ChemComm Published on 18 April 2013 Downloaded by University of Missouri at Columbia on 01/10/2014 15:33:58 COMMUNICATION Cite this: Chem Commun., 2013, 49, 5393 Received 27th February 2013, Accepted 18th April 2013 DOI: 10.1039/c3cc41513a View Article Online View Journal | View Issue Red blood cells decorated with functionalized core–shell magnetic nanoparticles: elucidation of the adsorption mechanism† ´,b Christine Me ´nager,a Anne Varenne*b and Thanh Duc Mai,abc Fanny d’Orlye a Jean-Michel Siaugue* www.rsc.org/chemcomm The decoration of red blood cells (RBCs) with aminated and carboxylated core–shell magnetic nanoparticles (CSMNs) was studied and elucidated It was demonstrated that only aminated CSMNs could decorate the RBCs and their adsorption interaction is mainly ruled by electrostatic attraction between the positively charged amino groups on CSMNs and the abundant sialic acid groups on the outer surface of RBCs The use of ferric oxide nanoparticles for theranostic applications has gained much interest in recent years, notably as contrast agents for magnetic resonance imaging, colloidal mediators for heat generation and magnetic drug cargos with controllable release in time and space.1 Magnetic nanoparticles, nevertheless, still suffer from the major problem of limited bio-distribution Some notable strategies to achieve these nanoparticles with a longer-circulating attribute are PEG functionalization and/or conjugation with polymersomes, microgels and liposomes (consult ref for a typical example) A more natural and biological approach to prolong the in vivo circulation half-life of nanoparticles, inspired from life sciences, is to employ RBCs as theranostic vectors Recently, Kolesnikova et al gave an account of the preparation and application of RBC-based drug delivery vehicles in comparison with those of their synthetic polymeric counterparts.3 Applications of RBC-inspired delivery systems can be gleaned from some selected reviews4–7 whereas some recent biomedical uses of magnetized erythrocytes can be referred to in ref 8–10 To the best of our knowledge, magnetized red blood cells so far have been produced by the trapping of magnetic nanoparticles inside RBCs, a process in which the cell membrane is forced to distort by osmotic stress (see ref 11 for example) The modification of the a UPMC University of Paris 06-CNRS-ESPCI Laboratoire Physicochimie des Electrolytes, Colloădes et Sciences Analytiques PECSA UMR 7195, place Jussieu, 75252 Paris, France E-mail: christine.menager@courriel.upmc.fr b Chimie ParisTech, Ecole Nationale Supe´rieure de Chimie de Paris, Imagery, Chemical and Genetic Pharmacology Unit (UPCGI), UMR CNRS 8151 – U INSERM 1022, 11, rue Pierre et Marie Curie, 75231 Paris Cedex 05, France E-mail: anne-varenne@chimie-paristech.fr c Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi, Viet Nam E-mail: maithanhduc83@gmail.com † Electronic supplementary information (ESI) available: More experimental details and supporting figures See DOI: 10.1039/c3cc41513a This journal is c The Royal Society of Chemistry 2013 cell’s nature after this encapsulation, notably the release of hemoglobin and the uptake of unwanted compounds into its inner compartment, as a result, is inevitable and undesirable On the other hand, the decoration of RBCs with magnetic nanoparticles seems to be a more gentle technique With the ultimate goal of constructing a novel biocompatible, magnetically controllable platform for diagnostic and therapeutic applications, our group has very recently laid the groundwork for this technique by establishing some preliminary multimodal imaging demonstrations of CSMN-decorated RBCs.12 Following this pioneering work, important insights into the mechanism of grafting RBCs with CSMNs are reported herein Based on the work describing the interaction of functionalized nanoparticles with giant unilamellar vesicles13 and with supported lipid bilayers,14 two mechanisms of adsorption of CSMNs onto RBCs are addressed The first one relies on the well-known strong affinity of the silanol groups to the phosphatidylcholine-rich cell membrane.15,16 The second direction, which has recently been evidenced by the decoration of RBCs with hydroxyapatite,17 chitosan18 or gold19 nanoparticles, focuses on the general electrostatic interaction between the oppositely-charged functional groups on the concerned objects To unveil this mechanism for RBCs–CSMNs, a series of interrelated experiments, in which the charge of CSMNs was modulated, were carried out The detailed procedure to synthesize cationic and anionic CSMNs, as well as their charge and size characterisation, were described previously.12,13,20 Briefly, maghemite nanoparticles (g-Fe2O3, nm mean physical diameter) were embedded in a fluorescent silica shell by co-condensation of tetraethylorthosilicate (TEOS)† and 3-aminopropyltriethoxysilane (APTS)† reacted with fluorescein isothiocyanate (FITC).† The silica shell functionalization was then implemented by co-condensation of 2-(methoxy(polyethyleneoxy)propyl)-trimethoxysilane (PEOS)† and APTS.