Tailoring the Properties of Polydiacetylene Nanosystems for Enhanced Cell Tracking Through Poly(glycerol Adipate) Blending: an In Vitro and In Vivo Investigation

ABSTRACT This study presents novel mixed nanosystems composed of 10,12‐pentacosadiynoic acid (PCDA) and biodegradable, enzymatically synthesized polymers—PGA, PGA HEX , or PGA BDM — designed to alter and exploit PCDA's chromatic properties. These nanoparticles offer a cost‐effective, non‐leachable tracking system suitable for both in vitro and in vivo biological assays. Notably, they enable real‐time monitoring without the need for additional fluorescent dyes, a common requirement in conventional systems. Fabricated via a simple nanoprecipitation method, the nanoparticles exhibit a narrow size distribution, zeta potential, and spherical morphology. Their enhanced stability in response to temperature and phosphate‐buffered saline (PBS) is attributed to hydrogen bonding, hydrophobic interactions, and π – π stacking within the polymeric matrix. Preliminary cytotoxicity assessments using MCF‐7 human breast cancer cells and the invertebrate model Caenorhabditis elegans —aligned with the 3R principle of replacing higher animals—demonstrated good biocompatibility. Importantly, both cellular and nematode uptake of PCDA and PCDA/PGA nanoparticles was successfully achieved, confirming their utility for both in vitro and in vivo tracking without the need for additional dyes.