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In JoVE (1)

Other Publications (7)

Articles by Iren Constantinescu in JoVE

 JoVE Immunology and Infection

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

1Centre for Blood Research, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia, 3Canadian Blood Services, University of British Columbia, 4Department of Chemistry, Life Sciences Centre, University of British Columbia


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The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Other articles by Iren Constantinescu on PubMed

Liposomes and Blood Cells: a Flow Cytometric Study

To clarify the interactions of liposomes with blood cells, this study examined the behaviour of liposomes of a range of compositions in the presence of purified human blood cells in buffer or plasma; or in whole blood, or in mice in vivo. Liposomes, labeled with the hydrophilic fluorochrome, carboxy fluorescein (CF), or with membrane-sequestering R18 or FITC-labeled phospholipids, were mixed with blood cells and the appearance of the fluorochromes in the blood cell population was monitored by flow cytometry. Irrespective of composition, with or without poly(ethylene glycol), all types of liposomes were found to interact rapidly and dose-dependently with red cells, leukocytes and platelets, both in vitro and in vivo. This took place equally in the presence and the absence of plasma proteins and functional enzyme cascades, suggesting that the prime facie interaction is opsonization-independent and is consistent with liposome-blood cell fusion.

Influence of the Lipid Composition on the Kinetics of Concerted Insertion and Folding of Melittin in Bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the 19Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the 19Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an alpha-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of 19Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the alpha-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.

Rational Design of Antithrombotic Peptides to Target the Von Willebrand Factor (vWf)--GPIb Integrin Interaction

Conventional antithrombotic drug discovery requires testing of large numbers of drug candidates. We used computer-aided macromolecular interaction assessment (MIAX) to select antithrombotic molecules that mimic and therefore block platelet GPIb's binding to von Willebrand factor (vWf), an early step in thrombus formation. We screened a random array of 15-mer D-amino acid peptides for binding vWf. Structures of 4 candidate peptides were inferred by comparison to sequences in protein databases, conversion from the L to D conformations and molecular dynamics (MD) determinations of those most energetically stable. By MIAX, we deduced the amino acids and intermolecular hydrogen bonds contributing to the GPIb-vWf interaction interface. We docked the peptides onto vWf in silico to localize their binding sites and consequent potential for preventing GPIb-vWf binding. In vitro inhibition of ristocetin-initiated platelet agglutination confirmed peptide function and suitability for antithrombotic development, thereby validating this novel approach to drug discovery.

Enhanced Cell Surface Polymer Grafting in Concentrated and Nonreactive Aqueous Polymer Solutions

Macromolecular cell surface modification techniques have shown tremendous utility in various biomedical applications. However, a major drawback concerns inefficient cell surface modification caused by the poor association of hydrophilic macromolecules with cell surfaces. Here, a novel, highly efficient, and universal strategy in which nonreactive "additive" macromolecules are used to modulate the grafting efficiency of cell surface reactive, hydrophilic macromolecules is described. Unprecedented enhanced cell surface modifications by up to 10-fold were observed when various concentrations of a suitable "additive" polymer was present with a constant and low concentration of a "reactive" macromolecule. The importance of this increased efficiency and the possible mechanisms involved are discussed. The cell compatible technique is demonstrated in the case of four different cell types--red blood cells (RBC), leukocytes, platelets, and Jurkat cells. A practical application of grafting macromolecules to cell surfaces in concentrated polymer solutions is demonstrated by the enhanced camouflage of RBC surface antigens for the development of RhD null RBC. In principle, the technique can be adapted to various macromolecular systems and cell types, with significant potential for biomedical applications such as live cell based technologies.

Red Blood Cell Membrane Grafting of Multi-functional Hyperbranched Polyglycerols

The covalent attachment of hydrophilic polymers or biopharmaceuticals to the surface of red blood cells (RBCs) has previously been shown as a relatively compatible and effective method for a range of applications. Here, the first example of cell-surface grafting with a hyperbranched and multi-functional macromolecule is described. A range (3 kDa-101 kDa) of dense, globular, and blood compatible hyperbranched polyglycerols (HPG) were synthesized and functionalized with cell-surface reactive, succinimidyl succinate groups (1-12 groups per polymer). Subsequently, HPG was grafted to the RBCs, which were analyzed using physical characterization techniques such as aqueous two-phase partitioning and particle electrophoresis. It was found that the extent of grafting was enhanced by increasing HPG molecular weight, the number of reactive groups per HPG, HPG concentration, and reaction time. Good in vitro cell viability - as measured by lipid peroxidation, hemoglobin oxidation, cell lysis, osmotic fragility, stability in fresh serum and aggregation behavior - was observed for grafting concentrations up to 4.8 mm. The multi-functional aspect of HPG is highlighted by the following observations: using fluorescein-labeled Anti-D (monoclonal) antibody and flow cytometry, the detection of cell-surface Rhesus (RhD) antigens were significantly reduced upon HPG grafting. Secondly, the potential for using HPG as a multi-functional, delivery agent was demonstrated by attaching fluorescent markers to the HPG via degradable linkages prior to grafting.

In vivo Circulation, Clearance, and Biodistribution of Polyglycerol Grafted Functional Red Blood Cells

The in vivo circulation of hyperbranched polyglycerol (HPG) grafted red blood cells (RBCs) was investigated in mice. The number of HPG molecules grafted per RBC was measured using tritium labeled HPGs ((3)H-HPG) of different molecular weights; the values ranged from 1 × 10(5) to 2 × 10(6) molecules per RBC. HPG-grafted RBCs were characterized in vitro by measuring the electrophoretic mobility, complement mediated lysis, and osmotic fragility. Our results show that RBCs grafted with 1.5 × 10(5) HPG molecules per RBC having molecular weights 20 and 60 kDa have similar characteristics as that of control RBCs. The in vivo circulation of HPG-grafted RBCs was measured by a tail vain injection of (3)H-HPG60K-RBC in mice. The radioactivity of isolated RBCs, whole blood, plasma, different organs, urine and feces was evaluated at different time intervals. The portion of (3)H-HPG60K-RBC that survived the first day in mice (52%) remained in circulation for 50 days. Minimal accumulation radioactivity in organs other than liver and spleen was observed suggesting the normal clearance mechanism of modified RBCs. Animals gained normal weights and no abnormalities observed in necropsy analysis. The stability of the ester-amide linker between the RBC and HPG was evaluated by comparing the clearance rate of (3)H-HPG60K-RBC and PKH-26 lipid fluorescent membrane marker labeled HPG60K-RBCs. HPG modified RBCs combine the many advantages of a dendritic polymer and RBCs, and hold great promise in systemic drug delivery and other applications of functional RBC.

Influence of Polymer Architecture on Antigens Camouflage, CD47 Protection and Complement Mediated Lysis of Surface Grafted Red Blood Cells

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic delivery applications. The intermediate sized polymers (PEG or HPG) which showed greater antigen camouflage at lower grafting concentrations have significant potential in transfusion as universal red blood donor cells.

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