This research was funded by the Canadian Blood Services (CBS) and the Canadian Institutes of Health Science (CIHR) Research Partnership Fund. The authors thank the LMB Macromolecule Hub at the UBC Centre for Blood research for the use of their research facilities. The infrastructure facility is supported by the Canada foundation for Innovation (CFI) and the Michael Smith Foundation for Health Research (MSFHR). R. Chapanian is a recipient of (CIHR/CBS) postdoctoral fellowships in Transfusion Science and a recipient of MSFHR research trainee post doctoral fellowship. J.N. Kizhakkedathu is a recipient of MSFHR Career Investigator Scholar Award.

A. Hyperbranched Polyglycerol Modification (SS-HPG)
<ol>
 <li>Place lyophilized HPG 60 kDa (0.5 g, 0.0083 mmol) in a round bottom flask and dry it overnight under vacuum at 90 &deg;C.</li>
 <li>Refrigerate the flask to room temperature, and dissolve the dried HPG in anhydrous pyridine (3 ml).</li>
 <li>To functionalize approximately eight hydroxyl groups on HPG with carboxyl groups, add catalytic amount of dimethylaminopyridine (one drop of 5 mg/ml solution in pyridine) to the HPG solution. To this mixture, add succini...

<ol>
	<li>Bradley, A. J., Murad, K. L., Regan, K. L., Scott, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+consequences+of+linker+chemistry+and+polymer+size+on+stealth+erythrocytes:+size+does+matter.">Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter.</a> Biochim. Biophys. Acta. 1561 (2), 147-158 (2002).</li><li>Murad, K. T., Mahany, K. L., Brugnara, C., Kuypers, F. A., Eaton, J. W., Scott, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+consequences+of+antigenic+modulation+of+red+blood+cells+with+methoxypoly(ethylene+glycol.">Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol.</a> Blood. 93 (6), 2121-2127 (1999).</li><li>Scott, M. D., Murad, K. L., Koumpouras, F., Talbot, M., Eaton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+camouflage+of+antigenic+determinants:+Stealth+erythrocytes.">Chemical camouflage of antigenic determinants: Stealth erythrocytes.</a> Proc. Natl. Acad. Sci. U.S.A. 94 (14), 7566-7571 (1997).</li><li>Daniels, G., Reid, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+groups:+the+past+50+years.">Blood groups: the past 50 years.</a> Transfusion. 50 (2), 281-289 (2010).</li><li>Murad, K. L., Gosselin, E. J., Eaton, J. W., Scott, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stealth+cells:+Prevention+of+major+histocompatibility+complex+class+II-mediated+T-cell+activation+by+cell+surface+modification.">Stealth cells: Prevention of major histocompatibility complex class II-mediated T-cell activation by cell surface modification.</a> Blood. 94 (6), 2135-2141 (1999).</li><li>Kainthan, R. K., Hester, S. R., Levin, E., Devine, D. V., Brooks, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+biological+evaluation+of+high+molecular+weight+hyperbranched+polyglycerols.">In vitro biological evaluation of high molecular weight hyperbranched polyglycerols.</a> Biomaterials. 28 (31), 4581-4590 (2007).</li><li>Kainthan, R. K., Janzen, J., Levin, E., Devine, D. V., Brooks, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+testing+of+branched+and+linear+polyglycidol.">Biocompatibility testing of branched and linear polyglycidol.</a> Biomacromolecules. 7 (3), 703-709 (2006).</li><li>Chapanian, R., Constantinescu, I., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+circulation,+clearance,+and+biodistribution+of+polyglycerol+grafted+functional+red+blood+cells.">In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.</a> Biomaterials. 33 (10), 3047-3057 (2012).</li><li>Chapanian, R., Constantinescu, I., Rossi, N. A. A., Medvedev, N., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+polymer+architecture+on+antigens+Camouflage,+CD47+protection+and+complement+mediated+lysis+of+surface+grafted+red+blood+cells.">Influence of polymer architecture on antigens Camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.</a> Biomaterials. 33 (31), 7871-7883 (2012).</li><li>Rossi, N. A. A., Constantinescu, I., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+cell+surface+polymer+grafting+in+concentrated+and+nonreactive+aqueous+polymer+solutions.">Enhanced cell surface polymer grafting in concentrated and nonreactive aqueous polymer solutions.</a> J. Am. Chem. Soc. 132 (10), 3423-3430 (2010).</li><li>Rossi, N. A. A., Constantinescu, I., Kainthan, R. K., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+membrane+grafting+of+multi-functional+hyperbranched+polyglycerols.">Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols.</a> Biomaterials. 31 (14), 4167-4178 (2010).</li><li>Muzykantov, V. R., Smirnov, M. D., Domogatsky, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+complement+activity+assay+in+microtitration+plates.">Hemolytic complement activity assay in microtitration plates.</a> J. App. Biochem. 7 (3), 223-227 (1985).</li><li>Walter, H., Brooks, D. E., Fisher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Partitioning in aqueous two-phase systems: theory, methods, uses, and applications to biotechnology. , (1985).</li><li>Rossi, L., Serafini, S., Pierige, F., Antonelli, A., Cerasi, A., Franternale, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte-based+drug+delivery.">Erythrocyte-based drug delivery.</a> Expert Opin. Drug Deliv. 2 (2), 311-322 (2005).</li><li>Walter, H., Krob, E. J., Brooks, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+surface+properties+other+than+charge+involved+in+cell+separation+by+partition+in+polymer,+aqueous+2-phase+systems.">Membrane surface properties other than charge involved in cell separation by partition in polymer, aqueous 2-phase systems.</a> Biochemistry. 15 (14), 2959-2964 (1976).</li><li>Muzykantov, V. R., Murciano, J. C., Taylor, R. P., Atochina, E. N., Herraez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+complement-mediated+elimination+of+red+blood+cells+modified+with+biotin+and+streptavidin.">Regulation of the complement-mediated elimination of red blood cells modified with biotin and streptavidin.</a> Anal Biochem. 241 (1), 109-119 (1996).</li></ol>

