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

Abstract

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][2][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 ions 3 . 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 antigens 10,11 . Video LinkThe video component of this article can be found at http://www.jove.com/video/50075/ Protocol A. Hyperbranched Polyglycerol Modification (SS-HPG)1. Place lyophilized HPG 60 kDa (0.5 g, 0.0083 mmol) in a round bottom flask and dry it overnight under vacuum at 90 °C. 2. Refrigerate the flask to room temperature, and dissolve the dried HPG in anhydrous pyridine (3 ml). 3. 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 succinic anhydride, (0.0067 g, 0.0664 mmol) dissolved in 0.5 ml pyridine drop wise over 10 min. Stir the mixture overnight at room temperature under argon. 4. Precipitate the mixture in 40 ml of cold acetone (4 °C) in a 50 ml centrifuge tube, and centrifuge using a Beckman J2-MC centrifuge at 27,000x g for 15 min. Decant the supernatant, and remove residual acetone by flushing with argon at room temperature. 5. To activate the carboxyl groups with succinimidyl succinate (SS) groups, dissolve the carboxyl -functionalized HPG in 3 ml of anhydrous DMF.Add N-hydroxysuccinimide (0.0077 g, 0.0664 mmol) and N, N'-diisopropylcarbodiimide (0.0084 g, 0.0664 mmol) to the HPG solution and stir the mixture overnight at room temperature under argon. 6. Purify the SS-HPG by precipitation in cold acetone. 7. Remove residual acetone by flushing with argon. 8. Determine the purity of modified HPG and the degree of carboxyl and SS functionalization by proton ( 1 H)-NMR analysis.

The effect of hyperbranched polyglycerol coatings on drug delivery using degradable polymer nanoparticles

“…HPG is a hyperbranched, hydrophilic polymer with a high density of hydroxyl groups on its surface: it is more hydrophilic than PEG and has been demonstrated to have better compatibility and non-specific resistance to biomolecules than PEG in certain applications [12, 14, 32]. HPG is well known to have a lower intrinsic viscosity than linear PEG, which decreases the possibility of red cell aggregation when present in the circulation [20] [33]. While HPG has been explored in a variety of biomedical settings—principally for coatings on implanted materials—it has never before been tested as a surface coating for drug delivery systems.…”

Engineering the Mammalian Cell Surface with Synthetic Polymers: Strategies and Applications

“…In an attempt to provide an alternative to PEG, previously shown to trigger the production of anti‐PEG antibodies, [ 52–54 ] hyperbranched polyglycerol was used to engineer the RBCs’ surface, providing an effective method for immunocamouflage. Similarly to PEG, HPG coating was successfully able to shield antigens at the surface of RBCs, providing a significant reduction in the level of antibody binding to cells.…”