Directly visualizing individual polyorganophosphazenes and their single-chain complexes with proteins

Abstract

Polyorganophosphazenes are water-soluble macromolecules with immunoadjuvant activity that self-assemble with proteins to enable biological functionality. Direct imaging by cryogenic electron microscopy uncovers the coil structure of those highly charged macromolecules. The successful visualization of individual polymer chains within the vitrified state is achieved in the absence of additives for contrast enhancement and is attributed to the high mass contrast of the inorganic backbone. Upon assembly with proteins, multiple protein copies bind at the single polymer chain level resulting in structures reminiscent of compact spherical complexes or stiffened coils. The outcome depends on protein characteristics and cannot be deduced by commonly used characterization techniques, such as light scattering, thus revealing direct morphological insights crucial for understanding biological activity. Atomic force microscopy supports the morphology outcomes while advanced analytical techniques confirm protein-polymer binding. The chain visualization methodology provides tools for gaining insights into the processes of supramolecular assembly and mechanistic aspects of polymer enabled vaccine delivery.

Figure 1.

Visualization of individual PCPP chains. (a) Chemical structure of PCPP repeat unit. (b) Cryo-EM image of a single PCPP chain (defocus 2.12. μm) and (c) projections of representative polymer chain trajectories obtained from cryo-EM images shown in Supplementary Figure 1, (d) AFM images of individual PCPP chains.

Figure 2.

Contrasting of PCPP with BSA. Cryo-EM images of (a) PCPP-BSA (defocus 2.01 μm) and (b) PCPP (defocus 0.10 μm) (2.5 mg mL⁻¹ PCPP, 0.25 mg mL⁻¹ BSA, 50 mmol phosphate buffer, pH 7.4).

Figure 3.

Interactions of PCPP with lysozyme. (a) Schematic presentations of electrostatic potential surfaces of hen egg lysozyme (images generated using PyMOL 2.5.5 (Schrödinger Inc., New York, NY); (b) Fluorescence of lysozyme - dotted line, PCPP-lysozyme complex (1:7 mole mole⁻¹) - solid line and PCPP - dashed line (0.5 mg mL⁻¹ PCPP, 0.06 mg mL⁻¹ lysozyme, 50 mM phosphate buffer, pH 7.4, excitation wavelength: 280 nm); (c) size-exclusion chromatography profiles of PCPP-lysozyme - solid line and PCPP - dotted line (0.5 mg mL⁻¹ PCPP, 0.06 mg mL⁻¹ lysozyme, excitation/emission wavelengths: 280 nm/340 nm; 50 mM phosphate buffer, pH 7.4) (d) zeta-potential distribution profiles for PCPP - dashed line, lysozyme - dotted line and their mixture -solid line (0.5 mg mL⁻¹ PCPP, 1 mg mL⁻¹ lysozyme, complex: 0.5 mg mL⁻¹ PCPP and 0.06 mg mL⁻¹ lysozyme, 50 mM phosphate buffer, pH 7.4)

Figure 4.

Visualization and characterization of PCPP complexes with lysozyme. (a-b) Cryo-EM images of PCPP-lysozyme complexes (1:7 mole ratio, 0.5 mg mL⁻¹ PCPP and 0.06 mg mL⁻¹ lysozyme, defoci: 0.10–2.50 μm) and projections of their chain trajectories. (c-d) complex size distribution as determined by measuring pervaded diameters of coils in cryo-EM images and DLS.

Figure 5.

Visualization and characterization of PCPP using AFM. (a) AFM images at various magnifications. (b) Schematic presentation of adsorbed complex with average dimensions. (c-d) Diameter (solid-colored squares) and (open circles) height distribution of complexes (AFM images are taken from bPEI-treated mica substrates).