Signal transduction by erythrocytes on specific binding of doxorubicin immobilized on nanodispersed magnetite

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Abstract

Two specific binding sites for doxorubicin were revealed at the plasma membrane of human erythrocytes on investigation of the binding of doxorubicin magnetic nanoconjugates. Free and conjugated doxorubicins modulated signal transduction in erythrocytes in a similar way. Both up-regulated nitric oxide and cyclic GMP (cGMP) and down-regulated cyclic AMP (cAMP) production and stabilize the membranes of damaged erythrocytes.

Introduction

Doxorubicin (DOX), whose principle target was previously thought to be DNA, has been shown to exert its cytotoxic action solely by cell surface interaction since extracellular drug was required to initiate the cytotoxic cascade [1], [2]. These findings initiated studies with DOX conjugates to find more efficient drug forms and to reduce the toxicity of free DOX. Increasing evidence firmly suggests that the underlying mechanism for anthracycline cytotoxicity is the induction of apoptosis through intracellular-mediated signaling pathways [3], [4]. It has also been shown that adriamycin initiates inhibition of the growth-related NADH oxidase in the plasma membrane of cancer cells associated with apoptosis [5]. Nevertheless, the events in DOX-induced apoptosis are poorly understood and determining the mechanisms of DOX membrane effects remains a challenge.

The purpose of this work was to investigate the binding of DOX, immobilized on a magnetic nanomaterial, to intact human erythrocytes, and to test the effects of this conjugate on signal transduction in human erythrocytes using quantitative determination of the intracellular cAMP and cGMP pools and the stable nitric oxide (NO) metabolites, NO2 and NO3. Human erythrocytes were chosen as a model cell system due to the known interactions of DOX with blood components. These interactions determine, to a great extent, DOX pharmacokinetics [6]. The DOX-erythrocyte binding mechanisms include the embedding of DOX into the lipid matrix, the insertion of DOX into the erythrocyte stroma using the ‘flip-flop’ mechanism [7], or, depending on the level of cholesterol in the membrane, using a specific transporter [8].

Section snippets

Experimental

All chemicals were purchased from Sigma-Aldrich. Nanodispersed magnetite was modified by oligomerization of γ-aminopropyltriethoxysilane and activated with 1,6-diisocyanatohexane as described elsewhere [9], followed by chemisorption of hydroxyethylstarch from aqueous solution and repeated activation with 1,6-diisocyanatohexane. The mean diameter of the particle core was 24±19 nm according to electron microscopy data (Fig. 1) and the average nanocrystal size was 30 nm, as calculated from the

Results and discussion

DOX immobilized at the surface of magnetite nanoparticles retained its ability to bind its target receptors in competition with free DOX. Fig. 3 shows the binding of immobilized DOX to human erythrocytes in the presence of 10−12–10−5 M free DOX. The introduction of free DOX inhibited immobilized DOX binding, providing evidence of specific DOX binding at the cell surface. As shown by the concurrent binding measurements (Fig. 3), there are two highly specific binding sites (receptor pools) for DOX

Conclusion

There are specific binding sites for DOX at the plasma membrane of human erythrocytes. The DOX-M conjugate does not penetrate into the cell for at least an hour. It has been established that free and conjugated DOX are antagonists of the erythrocyte receptors coupled to the phospholipid signal system. This is evidenced by an increase in NO and cGMP levels. On the basis of the experimental data obtained, one can concede the following sequence of events initiated by DOX interaction with

Acknowledgments

We thank Dr. L. Stechenko for performing the electron microscopic studies. We also thank Dr. G. Solyanik for testing the anticancer activity of DOX conjugates in vitro and Dr. J. Gerloff for valuable discussions.

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