Objective: The aim of this study is to evaluate the use of PEG/glycerides of different HLB; oleoyl macrogol-6-glycerides (Labrafil (R) M 1944 CS) and caprylocaproylmacrogol-8-glycerides (Labrasol (R)), compared to Labrafac lipophile (R) as PEG-free glyceride in the preparation of nanostructured lipid carriers (NLCs). PEG/glycerides are suggested to perform a dual function; as the oily component, and as the PEG-containing substrate required for producing the PEGylated carriers without physical or chemical synthesis. Methods: Lipid nanocarriers were loaded with simvastatin (SV) as a promising anticancer drug. An optimization study of NLC fabrication variables was first conducted. The effect of lyophilization was investigated using cryoprotectants of various types and concentrations. The prepared NLCs were characterized in terms of particle size (PS), size distribution (PDI), zeta potential (ZP), drug entrapment, in vitro drug release, morphology and drug-excipient interactions. The influence of glyceridesPEG on the cytotoxicity of SV was evaluated on MCF-7 breast cancer cells, in addition to the cellular uptake of fluorescent blank NLCs. Results: The alteration between different oil types had a significant impact on PS, ZP and drug release. Both sucrose and trehalose showed the lowest increase in PS and PDI of the reconstituted lyophilized NLCs. The in vitro cytotoxicity and cellular uptake studies indicated that SV showed the highest antitumor effect on MCF-7 cancer cells when loaded into Labrasol (R) NLCs demonstrating a high cellular uptake as well. Conclusion: The study confirms the applicability of PEG/glycerides in the development of NLCs. Encapsulating SV in Labrasol (R)-containing NLC could enhance the antitumor effect of the drug.