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The main objectives of the present study were (i) to evaluate the effect of the mucus layer on saquinavir-loaded nanostructured lipid carriers (SQV-NLCs) uptake and (ii) to evaluate the mucopenetrating properties of dextran–protamine (Dex–Prot) coating on NLCs as per SQV permeability enhancement. Three different NLC formulations differing on particle size and surfactant content were obtained and coated with Dex–Prot complexes. SQV permeability was then evaluated across Caco-2 cell monolayers (enterocyte-like model) and Caco-2/HT29-MTX cell monolayers (mucus model). In the Caco-2 monolayers, Dex–Prot–NLCs increased up to 9-fold SQV permeability in comparison to uncoated nanoparticles. In the Caco-2/HT29-MTX monolayers, Dex–Prot–NLCs presenting a surface charge close to neutrality significantly increased SQV permeability. Hence, Dex–Prot complex coating is a promising strategy to ensure successful nanoparticle mucus-penetration, and thus, an efficient nanoparticle oral delivery. To our knowledge, this is the first time that Dex–Prot coating has been described as a nanoparticle muco-penetration enhancer across the intestinal mucus barrier.