In this paper we report on three different hydrophilic copolymers based on alpha,beta-polyaspartylhydrazide (PAHy) bearing butyric groups in the side chain (C-4) (PAHy-C-4) or a combination of butyric groups and positive charged residues ((carboxypropyl)trimethylammonium chloride, CPTACI) (PAHy-C-4-CPTA) that were synthesized and used for the preparation of new supramolecular vesicular aggregates (SVAs) containing gemcitabine as an antitumor drug. Gemcitabine-loaded SVAs containing synthesized PAHy derivatives were characterized from the physicochemical and technological point of view and the in vitro toxicity and anticancer activity on two different human cancer cell lines, i.e., CaCo-2 (human colon carcinoma) and ARO (human anaplastic thyroid carcinoma) cells, were also evaluated. Moreover, considering that carrier-cell interaction is an important factor to achieve an improvement of anticancer drug activity, confocal laser scanning microscopy and flow cytometric experiments were carried out on the two different cancer cell lines.