Designing oligoarginine-associated PECA nanoparticles for enhanced cellular uptake

Abstract

Introduction: Polymeric nanoparticles can be used as carriers to improve oral bioavailability of therapeutic peptides or proteins. These polymeric carriers can be formulated with cell-penetrating peptides such as oligoarginine to further enhance uptake. Such a combined formulation could deliver bioactive effectively, using less oligoarginine than required for effective cell permeation using oligoarginine alone. The aims of this study were to formulate poly(ethylcyanoacrylate) (PECA) nanoparticles with oligoarginine, characterize the resulting nanoparticles and investigate their in vitro uptake. Methods: PECA nanoparticles were produced by in situ polymerization in a water-in-oil microemulsion. Various oligoarginines were dissolved in the aqueous phase of the microemulsion prior to the addition and polymerization of monomers, to produce different oligoarginine-associated nanoparticles. The resultant nanoparticles were characterized for size and zeta potential in ultra-pure water and the cell incubating medium (Hanks Balanced Salt Solution, HBSS). The nature of the association between oligoarginines and nanoparticles was investigated by MALDI-TOF mass spectrometry. The uptake of the oligoarginine-associated nanoparticles, loaded with a fluorescent probe, by Caco-2 cells was investigated using fluorescence-activated cell sorting (FACS) and confocal imaging. Uptake studies were conducted in both undifferentiated cells and fully differentiated cell monolayers. The uptake of radiolabeled oligoarginine nanoparticles by Caco-2 cells was quantified by scintillation counting and the accumulation of nanoparticles on the cell surface was evaluated with a mathematical simulation model. Results: PECA nanoparticles formulated with di-arginine-histidine (RRH) and tetra-arginine-histidine with an aminocaproic acid spacer (R4acaH) were cationic (zeta potential of +35 and +33.5 mV, respectively) and approximately 200 nm in diameter. Mass spectrometric studies revealed that RRH was covalently tagged to the PECA nanoparticles via histidine but R4acaH was not. Because these RRH-tagged nanoparticles aggregated in HBSS, poloxamer-407 surfactant was added to stabilize the colloidal system. However, the addition of surfactant was found to neutralize the positive zeta potential of the nanoparticles. RRH-tagged nanoparticles associated with a higher proportion of undifferentiated Caco-2 cells after 2 h incubation than unmodified nanoparticles and confocal imaging showed that they were mainly located on the cell surface. Association of RRH-tagged nanoparticles in fully differentiated Caco-2 cell monolayers was not increased compared to that of unmodified nanoparticles. The accumulation trend of PECA nanoparticles on the cell surface predicted by a mathematical simulation model was consistent with the cellular experimental data. Conclusions: PECA nanoparticles associated covalently with RRH via histidine anchoring to produce cationic nanoparticles, which were neutralized in the presence of surfactant. These nanoparticles adhered to undifferentiated Caco-2 cells to a greater extent than unmodified PECA nanoparticles. However, scintillation counting data revealed that the greater tendency to adhere did not result in greater uptake of the RRH-tagged nanoparticles in fully differentiated Caco-2 cell monolayers, which was consistent with the findings of flow cytometry. Mathematical simulation modelling was able to predict the low accumulation of the nanoparticles at the bio-interface but did not account for the adherence tendency of the nanoparticles and the initial contact adherence that occurred during convection mixing upon introduction of the formulation to the cells. Further surface characterization of the RRH-tagged nanoparticles is required to gain deeper insight into the nature of the interaction at the cellular bio-interface. Although fluorescence analysis (such as FACS and confocal imaging) was able to quantify the proportion of cells associated with the polymeric nanoparticles and verify internalization, scintillation counting data complemented the cellular association with invaluable information on the proportion of nanoparticles associated with the cells. Therefore, these techniques should be used together to critically assess cellular association and uptake.