We have designed series of monodispersed nanohydrogels (NHGs) with sizes ranging from 20 to 400 nm, generated from mixtures of N-isopropylacrylamide, di-block (hydrophilic-hydrophobic), and tri-block (hydrophobic-hydrophilic-hydrophobic) copolymer acrylamide macro-monomers. When the monomers are mixed at high temperature they collapse into well-defined self-assemblies, which can be further polymerized leading to cross-linked NHGs with sizes matching the intermediate self-assemblies. The size of the self-assemblies can be tuned/calibrated by combining different ratios of the starting monomeric mixtures at high temperature. Herein, we defined the concept of” phantom monomers” which are the closest structure that mimic a selected monomer but lacks the active function for polymerization. The phantom monomer co-formulated with other monomers will be present in the intermediate self-assemblies due to its similarity with one of the active monomers. However, upon polymerization, the phantom monomer, lacking of a polymerizable function, will be excluded and a new NHGs will be generated. The comparative analyses of our previously obtained standard NHGs (G1) with those obtained here using phantom monomers (G2 and G3) not only put in evidence the self-assembly mediated mechanism but also prove the generation of new monodispersed NHG’s with improved drug-loading properties. As proof of concept, the NHGs were loaded with doxorubicin (DOX) and tested in cells. Results indicate that the drug-loading of the NHGs increases from G1 to G3 and while using the same drug concentration, different size NHGs affect differently the treated cells and disclose a different activity and localization as compared to free DOX by confocal microscopy.