No conflicts of interest declared.

Universal donor RBCs have great potential in enhancing blood availability and safety for blood transfusion therapy. RBCs are also considered promising drug delivery vehicles due to their long circulation and inherent biocompatibility 14, 15. Experiments presented in this paper evaluate the in vitro characteristics of HPG modified RBCs. The in vitro properties and in vivo circulation of HPG modified RBCs have been investigated in our group recently 8, 11. The partition...

<table border="1">
 <tbody>
 <tr>
 <td>Glycidol </td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>Trimethylolpropane </td>
 <td>Fluka </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>Potassium methylate</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Anhydrous pyridine</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>4-Dimethylaminopyridine</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Succinic anhydride</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Anhydrous dimethyl formamide</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>N-Hydroxysuccinimide </td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>N,N'-Diisopropylcarbodiimide </td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>MTS cards</td>
 <td></td>
 <td></td>
 <td>Micro Typing System (MTS) cards (FL, USA)</td>
 </tr>
 <tr>
 <td>Dextran 500 kDa</td>
 <td>Pharmacia Fine Chemicals</td>
 <td></td>
 <td>(Sweden)</td>
 </tr>
 <tr>
 <td>PEG 8 kDa</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>FITC monoclonal anti-Rhesus D (RhD)</td>
 <td>Quotient Biodiagnostics</td>
 <td></td>
 <td>(PA, USA)</td>
 </tr>
 <tr>
 <td>PE monoclonal anti-CD47 </td>
 <td>BD Biosciences </td>
 <td></td>
 <td>(NJ, USA) </td>
 </tr>
 <tr>
 <td>Drabkin's reagent </td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table. Chemicals and reagents used for the grafting of HPG polymers to RBC membrane and their analysis.</td>
 </tr>
 </tbody>
 </table>

antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

Camouflage of Rhesus D antigen and CD47 RBC surface protein were quantified by flow cytometry using fluorescent labelled monoclonal antibodies, and a representative result is given in Figure 1. In case of HPG-grafted RBCs (grey), the intensity of the signal decreased (peak shifted to left) compared to the control RBCs (red &amp; green) indicating a reduction in binding to antibodies to cell surface which indicate the masking of surface proteins.
	<p class="jove_content" fo:keep-together.within-...

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

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.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

antigens-protected-functional-red-blood-cells-membrane-grafting

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Immunology and Infection

18.0K visualizações.  University of British Columbia. A modificação da membrana celular das células vermelhas do sangue (RBC) com poliglicerol hiper-ramificado (HPG) é apresentada. Hemácias modificadas foram caracterizadas por partição de fase aquosa dois, fragilidade osmótica e lise mediada pelo complemento. A camuflagem de proteínas de superfície e antígenos foi avaliada usando a citometria de fluxo e Sistema de Digitação Micro (MTS) cartões de fenotipagem de sangue.