Studies on the Mechanism of Interaction of a Bioresponsive Endosomolytic Polyamidoamine with Interfaces. 1. Micelles as Model Surfaces

Abstract

Polymers are appealing as pH-responsive elements of multicomponent systems designed to promote cytosolic delivery of macromolecular drugs (including proteins and genes), but so far the delivery efficiency achieved has been relatively modest. Therefore, the aim of this study was to apply several physicochemical techniques that are well established in the colloid field (surface tension measurements, small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR)) to probe the mechanism of endosomolytic polymer-surface interaction over the pH range 7.4 to 5.5 using the poly(amidoamine) (PAA) ISA23 x HCl and a series of "model" micelle surfaces. These micellar models were chosen to represent increasing complexity from simple, single surfactant sodium dodecylsulfate (SDS) micelles, surfactant mixtures containing bulky malono-bis-N-methylglucamide headgroups, or highly extended ethylene oxide headgroups. Spherical micelles composed of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) were also used. Changes in the onset of micellization, micelle surface fluidity, and in selected cases, the overall micelle shape and size were all quantified as a function of pH in the presence and absence of ISA23 x HCl. This amphoteric PAA is negatively charged at pH 7.4 and becomes gradually more protonated on exposure to lower pH values representative of the endosomal-lysosomal pathway. As expected, the strength of polymer interaction with anionic micelles increased with a decrease in pH, while for cationic micelles the opposite was observed. Addition of bulky, nonionic surfactant headgroups led to weaker interactions. The observations from surface tension and SANS studies showed a complex pattern of interaction with both an electrostatic and hydrophobic component. Using EPR it was confirmed that ISA23 x HCl perturbed the micelle palisade layer leading to a decrease in fluidity of the interface with a lower degree of headgroup hydration, and a significant change in micelle morphology. Surprisingly, there was no interaction between ISA23 x HCl and globular micelles formed from lyso-PC (a more biologically relevant model), and this suggests that the PAA structure could be better optimized to promote rapid interaction with endosomal membranes at the physiologically relevant pH 6.5.

Interaction of an Endosomolytic Polyamidoamine ISA23 with Vesicles Mimicking Intracellular Membranes: A SANS/EPR Study

“…[9] Addition of bulky, non-ionic surfactant headgroups led to weaker interactions, and EPR confirmed that ISA23 Á HCl perturbed the micelle palisade layer leading to a decrease in fluidity of the interface with a lower degree of headgroup hydration, with a significant change in micelle morphology. Surprisingly, in these studies, [9] no interaction was seen at any pH (7.4-5.5) with globular, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) micelles that are arguably a more biologically relevant model. These observations confirmed a complex pattern of ISA23/micelle interaction with both an electrostatic and a significant hydrophobic component.…”

“…[5,[7][8][9] Techniques more commonly applied in colloidal science such as pulsed-gradient spin-echo NMR, electron paramagnetic resonance (EPR) and small-angle neutron scattering (SANS) were used to study solution conformation and model surface interaction. Initial studies on the solution behavior of ISA23 Á HCl indicated a Gaussian-coil conformation with pH-induced coil expansion.…”

Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo

“…PAA shape, size, and pH-responsive changes in conformation were determined using small angle neutron scattering (SANS) [17,18], and the rate of diffusion in solution was defined using pulsed-gradient spin-echo NMR [17]. Most recently PAA interaction with model micelles [19], and model membranes whose composition was chosen to mimic the plasma, endosomal and lysosomal membrane was measured using surface tension and electron plasmon resonance techniques in combination with SANS [19].…”

Poly(amidoamine) Conjugates Containing Doxorubicin Bound via an Acid‐Sensitive Linker

“…A recent study using small angle neutron scattering (SANS) suggested that this difference might be explained by different conjugates conformation 48. Such experiments have already shown the pH‐dependent changes in conformation of poly(amidoamine)s,20–22 experiments with the present conjugates could lead to further insight into the mechanism of Dox release.…”

“…Poly(amidoamine)s also present the advantage to degrade to oligomeric products in aqueous media within days or weeks, depending on their structures 18, 19. Owing to their capacity to undergo a conformational change from a coiled structure at pH = 7.4 to a more extended one when exposed to acidic pH,20–22 most recent effort have been directed to develop stimuli‐responsive constructs for biomacromolecules intracellular delivery 23–26. However, these polymers have also the potential to be used as drug carrier.…”