Complex coacervates as a foundation for synthetic underwater adhesives

Abstract

Complex coacervation was proposed to play a role in the formation of the underwater bioadhesive of the Sandcastle worm (Phragmatopoma californica) based on the polyacidic and polybasic nature of the glue proteins and the balance of opposite charges at physiological pH. Morphological studies of the secretory system suggested that the natural process does not involve complex coacervation as commonly defined. The distinction may not be important because electrostatic interactions likely play an important role in the formation of the sandcastle glue. Complex coacervation has also been invoked in the formation of adhesive underwater silk fibers of caddisfly larvae and the adhesive plaques of mussels. A process similar to complex coacervation, that is, condensation and dehydration of biopolyelectrolytes through electrostatic associations, seems plausible for the caddisfly silk. This much is clear, the sandcastle glue complex coacervation model provided a valuable blueprint for the synthesis of a biomimetic, water-borne, underwater adhesive with demonstrated potential for repair of wet tissue.

Figure 1. Complex coacervates as adhesives

The top row represents the complex coacervation and solidification of mixed polyphosphates, polyamines, and divalent cations. The bottom row connects the features of the polyelectrolyte phase behavior to the properties of an underwater glue.

Figure 2. Sandcastle glue

A.) A tube rebuilt in captivity on top of the natural tube with 0.5 mm glass beads. B.) The recently secreted glue spots are white. The concavity of the sides and low contact angle suggested the glue was fluid when secreted and wetted the surface of the glass bead. C.) The glue spots turned brown over several hours after secretion due to oxidative crosslinking through o-quinones. D.) The glue was auto-fluorescent. An optical section acquired by laser scanning confocal microscopy revealed a gradient micro-foam structure.

Figure 3. Representative glue protein sequences

A.) Sequence of polyacidic Pc3B. B.) The serine residues (S) are more than 95% phosphorylated on the hydroxyl sidechain. The tyrosines (Y) are hydroxylated into dopa residues. C.) Sequence of polybasic Pc2. D.) Structures of histidine (H) and lysine (K) residues with amine sidechains.

Figure 4. Synthetic polyphosphate solubility

The experimental conditions (14°C, 470 mM NaCl) simulated the Sandcastle worm’s ocean environment. At approximately the pH of secretory granules (pH 5.0, gray squares) the polyphosphate (MW 60.4 kDa, PDI 2.5) was soluble at Mg²⁺ to PO₄ ratios up to 3. At the pH of the ocean (pH 8.2, black diamonds) the polyphosphate fully precipitated at a 1:1 ratio of Mg²⁺ to PO₄.

Figure 5. Structure of the adhesive gland

A.) Sandcastle worm out of its tube. The adhesive gland occurs in the first three parathoracic segments. B.) The adhesive precursors are auto-fluorescent providing an overview of the secretory system. The worm is outlined with a nuclei stain (dapi). C.) The adhesive precursors are packaged into two types of secretory granules. One type (heterogeneous) stained strongly (dark) for divalent cations. The other type (homogeneous) stained weakly. D.) The granules were transported in separate channels toward the building organ (BO). E.) The granules remained intact and unmixed when they arrived at the BO.

Figure 6. Elemental analysis of secretory granules by energy dispersive x-ray spectroscopy

A.) Scanning electron micrograph of a thin section of adhesive gland with a backscattered electron detector. The heterogeneous granules contain bright substructures. B.) Relative concentration of P and Mg²⁺ along the diagonal line in A. The peaks in P and Mg²⁺ are coincident with the dense substructures in the heterogeneous granules. Homogeneous granules contain background levels of P and Mg²⁺.

Figure 7. Synthetic analogs of glue proteins

A.) Structure of the Pc3B polymethacrylate analog copolymer. B.) Structure of the polyamine analog copolymer. The analog polymers are random copolymers synthesized by free radical polymerization.

Figure 8. Complex coacervation of synthetic glue protein analogs

A.) At pH 6.0 the synthetic glue protein analogs formed a stable colloidal solution of polyelectrolyte complexes. B.) At pH 7.4 the polyelectrolytes condensed into a complex coacervate. C.) The complex coacervate was pipettable and D.) could be delivered underwater without dissolving, readily spread on the wet surface of the glass container, and did not adhere to the plastic pipette. E.) Schematic diagram of complex coacervation. Formation of the complex coacervate macrophase is preceded by formation of nanocomplexes. When the nanocomplexes are electrically neutral they associate into a liquid, interconnected network of copolyelectolyte complexes. Counter microions and water are released in the process.

Figure 9. Bond strengths of synthetic complex coacervates

A.) Underwater test configuration with temperature controlled water bath. The bonds were formed on wet substrates and cured fully submerged in water. The bonds were never allowed to dry before testing underwater. B.) The load required to separate the bonded aluminum adherends increased as the ratio of Mg²⁺ to PO₄ increased at a fixed amine/PO4 ratio.

Figure 10. Repeating structure of caddisfly H-fibroin

Partial sequence (441 aa) from the C-terminus of L. decipiens (AB214509). The negatively charged phosphoserine blocks (gray or red) are separated by a hydrophobic region with a central proline (P) in the D repeats. A positively charged block (dark gray or blue) occurs in all three types of repeats. A (SX)₄ motif occurs in the F blocks but corresponding phosphorylated tryptic peptides were not found in the tandem MS spectra.

Figure 11. Distribution of foot proteins in mussel adhesive plaques

The compositional, graded structural complexity, and assembly sequence of the mussel adhesive plaque is presumably the result of adaptation to the difficult task of bonding a thread attached to its soft body to a wet hard rock. By comparison, the sandcastle worm has the simple task of externally joining together two hard mineral particles and its glue is correspondingly less complex.