† The positive charge of cationic CSMNs can be tuned by varying the APTS to PEOS molar ratios (A/P) up to 3.5 The exclusion of APTS (A/P = 0) results in the formation of CSMNs containing only PEG with silanol functional groups on the surface Carboxylated nanoparticles were produced by conversion of amino to carboxylic functions (denoted by a negative value of the A/P ratio) The mean physical and hydrodynamic diameters of CSMNs are 40 nm and 75 nm, respectively In MOPS ((ionic strength I = 100 mM)† and sucrose Chem Commun., 2013, 49, 5393 5395 5393 View Article Online Published on 18 April 2013 Downloaded by University of Missouri at Columbia on 01/10/2014 15:33:58 Communication ChemComm Fig TEM micrographs of RBCs after being in contact for 15 with aminofunctionalised CSMNs of (A) 1.00 A/P; (B) 1.75 A/P Fig z Potentials of cationic (positive A/P ratio) and anionic (negative A/P ratio) CSMNs, as well as intact and CSMN-grafted RBCs according to the A/P ratio Dispersion medium: MOPS (I = 100 mM) and sucrose (C = 50 mM).† Each experimental point is the mean of replicates, and the error bars stand for Ỉone standard deviation (concentration C = 50 mM)† (pH 7.4)), CSMNs are stable for months The average concentration of CSMNs is  1017 particles per L CSMNs bearing different functional groups exhibit a variation in z potentials from À14 to +13 mV as shown in Fig A higher A/P ratio leads to a more positive charge,20 which results in a higher z potential The z potential of intact RBCs is around À10 mV The decoration of RBCs with cationic CSMNs leads to a higher z potential A level-off of the RBCs–CSMNs’ z potential curve was observed at A/P ratios higher than 1.75, leading to a value of +18 mV The presence of CSMNs bearing PEG–silanol or carboxylic groups (negative charges) does not affect the charge status of the surface of the erythrocytes The hybrid objects were then visualized using fluorescent microscopy The inclusion of FITC† into the shell layer of nanoparticles allows the tracking and localization of CSMNs on the surface of erythrocytes by observing the green fluorescence The RBCs after having been in contact with CSMNs for about 15 were pictured as in Fig The absence of interaction between anionic CSMNs and the cell membrane was evidenced by the lack of illumination (Fig 2A and B) On the other hand, the decoration of RBCs with cationic nanoparticles results in an observable green coating over the cell surface (Fig 2C and D) The obtained light intensity is correlated to the number of CSMNs on RBCs It thus indicates that the RBCs–CSMNs interactivity is proportional to the density of amino groups on the CSMNs The exterior of grafted RBCs was then zoomed in using transmission electron microscopy (TEM) The more CSMNs are adsorbed on the membrane faỗade, the better electronic contrast can be achieved As can be seen in Fig 3, the distribution of CSMNs of smaller A/P is more scattered, reflected by a less contrast imaging capture Fig Optical fluorescence images of RBCs after being in contact for 15 with (A) carboxylic-functionalized CSMNs, (B) PEG-functionalized CSMNs (A/P = 0), (C and D) amino-functionalized CSMNs (A/P = and 1.75, respectively) 5394 Chem Commun., 2013, 49, 5393 5395 Clearly, the RBC–CSMN interaction occurs only with nanoparticles possessing a positive charge due to the amino groups whereas those with negative charge induce no adsorption The wellstudied high affinity of silanol groups to phosphatidylcholine on the cell membrane does not trigger any decoration in this case It is very possibly because such affinity is suppressed due to the presence of PEG on the outer layer of CSMNs The addition of PEG to the shell of nanoparticles, albeit limiting the hemolytic activity thanks to its biocompatibility,21–23 and preventing these tiny particles from translocation into the bilayer membrane,13 diminishes the accessibility of surface silica-moieties to the outer layer of the erythrocytes, as already described elsewhere.16,24 Although phosphatidylcholine (PC), sphingomyelin (SPH) and cholesterol are the main components of the outer lipid membrane of erythrocytes, their surface charge is mainly due to the carboxyl groups of N-acetylneuraminic (sialic) acid residues in glycoproteins of the external surface.25,26 This negatively charged surface of RBCs facilitates the immobilization of cationic nanoparticles, and at the same time hinders any approach of carboxylic-functionalized counterparts The interaction was then carried out with both intact erythrocytes and those with a diminished density of sialic groups The diminution of sialic groups was implemented by enzymatic treatment of RBCs with neuraminidase – a selective enzyme for breakdown of the sialic group binding.26,27 After enzymatic treatment, an increase in z potential from À10 mV to about À6 mV was achieved The interaction of these enzyme-treated RBCs with cationic CSMNs, referenced to that of intact RBCs, is interpreted in terms of z potential as shown in Fig The enzymatic treatment leads to a less decoration degree, as observed with confocal microscopy in Fig The fluorescence intensity was higher intact RBC–CSMN Fig z Potentials of intact and enzyme-treated erythrocytes before and after adsorption of