Background: Insulin, vital for diabetes management, faces challenges with poor permeability and stability, demanding enhanced effectiveness and safety measures. Various oral peptide delivery systems have emerged, but non-targeted ones, while preventing degradation, lack the ability to facilitate peptide transport. The limitation lies in their non-selectivity. Intestinal macrophage cells in gut-associated lymphoid tissue express carbohydrate-binding receptors (CBRs), ideal for receptor-mediated endocytosis and peptide delivery.

Objective: This study aimed to create mannose ligand conjugated nanoparticles (MNPs) using a quality-by-design (QbD) approach to tackle oral administration challenges. The secondary objective was to identify factors influencing product quality. Lymphatic uptake of NPs was the quality target product profile (QTPP), and the study focused on critical formulation attributes (CFAs) like Mannosylated Chitosan concentrations (MCs) and TPP concentrations, as well as critical process parameters (CPP) such as stirring speed.

Methods: MNPs, produced via inotropic gelation, were encapsulated in enteric-coated capsules for protection. The impact of CFAs and CPP on particle size and entrapment was observed through Box-Behnken design (BBD), statistically analyzed with ANOVA (p<0.05). Optimal conditions were determined using desirability values. Characterization involved HPLC, zeta-seizer analysis, SEM for morphology, MTT assay for safety, and cell-line studies for M-cell targeting.

Results: SEM revealed oval-shaped NPs with an average size of 245.52±3.37 nm, surface potential of 22.12±2.13, and 76.15±1.3% drug entrapment. MTT assay affirmed safety, while confocal imaging indicated selective M-cell uptake.

Conclusion: BBD facilitated optimized NP formulation, providing clear insights into variable significance. Cell-line studies confirmed safety and effective cellular uptake of the NPs.