Newer non-ionic amphiphiles have been synthesized using biocompatible materials and by following a greener approach i.e., D-glucitol has been used as a template, and hydrophobic and hydrophilic segments were incorporated on it by using click chemistry. The hydrophilic segments in turn were prepared from glycerol using an immobilized Candida antarctica lipase (Novozym-435)-mediated chemoenzymatic approach. Surface tension measurements and dynamic light scattering studies reflect the self-assembling behavior of the synthesized amphiphilic architectures in the aqueous medium. The results from UV-Vis and fluorescence spectroscopy establish the encapsulation of guests in the hydrophobic core of self-assembled amphiphilic architectures. The results of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay indicate that the amphiphiles are well tolerated by the used A549 cell lines at all tested concentrations.