Oral bioavailability of loperamide is restricted by its limited absorption in the gastrointestinal tract due to its poor aqueous solubility and its P-glycoprotein (Pgp) substrate characteristic. In addition, ammonium methacrylate copolymers have shown to have mucoadhesive properties, whereas poloxamer 188, has been suggested as a Pgp inhibitor. Thus, in this work, we evaluate conditions that affect physicochemical parameters of ammonium methacrylate/poloxamer 188-based nanocarriers loaded with loperamide hydrochloride. Nanocarriers were synthesized by nanoprecipitation, enhancing loperamide encapsulation efficiency by modifying the aqueous phase to basic pH. The isolation of the non-encapsulated drug fraction from the nanocarriers-incorporated fraction was conducted by centrifugation, ultrafiltration, vacuum filtration and diafiltration. The last method was effective in providing a deeper understanding of drug-nanocarrier loading and interactions by means of modeling the data obtained by it. Through diafiltration, it was determined an encapsulation efficiency of about 93%, from which a 38% ±6 was shown to be reversibly (thermodynamic interaction) and a 62% ±6 irreversibly (kinetic interaction) bound. Finally, release profiles were assessed through empirical and semi-empirical modeling, showing a biphasic release behavior (burst effect 11.34% and total release at 6 h = 33% ±1). Thus, encapsulation efficiency and release profile were shown to have a strong mathematical modeling-based correlation, providing the mechanistic approach presented in this article a solid support for future translational investigations.