Chitosan nanoparticles as delivery systems for doxorubicin
Introduction
Doxorubicin (DOX) and its bioactive derivatives are among the most widely used anticancer drugs in chemotherapy treatment [1]. However, problems related to the development of multidrug resistance [2] and acute cardiotoxicity [3] have led researchers to investigate alternative forms of administering DOX for the treatment of cancer, with both prodrug [4] and particulate [5] methods involved as active fields of DOX research for the past two decades.
DOX microencapsulation has shown some applications for the controlled release of DOX over extended periods of time [6]. Though relevant for solid, accessible tumors, these particles are too large to be endocytosed by most cells or circulate freely in the bloodstream. Consequently, the association of DOX to submicron carriers, such as liposomes [7], nanoparticles [8], or micelles [9], has drawn greater interest.
The majority of attempts to associate DOX to nanoparticulate carriers have used anionic or neutral polymers. Akiyoshi et al. [10] achieved DOX encapsulation in cholesterol-bearing pullulan hydrogel nanoparticles, though loading levels were very low (<0.1 wt.%) and cytotoxic effects of the nanoparticles were lower than that of free DOX. Combining prodrug and encapsulation strategies, Yoo et al. [11] covalently linked DOX to the terminus of poly(d,l-lactic-co-glycolic acid) (PLGA), then formed nanoparticles with the conjugate by an emulsion-solvent diffusion method. The group was able to obtain considerable loadings (3.45 wt.%), achieve a controlled release of DOX over nearly 1 month, and maintain antiproliferative activity relative to free DOX, though these results were possible only by forming the covalent linkage between the polymer and the drug.
The vast majority of work involving nanoparticulate DOX association, however, has been with polyacrylates, exploiting charge interactions of the polymer with the drug to achieve high association efficiencies. Polymethacrylate nanoparticles with adsorbed DOX were administered intravenously to hepatoma patients and demonstrated prolonged plasma levels, as well as reduced total clearance of DOX relative to a control DOX solution [12]. DOX associated to polyalkylcyanoacrylate nanoparticles [13] have demonstrated reduced cardiotoxicity following intravenous administration in mice [14] as well as increased cytoxicity against multidrug resistant cell lines in vitro [15]. Later work showed that coating of these particles with polysorbate 80 significantly increased DOX accumulation in brain tissue [16]. However, these DOX loaded particles have demonstrated acute renal toxicity [17] as well as decreased permeability of the drug across artificial membranes with respect to free DOX [18].
An alternative approach would be to entrap DOX into a positively charged carrier. Cell adhesion and potentially cell uptake of such particles should be favored due to their attraction to negatively charged cell membranes, an attractive feature for the treatment of solid tumors. From the perspective of intravenous administration, positively charged particles would interact with different blood components as compared to negatively charged particles. These changes could potentially create a different biodistribution and/or organ accumulation pattern following intravenous administration. Additionally, a positively charged system that would be expected to interact with cells and/or membranes would be desirable for testing alternative modes of administration of DOX, i.e. mucosal administration.
We believed that an interesting candidate with which to test these hypotheses was the cationic polysaccharide, chitosan. This biopolymer has shown favorable biocompatibility characteristics [19] as well as the ability to increase membrane permeability, both in vitro [20] and in vivo [21], and be degraded by lysozyme in serum [22]. Consequently, the aim of this paper was to encapsulate appreciable quantities of DOX in chitosan nanoparticles made by ionotropic gelation with sodium tripolyphosphate (TPP) and test the effects of DOX encapsulation and/or release on cytotoxic activity relative to free DOX. To achieve this aim, we tried two approaches: ionic bridging with a coincorporated polyanion and polymer/drug complexation.
Section snippets
Materials
Chitosan hydrochloride salt, Protasan CL 110 (Mw>100 kDa), was purchased from Pronova Biopolymers (Oslo, Norway). Doxorubicin hydrochloride was obtained as a 2 mg/ml solution in 0.9% (w/v) sodium chloride from Tedec-Meiji Farma (Madrid, Spain). TPP, type B gelatin (75 bloom), polyphosphoric acid, and dextran sulfate (Mw=10 kDa) were all purchased from Sigma-Aldrich S.A. (Madrid, Spain). Phosphorylated glucomannan was a gift from Industrial Farmacéutica Cantabria (Madrid, Spain). Unless
Results
The molecular structures of DOX and the complexing agents, polyphosphoric acid and dextran sulfate, are shown in Fig. 1. The protonable groups in the DOX molecule were expected to interact with the deprotonable groups of polyphosphoric acid and dextran sulfate.
The interaction of DOX with different polyanions and chitosan was first investigated spectrophotometrically. As seen in Fig. 2A and B, the DOX peak at 480 nm was reduced by ∼53% upon incubation with either polyphosphoric acid (Fig. 2A) or
Discussion
The major goal of this work was to develop a chitosan nanoparticulate system as a novel, positively charged, colloidal carrier for DOX. The greatest challenge was to encapsulate appreciable quantities of DOX, overcoming the charge repulsion between the cationic polymer (pKa=6.5) [26] and the predominantly positively charged anthracycline drug (pKa=8.2) [27]. To begin, we selected a chitosan–TPP nanoparticle formulation [23] which could accommodate a large quantity of TPP (only ∼25% molar excess
Conclusion
In this paper, we describe the feasibility of using chitosan nanoparticles as colloidal carriers for the delivery of the small, cationic anthracycline drug, doxorubicin (DOX). By incorporating the polyanion, dextran sulfate, we were able to encapsulate considerably high quantities of DOX considering the inherent polymer–drug charge repulsion. These particles demonstrated a minimal burst release and retained the cytotoxic activity of DOX in vitro. Additionally, we showed that DOX can be
Acknowledgments
We would like to thank Mónica Hombreiro Pérez for her valuable help in the preparation of the samples for the confocal studies. This work was supported by a grant from the Spanish government (SAF97-0169). K.A.J. and M.P.F. would like to additionally thank the USIA Fulbright Association and the UE Socrates Program, respectively, for financial support.
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