band 3 molecules revealed a gradual reduction in mobility of the anion transporter as erythroblasts differentiated. Evidence for this progressive immobilization included a gradual decline in diffusion coefficients as determined at a video acquisition rate of 120 frames/s and a decrease in the percentage of compartment sizes >100 nm. Because complete acquisition of the properties of band 3 seen in mature erythrocytes is not observed until circulating erythrocytes are formed, we suggest that membrane maturation involves a gradual and cooperative assembly process that is not triggered by the synthesis of any single protein.

JTD Keywords: Band 3 diffusion, Erythrocyte, Progenitor cells, Single particle tracking, Streptavidin quantum dot

Non-invasive single cell analyses are increasingly required for the medical diagnostics of test substances or the development of drugs and therapies on the single cell level. For the non-invasive characterisation of cells, impedance spectroscopy which provides the frequency dependent electrical properties has been used. Recently, microfludic systems have been investigated to manipulate the single cells and to characterise the electrical properties of embedded cells. In this article, the impedance of partially embedded single cells dependent on the cellular behaviour was investigated by using the microcapillary. An analytical equation was derived to relate the impedance of embedded cells with respect to the morphological and physiological change of extracellular interface. The capillary system with impedance measurement showed a feasibility to monitor the impedance change of embedded single cells caused by morphological and physiological change of cell during the addition of DMSO. By fitting the derived equation to the measured impedance of cell embedded at different negative pressure levels, it was able to extrapolate the equivalent gap and gap conductivity between the cell and capillary wall representing the cellular behaviour.

JTD Keywords: Frequency-domain, Spectroscopy, Erythrocytes, Biosensor, Membrane, System