Aim: MUC-1 lipopeptide vaccine exhibited immense potential in the treatment of non-small cell lung cancer (NSCLC) in both preclinical and clinical trials. However, it lacks triggering of mucosal immunity at the site of action. Therefore, in present investigation, MUC-1 peptide-loaded poly(lactide-co-glycolide) nanoparticles (MUC-1 peptide-PLGA-NPs) and MUC-1 peptide-loaded poly(lactide-co-glycolide) non-aggregated nanoparticles (MUC-1 peptide-PLGA-NA-NPs) using Central Composite Design (CCD) were customised. Methods and Results: The mean particle size of MUC-1 peptide PLGA-NPs was estimated to be 176.7 +/- 32.7 nm, significantly (p < 0.05) higher than 100.3 +/- 24.3 nm of MUC-1 peptide-PLGA-NA-NPs. Furthermore, integrity and stability of MUC-1 were maintained in MUC-1 peptide PLGA-NA-NPs. MUC-1 peptide-PLGA-NA-NPs exhibited augmented cellular uptake in mouse RAW264.7 macrophages preferably by clathrin-mediated endocytosis pathway as compared to phagocytosis followed by MUC-1-peptide PLGA-NPs owing to size <= 100 nm, and spherical shape. Conclusion: MUC-1 peptide-PLGA-NA-NPs may be a potential candidate to study antitumor potential in xenograft model of NSCLC through inhalation route of administration.