cationic CSMNs This journal is c The Royal Society of Chemistry 2013 View Article Online Published on 18 April 2013 Downloaded by University of Missouri at Columbia on 01/10/2014 15:33:58 ChemComm Communication Fig Confocal micrographs of an enzyme-treated and an intact erythrocyte after decoration with cationic CSMNs Intact RBCs–CSMNs of (A) 1.75 A/P and (B) 1.00 A/P; neuraminidase-treated RBCs–CSMNs of (C) 1.75 A/P and (D) 1.00 A/P (Fig 5A and B) whereas weak illumination was observed from enzyme-treated RBC–CSMN (Fig 5C and D) If the brightness from intact RBC–CSMN of 1.75 A/P is assigned as 100%, then those from intact RBC–CSMN of A/P, enzyme-treated RBC–CSMN of 1.75 A/P, and enzyme-treated RBC–CSMN of A/P are 50%, 56% and 3%, respectively (data obtained from the image-processing package ImageJ) The reduced interaction of cationic CSMNs with neuraminidase-treated RBCs confirms the theory that such decoration is mainly ruled by electrostatic attraction between sialic acid and amino functional groups The interaction between these functional groups was also evidenced by Delcea et al., using Raman spectroscopy.19 The in vitro stability of the hybrid object, in terms of desorption of CSMNs from RBCs and hemolytic activity, was then evaluated Our experimental data showed that within hours in the dispersion medium, CSMNs were still tightly attached to the erythrocyte membrane (see Fig S1, ESI†) A drastic reduction of the hybrid object’s z potential was observed after hours due to self-stripping of CSMNs from RBCs On the other hand, when the hybrid object was stored with a surfeit of CSMNs, no significant change in z potential was observed over 24 hours (data not shown) These results indicate that the interaction equilibrium is shifted to the decoration process in the presence of an excess of CSMNs in the medium The hemolysis of these magnetic RBCs was accordingly tested over hours during which the decoration is still observable Our results (see Fig S2, ESI†) showed that no hemolysis was induced by CSMNs of A/P = during this period, which is relevant to the results reported by Laurencin et al.12 However, CSMNs of higher A/P ratios were found to cause hemolysis after hours of incubation Cationic nanoparticles with a higher amino functional density provoked a more significant lysis of cells It seems that the more the adsorption process occurs, the more the rupture of the RBC membrane is induced Indeed, a similar phenomenon was already observed for the interaction of silica-based moieties with RBCs (see ref 28 and some references listed therein) Thus the subsequent design of the cationic-functionalized CSMNs for decoration should be improved according to the following objectives: (1) high imaging contrast by employing large magnetic cores and a high FITC concentration in the shell, (2) high magnetization degree by using large magnetic cores, and (3) high stability and low hemolysis of the magnetic RBCs by tuning the CSMNs amino group density and nature This journal is c The Royal Society of Chemistry 2013 It was demonstrated that the interaction between RBCs and CSMNs is mainly ruled by electrostatic attraction The facile decoration opens the floor for some possible applications at hand, such as quick detection of haemorrhage and monitoring of the healing processes Quantification of this adsorption interaction, i.e determination of the binding constant, as the ground work for any further optimization, will be soon carried out This work was supported by the fellowship for prospective researchers (Grant No PBBSP2_141401) from the Swiss National Science Foundation The authors thank Aude Michel for technical assistance and electron microscopy operation Notes and references S Laurent, D Forge, M Port, A Roch, C Robic, L V Elst and R N Muller, Chem Rev., 2008, 108, 2064–2110 M R Preiss and G D Bothun, Expert Opin Drug Delivery, 2011, 8, 10251040 ăhwald, Expert Opin Drug T A Kolesnikova, A G Skirtach and H Mo Delivery, 2013, 10, 47–58 R H Fang, C M J Hu and L F Zhang, Expert Opin Biol Ther., 2012, 12, 385–389 C.-M J Hu, R H Fang and L Zhang, Adv Healthcare Mater., 2012, 1, 537–547 S Biagiotti, M F Paoletti, A Fraternale, L Rossi and M Magnani, IUBMB Life, 2011, 63, 621–631 V R Muzykantov, Expert Opin Drug Delivery, 2010, 7, 403–427 C Cinti, M Taranta, I Naldi and S Grimaldi, PLoS One, 2011, 6, e17132 D E Markov, H Boeve, B Gleich, J Borgert, A Antonelli, C Sfara and M Magnani, Phys Med Biol., 2010, 55, 6461–6473 10 A Antonelli, C Sfara, E Manuali, I J Bruce and M 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This negatively charged surface of RBCs facilitates the immobilization of cationic nanoparticles, and at the same time hinders any approach of carboxylic -functionalized counterparts The interaction... respectively (data obtained from the image-processing package ImageJ) The reduced interaction of cationic CSMNs with neuraminidase-treated RBCs confirms the theory that such decoration is mainly... shifted to the decoration process in the presence of an excess of CSMNs in the medium The hemolysis of these magnetic RBCs was accordingly tested over hours during which the decoration is still