Reversion of multidrug resistance by co-encapsulation of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles.

Individual and combined polyalkylcyanoacrylate nanoparticle formulation of cyclosporin A and doxorubicin were prepared and evaluated in an attempt to show improved growth inhibition efficacy in a resistant cell culture line. The drug loaded nanoparticles were prepared using the well established emulsion polymerization process without using any modification for the hydrophilic doxorubicin drug whereas the incorporation of cyclosporin A needed to wait a moment after the polymerization reaction started. This was necessary to avoid cyclosporin A precipitation and polymer aggregation. Cyclosporin A release from the nanoparticles was rapid probably because the drug was adsorbed onto the nanoparticles surface rather than embedded into the polymeric core. Doxorubicin displayed also a burst effect but with a slower second phase probably related with the nanoparticles bioerosion rate owing to its entrapment in the polymeric network. Finally, it was shown in resistant cell culture experiments that the association of both cyclosporin A and doxorubicin within a single nanoparticle formulation elicited the most effective growth rate inhibition as compared to other combinations of both drugs while using a lower amount of polymer compared to separated nanoparticle formulations. This result was probably due to the synergistic effect achieved by combining the chemo-sensitizing compound cyclosporin A, with an effective cytotoxic drug like doxorubicin.

Investigation of the role of macrophages on the cytotoxicity of doxorubicin and doxorubicin-loaded nanoparticles on M5076 cells in vitro.

Doxorubicin-loaded PACA nanoparticles have been shown to be more efficient than free drug in mice bearing hepatic metastasis of the M5076 tumour. Due to the high phagocytic activity of Küpffer cells in the liver, it may be that these cells played a role of drug reservoir after nanoparticle phagocytosis. Therefore, the objective of this study was to assess the role of macrophages in mediating the cytotoxicity of doxorubicin-loaded nanoparticles on M5076 cells. The growth inhibition of tumour cells was evaluated in two ways: firstly, the cells were incubated in a coculture system consisting of special wells with two compartments separated by a porous membrane. M5076 cells were seeded into the lower compartment and the macrophages J774.A1 were introduced into the upper part. The macrophages were activated or not by IFN-gamma. The drug preparations were added only in the macrophage insert. Secondly, growth inhibition was also assessed in the conventional way, i.e. in direct contact with the tumour cells to serve as a reference. After direct contact, free doxorubicin (Dox) and doxorubicin-loaded nanoparticles (NP-Dox) had the same efficacy against M5076 cell growth. The coculture experiments led to a 5-fold increase in the IC(50) for both Dox and NP-Dox. The activation of macrophages by IFN-gamma in coculture significantly decreased the IC(50) values. In conclusion, after phagocytosis of doxorubicin-loaded nanoparticles, J774.A1 cells were able to release active drug, allowing it to exert its cytotoxicity against M5076 cells. Drug efficacy was potentiated by the activation of macrophages releasing cytotoxic factors such as NO, which resulted in increased tumour cell death. Thereby, the coculture system permitted us to investigate the macrophage-mediated cytotoxicity of colloidal carriers loaded with an anticancer drug, which is of great interest when further i.v. administration is envisaged.

Ability of doxorubicin-loaded nanoparticles to overcome multidrug resistance of tumor cells after their capture by macrophages.

PURPOSE: Investigation of the ability of doxorubicin-loaded nanoparticles (NP/Dox) to overcome multidrug resistance (MDR) when they have first been taken up by macrophages. METHODS: The growth inhibition of P388 sensitive (P388) and resistant (P388/ADR) tumor cells was evaluated in a coculture system consisting of wells with two compartments. The tumor cells were seeded into the lower compartment, the macrophages were introduced into the upper part in which the drug preparations were also added. RESULTS: Doxorubicin exerted lower cytotoxicity on tumor cells in coculture compared with direct contact. In P388/ADR, NP/Dox cytotoxicity was far higher than that of free doxorubicin (Dox). Three different formulations of cyclosporin A (either free (CyA), loaded to nanoparticles (NP/CyA) or in a combined formulation with doxorubicin (NP/Dox-CyA)), were added to modulate doxorubicin efficacy. The addition of cyclosporin A to Dox increased drug cytotoxicity. Both CyA added to NP/Dox and NP/Dox-CyA were able to bypass drug resistance. CONCLUSIONS: Despite the barrier role of macrophages, NP/Dox remained far more cytotoxic than Dox against P388/ADR. Both NP/Dox + CyA and NP/Dox-CyA allowed to overcome MDR, but the last one should present greater advantage in vivo by confining both drugs in the same compartment, hence reducing the adverse effects.