Understanding the Mechanism of Action of Poly(amidoamine)s as Endosomolytic Polymers:  Correlation of Physicochemical and Biological Properties

Abstract

Bioresponsive poly(amidoamine)s (PAA)s are currently under development as endosomolytic polymers for intracellular delivery of proteins and genes. Here for the first time, small-angle neutron scattering (SANS) is used to systematically investigate the pH-dependent conformational change of an endosomolytic polymer, the PAA ISA 23. The radius of gyration of the ISA23 was determined as a function of pH and counterion, the aim being to correlate changes in polymer conformation with membrane activity assessed using a rat red blood cell haemolysis assay. With decreasing pH, the ISA23 radius of gyration increased to a maximum (R(g) approximately 80 A) around pH = 3, before subsequently decreasing once more. At high pH and therefore high ionic strengths, the polymer is negatively charged and adopts a rather compact structure (R(g) approximately 20 A), presumably with the dissociated carboxylic groups on the exterior of the polymer coil. At low pH, the coil again collapses (R(g) < 20 A), presumably due to the effects of the high ionic strength. It is concluded that the nature of the salt form has no direct bearing on the size of the polymer coil, but it does indirectly determine the prevailing pH and, hence, polymer conformation. Pulsed-gradient spin-echo NMR measurements were in good agreement with the SANS estimates of the radius of gyration, although ISA23 polydispersity does complicate the data interpretation/comparison. These results support the proposed mode of action of PAAs, namely a coil expansion on passing from a neutral pH (extracellular) to an acidic pH (endosomal and lysosomal) environments. The results do, however, suggest that the charge on the polymer shows a closer correlation with the haemolysis activity rather than the polymer conformation.

Synthesis and Endosomolytic Properties of Poly(amidoamine) Block Copolymers

“…Thus, we are currently trying to define more clearly the solution properties of PAAs at intracellular pH [11,12] and their mechanisms of membrane destabilisation. [43] Only with this information will it become possible to tailor the most effective hybrid structures. Figure 4.…”

“…This was confirmed by a pH-dependent haemolysis assay [9,10] and more recently by small angle neutron scattering (SANS). [11,12] In the context of Summary: The poly(amidoamine)s (PAAs) ISA 1 and ISA 23 display pH-dependent conformational change and pHdependent membrane perturbation. These properties confer potential for use as endosomolytic polymers for intracytoplasmic delivery of toxins and genes.…”

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

“…5) suggest little membrane interaction. Previously reported SANS studies showed that ISA23 coil expansion resulting from polymer backbone protonation only occurred at pH 5 and below, pH values not normally associated with the endosomal compartment [18]. Although potentially interesting, further dissection of the endocytic pathways utilised by these PAAs in vitro was not undertaken as such studies are likely to be irrelevant to increased understanding intracellular fate in vivo.…”

“…1; Table 1). 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].…”

Selected Topics of Computational and Experimental Fluid Mechanics

“…This is modified by pH and ionic strength effects. Experimental studies of small-angle neutron scattering (SANS) have been published in order to illustrate the pH-dependency and conformational change of PAA ISA 23 [29]. Linear poly(amidoamine) polymers (PAAs) have amido-and tertiary amino-groups along the main polymer, which gives rise to an interesting pHdependent conformational change and thus offers a perfect prospect for devising polymers that present membrane activity at low pH.…”