Nanostructured lipid carriers decorated with polyphosphate coated linear and loop cell-penetrating peptides

Aim: This study aimed to evaluate the cellular uptake of nanostructured lipid carriers (NLCs) decorated with polyphosphate coated linear and loop cell-penetrating peptides (CPPs). Methods: Linear-CPPs and loop-CPPs were synthesized via ring-opening polymerization and anchored on the surface NLCs, followed by coating with polyphosphate (PP). These nanocarriers (NCs) were characterized in terms of particle size, polydispersity index (PDI), and zeta potential. Cell viability and hemolysis, as well as enzyme-induced charge conversion via phosphate cleavage by free and membrane-bound intestinal alkaline phosphatase (IAP) were investigated. Cellular uptake studies by Caco-2 and HEK cells were quantitatively analyzed by flow cytometry and visualized by confocal microscopy. Results: A shift in charge from positive to negative was obtained for both linear- and loop-CPPs-NLCs by coating with PP. PP-linear-CPPs-NLCs and PP-loop-CPPs-NLCs exhibited a particle size < 270 nm and a PDI of approximately 0.3. They had a minor effect on cell viability and caused in a concentration of 0.1 % (m/v) around 10 % hemolysis within 24 h. IAP triggered the cleavage and release of monophosphate from the surface of NLCs causing charge conversion from -22.2 mV to + 5.3 mV (Delta 27.5 mV) for PP-linear-CPPs-NLCs and from -19.2 mV to + 11.9 mV (Delta 31.1 mV) for PP-loop-CPPs-NLCs. Inhibition of alkaline phosphatase activity on Caco-2 and HEK cells confirmed the involvement of this enzyme in charge conversion. PP-linear-CPPs-NLCs showed on Caco-2 cells a higher uptake than PP-loop-CPPs-NLCs, whereas on HEK cells uptake of both types of NLCs was on the same level. The results of cellular uptake were confirmed visually by confocal microscopy. Conclusion: CPPs-NLCs coated with polyphosphate are a promising approach to overcome the polycationic dilemma and to enhance cellular uptake.