Dual pH-Responsive Calcium Phosphate Nanoparticles Conjugated with Folate by CuAAC Click Chemistry for Targeted Gemcitabine Delivery to Cancer Cells

Calcium phosphate nanoparticles (CaP NPs) are biocompatible carriers widely studied for drug delivery due to their pH-responsive degradation and controlled release properties. In this study, CaP NPs stabilized with carboxymethyl cellulose (CMC) and coated with a silica layer were designed for gemcitabine (GEM) loading and folate (FA) conjugation, targeting cancer cells overexpressing folate receptor alpha (FRα). GEM was covalently coupled to CMC via an amide bond before CaP precipitation, creating a prodrug system. The NPs exhibited dual pH-responsive release, in which CaP dissolution combined with polymer-drug cleavage through acid-catalyzed hydrolysis of CMC-GEM within endolysosomes ensured intracellular bioavailability of free GEM molecules. FA conjugation by strong covalent bonds via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction enhanced the uptake of CaP NPs in FRα-positive breast cancer cells (MCF-7), whereas both FA-conjugated and nonconjugated NPs exhibited similar uptake in normal human mesenchymal stem cells (hMSCs). GEM-loaded CaP NPs showed high cytotoxicity in FRα-overexpressing cancer cell lines (MCF-7, MDA-MB-231, HeLa), while FA conjugation significantly reduced toxicity in hMSCs without compromising anticancer activity. These findings demonstrate the potential of FA-conjugated and GEM-loaded CaP NPs as a nanoplatform for targeted cancer therapy with reduced toxicity in healthy cells.