Since biocatalysts manoeuvre most of the physiological activities in living organisms and exhibit extreme selectivity and specificity, their use to trigger physicochemical change in polymeric architectures has been successfully used for targeted drug delivery. Our major interest is to develop lipase responsive nanoscale delivery systems from bio-compatible and biodegradable building blocks. Herein, we report the synthesis of four novel non-ionic Gemini amphiphiles using a chemo-enzymatic approach. A symmetrical diglycerol has been used as a core that is functionalised with alkyl chains for the creation of a hydrophobic cavity, and for aqueous solubility (polyethylene glycol) monomethyl ether (mPEG) is incorporated. Such systems can exhibit a varied self-assembly behaviour leading to the observance of different morphological structures. The aggregation behaviour of the synthesised nanocarrier was studied by dynamic light scattering (DLS) and critical aggregation concentration (CAC) measurements. The nanotransport potential of amphiphiles was investigated for hydrophobic guest molecules, i.e. Nile red, nimodipine and curcumin. Cytotoxicity of the amphiphiles was studied using HeLa and MCF7 cell lines at different concentrations, i.e. 0.05, 0.1, and 0.5 mg mL−1. All nanocarriers were found to be non-cytotoxic up to a concentration of 0.1 mg mL−1. Confocal laser scanning microscopy (cLSM) study suggested the uptake of encapsulated dye in the cytosol of the cancer cells within 4 h, thus implying that amphiphilic systems can efficiently transport hydrophobic drug molecules into cells. The biomedical application of the synthesised Gemini amphiphiles was also investigated for dermal drug delivery. In addition, the enzyme-mediated release study was performed that demonstrated 90% of the dye is released within three days. All these results supported the capability of nanocarriers in drug delivery systems.