Biopolymers comprise a large variety of molecules of natural origin such as bacteria, algae, fungi, plants and animals, which have been increasingly studied for distinct applications due to their sustainability, low cost, biodegradability and biocompatibility (Biswas, Jony, Nandy, Chowdhury, Halder, Kumar, Ramakrishna, Hassan, Ahsan, Hoque & Imam, 2022; De Dier, Mathues & Clasen, 2013; Dodero, Williams, Gagliardi, Vicini, Alloisio & Castellano, 2019; Duxenneuner, Fischer, Windhab & Cooper-White, 2008; Gericke, Schlufter, Liebert, Heinze & Budtova, 2009; Haward, Sharma, Butts, McKinley & Rahatekar, 2012; Jaishankar, Wee, Matia-Merino, Goh & McKinley, 2015; Morris, Cutler, Ross-Murphy, Rees & Price, 1981; Wang, Geri, Chen, Huang, McKinley & Chen, 2022). These intrinsic properties of biopolymers have received much attention in the biomedical field, namely for the development of controlled drug release systems, novel wound dressing materials, antimicrobial coatings for medical devices and scaffolds for tissue engineering (Biswas et al., 2022). Toxicity issues raised by using synthetic polymers as encapsulating agents of numerous therapeutic compounds—usually administered through oral or intravenous routes—have led to their replacement by natural polymers (Gheorghita, Anchidin-Norocel, Filip, Dimian & Covasa, 2021). Nonetheless, it is important that the biopolymer used is able to release the active compound to the target area at the right time, safely and without side effects (Gheorghita et al., 2021).
Cyanoflan is a biopolymer secreted by the marine unicellular cyanobacterium Crocosphaera chwakensis CCY0110. Biological polysaccharides possess a polydisperse distribution of polymer chain lengths, producing a multi-scale microstructure whose experimental response is described by a seemingly continuous distribution of relaxation modes (Wagner, Barbati, Engmann, Burbidge & McKinley, 2017). This structurally complex heteropolysaccharide is no different, being composed of nine different monosaccharide residues (including two uronic acids), sulfate groups and peptides, and characterised by a high molecular weight (over 1 MDa) with broad distribution (Mota, Vidal, Pandeirada, Flores, Adessi, De Philippis, Nunes, Coimbra & Tamagnini, 2020). The main chemical and physical properties of Cyanoflan were obtained in previous work (Mota et al., 2020), which in part motivated us to perform our study. The versatility of Cyanoflan for therapeutic applications has been described in recent studies: it can be used as a vehicle for controlled delivery of functional proteins and/or vitamins (Estevinho, Mota, Leite, Tamagnini, Gales & Rocha, 2019; Leite, Mota, Durão, Neves, Barrias, Tamagnini & Gales, 2017), to develop infection-preventive antiadhesive coatings (Costa, Mota, Parreira, Tamagnini, Martins & Costa, 2019; Costa, Mota, Tamagnini, Martins & Costa, 2020; Matinha-Cardoso, Mota, Gomes, Gomes, Mergulhão, Tamagnini, Martins & Costa, 75 2021) and wound dressings (Costa, Costa, Rodrigues, Meireles, Soares, Tamagnini & Mota, 2021), or as thickener and emulsifying agent (Mota et al., 2020). Moreover, the biocompatibility of Cyanoflan has been studied both in vitro and in vivo (Costa et al., 2019, 2021; Leite et al., 2017), and regarding blood compatibility its use as a catheter coating did not induce the activation of adhered platelets (Costa et al., 2019).
The chemical heterogeneity and high dispersity usually exhibited by natural polymers on the one hand open opportunities for engineering functional materials, but on the other hand demand careful evaluation of biocompatibility for medical applications. Preliminary in vitro biocompatibility tests are typically dedicated to assessing their immunogenic potential, without considering the effects on blood rheology. Incorporating long linear polymers into blood plasma—together with the plasma protein adsorption and/or coagulation events that it can give rise to—may induce microstructural changes that can be characterised and related to changes in rheology. Applied research into this area has hitherto been scarce, but rheological measurements have already been extended to investigate blood compatibility of biopolymers e.g. chitosan derivatives (Lee, Ha & Park, 1995).
For soft biomaterials, characterising the evolution of the viscoelastic properties, microstructure, degree of heterogeneity and kinetics of network formation through the liquid–solid (sol–gel) transition is important to get a full picture of the rheological properties. Hence, multiple particle tracking (MPT) microrheology in which the thermal motion of dispersed beads is used to probe the material rheology—is particularly useful for such studies (He, Pascucci, Caggioni, Lindberg & Schultz, 2021; Larsen & Furst, 2008; Papagiannopoulos, Sotiropoulos & Pispas, 2016; Rich, McKinley & Doyle, 2011; