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The preparation, in-vitro release, in-vitro cytotoxicity, and in-vivo drug delivery of doxorubicin (DOX)-loaded chitosan (CS)-poly(acrylic acid) (PAA) hollow nanospheres were investigated. The loading was done by dissolving a certain amount of DOX in non-cross-linked CS-PAA nanospheres aqueous solution followed by cross-linking chitosan with glutaraldehyde. The drug-loading content was up to 4.3% and the size of drug-loaded hollow nanospheres, determined by dynamic light scattering, was 118 nm. The nanospheres showed a continuous release of the entrapped DOX up to 10 days in vitro and showed comparable in-vitro cytotoxicity against HepG2 cells compared to the free DOX. In-vivo DOX delivery of DOX-loaded CS-PAA nanospheres showed that DOX concentration in blood can be maintained for a longer period than free DOX solution, and the DOX concentration in mice liver can be maintained constantly at relatively high level. The interesting feature of DOX-loaded CS-PAA hollow nanopspheres is that the loaded DOX can be delivered into the mice brain. The confocal laser scanning microscopy analysis reveals that fluorescein isothiocyanate (FITC)-labeled CS-PAA can deposit in different organs including liver, spleen, and brain. [ABSTRACT FROM AUTHOR]