The spiro-acridine derivative (E)-1'-(benzylideneamino)-5′-oxo-1′,5′-dihydro-10H-spiro[acridine-9,2′-pyrrole]-4′-carbonitrile (AMTAC-01) is a drug candidate with anticancer activity through DNA entanglement and inhibition of the enzyme topoisomerase IIα. However, due to its poor water solubility, it has unfavorable bioavailability. Therefore, nanocarriers, such as polymeric nanoparticles, are an available means to enable the application of AMTAC-01 in humans, and the delivery of the nanoparticle can be improved by the enhanced permeability and retention (EPR) effect in the tumor. Therefore, the aim of this study was to develop poly(lactic acid) (PLA) nanoparticles containing AMTAC-01 for two methods (nanoprecipitation and single emulsion/evaporation) and to investigate their physicochemical properties and anticancer activity against colon cancer cells. The morphology and encapsulation efficiency of the nanoparticles were evaluated. In addition, the anticancer activity was investigated using a colon cancer cell line (HCT-116). The nanoparticles were successfully prepared and showed a unimodal particle size distribution (110–180 nm) with a spherical morphology and negative zeta potential. The encapsulation efficiency (E.E.) varied depending on the preparation method: the nanoprecipitation method (NP-1) has an E.E. of 50.1 % and single emulsion/evaporation (NP-2) 84.5 %. Additional chemical and physical characterization suggested entrapment of AMTAC-01 in the nanoparticles. Thermal analysis also indicated good complexation of AMTAC-01 and the polymer matrix. Cytotoxicity assays showed increased activity of AMTAC-01 in the nanoparticles in colon cancer cells (HCT-116), and cell cycle assay showed that NP-2 induced apoptosis of HCT-116 cancer cells by inducing DNA fragmentation. In conclusion, PLA nanocarriers are a viable tool that enable the therapeutic use of AMTAC-01.