A newer class of Gemini amphiphiles was prepared using d-glucitol as a template. C18 alkyl chains are incorporated on the template to confer hydrophobicity while monomethoxy polyethylene glycol (mPEG) moiety of varied size imparts hydrophilicity. An acetonide group mounted on glucitol moiety is used to maintain rigidity in the amphiphilic architecture and to study its impact on aggregation and transport ability. The aggregation behavior of the synthesized amphiphiles was studied through fluorescence spectroscopy, dynamic light scattering (DLS), and cryo-transmission electron microscopy (cryo-TEM). Their nanotransport potential was demonstrated using hydrophobic model guests like the fluorescent dye Nile red and the pharmacologically active agent curcumin. Cytotoxicity and cellular uptake studies were carried out over HeLa cancer cells to evaluate their biological applicability. The ester functionalities in the amphiphiles enable them to exhibit sensitivity towards hydrolases. Candida antarctica lipase (Novozym-435) has been successfully utilized to facilitate the enzyme-assisted release of the encapsulated dye.