Previous results have shown that PEG-coated poly(hexadecylcyanoacrylate) (PEG-PHDCA) nanospheres displayed a significant accumulation within an orthotopic 9L gliosarcoma model, after i.v. administration to rats. Hence, the aim of the present study was to evaluate in the same model the pre-clinical efficacy of this carrier when loaded with Doxorubicin, an anticancer drug which poorly distributes in the CNS. Free and nanospheres-encapsulated Doxorubicin were administered with a multiple dose treatment. Their maximum tolerated dose (MTD) and increase in life span were respectively assessed in healthy and intracranially 9L-bearing rats. A comparative biodistribution study of Doxorubicin-loaded and unloaded PEG-PHDCA nanospheres was also performed in the tumor-bearing group. The results showed that the cumulative MTD of nanoparticulate doxorubicin was 1.5 times higher than this of free Doxorubicin. Nevertheless, encapsulated Doxorubicin was unable to elicit a better therapeutic response in the 9L gliosarcoma. Biodistribution study revealed that the Doxorubicin-loaded nanospheres accumulated to a 2.5-fold lesser extent in the 9L tumor as compared to the unloaded nanospheres and that they were mainly localized in the lungs and the spleen. Such a typical profile indicated aggregation with plasma proteins as a consequence of the positive surface charge of these loaded particles; this ionic interaction resulting from drug encapsulation was mainly responsible for 9L treatment failure.