José A. Jordán

In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross-linking or diamide treatment

Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red‐blood‐cell membrane surface, namely oxidant compounds, such as ascorbate/Fe+2 and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3‐cross‐linking reagents. We monitored the phagocytosis of oxidized or Band 3‐cross‐linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe3+, but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross‐linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS3) and 3,3′‐dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS3‐cross‐linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short‐term delivery to cells of the mononuclear phagocytic system.

Differential In vitro and In vivo Behavior of Mouse Ascorbate/Fe3+ and Diamide Oxidized Erythrocytes

Chemical oxidation of mouse erythrocytes has been carried out using two different oxidizing systems namely: Diamide and Ascorbate/Fe3+ together with different concentrations of the oxidant. These oxidation treatments produced different extents of modification in membrane proteins as was observed by electrophoretic analyses that showed a possible formation of high molecular weight aggregates. Lipid peroxidation was also observed as the result of these chemical treatments. The action of these two oxidation treatments produced different extents of lipid peroxidation in which the effect Ascorbate/Fe3+ reached higher values than that shown by diamide treatments. To study the resulting in vitro behavior of such oxidized erythrocytes, we have evaluated the recognition of oxidized erythrocytes by peritoneal macrophages. In the conditions used, diamide oxidized erythrocytes were more highly recognized by macrophages than Ascorbate/Fe3+ treated erythrocytes. However, in both cases an influence of serum factors in the recognition process can be inferred. Additionally, we have correlated on one side the action of different oxidation systems on mouse erythrocytes with different in vivo behavior and organ uptake of the oxidized erythrocytes. On the other side, differential targeting of oxidized erythrocytes to a liver or spleen was observed on dependence of the oxidant used.

Band‐3 crosslinking‐induced targeting of mouse carrier erythrocytes

Mouse band‐3 crosslinked carrier erythrocytes have been prepared. [125I]Carbonic anhydrase (CA) has been encapsulated into mouse erythrocytes. Then, loaded erythrocytes were labelled with 51Cr. Eventually, these doubly labelled cells were crosslinked with band‐3 crosslinking reagents. [125I]CA was shown to have cytosolic localization in crosslinked carrier erythrocytes. Estimation of the action of band‐3 crosslinkers on mouse carrier‐erythrocyte membranes rendered values around 17–21% of band‐3 monomer reduction. Crosslinked carrier erythrocytes were in vivo targeted to liver, as shown by chromium‐labelling localization. Also, encapsulated CA radioactivity was localized in vivo predominantly in liver, which is clearly in contrast with the behaviour shown by free CA injected into animals. These results support this model as a feasible system for the analysis of carrier‐erythrocyte survival and targeting as well as the in vivo efficacy of release and targeting of encapsulated compounds.

IN VIVO BEHAVIOUR OF RAT BAND 3 CROSS-LINKED CARRIER ERYTHROCYTES

Rat band 3 cross-linked carrier erythrocytes have been prepared. Iodinated carbonic anhydrase has been encapsulated into rat erythrocytes. Then, carrier erythrocytes were labeled with 51chromium. Eventually, these doubly labeled rat RBCs were treated with a band 3 cross-linking reagent, namely bis(sulfosuccinimidyl)suberate (BS3). 51Chromium labeling and 125I CA showed to have cytosolic localization in cross-linked carrier erythrocytes. Estimation of the band 3 cross-linking induced by BS3 on rat carrier erythrocytes has been done rendering values around 25% of band 3 monomer reduction. BS3-cross-linked carrier erythrocytes when injected into rats are mainly targeted to liver as shown by chromium labeling localization. Also, encapsulated CA radioactivity carried by cross-linked carrier rat erythrocytes when injected into rats is localized predominantly in liver as shown by in vivo experiments. Accordingly, cross-linked carrier erythrocytes are highly recognized by peritoneal macrophages as detected by in vitro analyses of macrophage recognition. Thus, our data revealed a targeting of carrier rat erythrocytes induced by cross-linking of band 3 protein by BS3. These results support claims in favor of this animal model as a feasible system to analyze cross-linked carrier erythrocytes survival and targeting as well as the in vivo efficacy of targeting of loaded compounds to liver.

Influence of Chemical Modification on “ In Vivo ” and “ In Vitro ” Mouse Carrier Erythrocyte Survival and Recognition

Several systems have been developed with therapeutical purposes for drug delivery. Among them, red blood cells (RBCs) have been claimed to be a physiological method to convey and deliver active compounds.4,6,8,11 The preparation of erythrocytes as carriers can require encapsulation procedures or/and chemical modification of erythrocyte surface.3,22 In fact, the efficacy of these systems can be dependent of the use of several chemical treatments which can react with cell membrane proteins. Crosslinking reagents can be applied to red blood cell modification. Glutaraldehyde (GA) has been the most extensively used crosslinker.7,21 Also, other crosslinkers can be applied to carrier erythrocytes preparation.12,16 Biotinylation is another alternative method for carrier preparation.15,19 Eventually, chemical modification can promote targeting of carrier erythrocytes to several organs.12,21 We have focused our attention on the action of crosslinking reagents which react with band 3 in mouse erythrocyte membrane. Additionally, biotinylation of mouse erythrocytes has been studied. “In vivo” behaviour of these chemically modified erythrocytes have been studied. These survival results have been compared to recognition by macrophages. Thus, we described conditions for using these chemical treatments for targeting to organs and macrophages.

Rat Carrier Erythrocytes Circulate and Arrive to Organs

Different delivery systems are currently used in therapy. They have the advantage of protecting the active substance from rapid clearance and avoiding toxic side effects. Among the many carrier systems proposed12, RBCs have many desirable properties: they are naturally biodegradable and may stay in circulation over prolonged periods of time11,25; RBCs are easily obtainable and large amounts of material can be entrapped in a small volume of cells by hypotonic dialysis; autologous cells elicit little or no immune response7,16,20.