Effect of Bottlebrush Poloxamer Architecture on Binding to Liposomes

Abstract

Poloxamers�triblock copolymers consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)�have demonstrated cell membrane stabilization efficacy against numerous types of stress. However, the mechanism responsible for this stabilizing effect remains elusive, hindering engineering of more effective therapeutics. Bottlebrush polymers have a wide parameter space and known relationships between architectural parameters and polymer properties, enabling their use as a tool for mechanistic investigations of polymer−lipid bilayer interactions. In this work, we utilized a versatile synthetic platform to create novel bottlebrush analogues to poloxamers and then employed pulsed-field-gradient NMR and an in vitro osmotic stress assay to explore the effect of bottlebrush architectural parameters on binding to, and protection of, model phospholipid bilayers. We found that the binding affinity of a bottlebrush poloxamer (BBP) (B-E 10 43 P 5 15 , M n ≈ 26 kDa) is about 3 times higher than a linear poloxamer with a similar composition and number of PPO units (L-E 93 P 54 E 93 , M n ≈ 11 kDa). Furthermore, BBP binding is sensitive to overall molecular weight, side-chain length, and architecture (statistical versus block). Finally, all tested BBPs exhibit a protective effect on cell membranes under stress at sub-μM concentrations. As the factors controlling membrane affinity and protection efficacy of bottlebrush poloxamers are not understood, these results provide important insight into how they adhere to and stabilize a lipid bilayer surface.

Discovery of Kinetic Trapping of Poloxamers inside Liposomes via Thermal Treatment

“…Furthermore, B -E 11 43 P 6 15 did not desorb from the bilayer at 10 °C, irrespective of the thermal history (Figure S17). This is likely because the bottlebrush occupies a bound conformation where the backbone lies parallel to the bilayer plane, enabling multiple PPO and PEO side chains to intercalate into the acyl region . Thus, for desorption to occur, every side chain must simultaneously pull out of the bilayer, an unlikely event that would also result in significant steric crowding.…”

“…This leads to decay of all NMR signals as a function of the magnetic field gradient strength and the diffusion coefficient of the species, according to eq : , ln true( I I 0 true) = − γ 2 δ 2 G 2 true( normalΔ − δ 3 true) D where I and I 0 are the integral of a chosen NMR signal at the given gradient strength and a gradient strength of 0, respectively, δ is the duration of the magnetic field gradient pulse, Δ is the time between magnetic field gradient pulses (diffusion time), G is the magnetic field gradient strength, γ is the gyromagnetic ratio of a proton (42.6 MHz/T), and D is the translational diffusion coefficient of the species associated with the chosen NMR signal. Previous studies have demonstrated that PFG-NMR is a robust technique for measuring the fraction of polymer chains that bind to a liposome. ,,, When multiple populations are present, as long as their characteristic sizes differ by at least a factor of ∼2, multiple rates of diffusion can be extracted by fitting the signal decay to a sum of exponentials given by eq : ln true( I I 0 true) = ln ( ∑ i f i exp true( prefix− γ 2 δ 2 G 2 ( Δ − δ 3 ) D i true) true) …”

“…Previous studies have demonstrated that PFG-NMR is a robust technique for measuring the fraction of polymer chains that bind to a liposome. 11,24,42,47 When multiple populations are present, as long as their characteristic sizes differ by at least a factor of ∼2, multiple rates of diffusion can be extracted by fitting the signal decay to a sum of exponentials given by eq 2: i k j j j j j y { z z z z z i k j j j j j j i k j j j j i k j j j y…”

“…52 ■ RESULTS AND DISCUSSION Impact of Thermal History on F127 Binding to Liposomes. We employed an established liposome binding assay based on PFG-NMR 11,24,41,42,53 to assess the impact of thermal history on F127 binding to POPC liposomes. Figure 1a shows the thermal history of two otherwise identical F127 + POPC samples at 1 mg/mL polymer and 10 mM POPC.…”

“…Poloxamers are nonionic ABPs with PEO–PPO–PEO triblock architecture that are commercially available over a range of molecular weights and compositions and are biocompatible. , Poloxamer binding to phospholipid bilayers is endothermic, driven by entropically dominated hydrophobic forces. , Because the hydrophobic effect and the hydrogen-bonded water shell of PEO and PPO are sensitive to temperature, it is important to understand how temperature and thermal history affect poloxamer–lipid bilayer interactions. However, most mechanistic studies of polymer–lipid bilayer interactions have been conducted at a single temperature, often room temperature, for experimental convenience or because the effects of other variables were the focus. ,,,,− …”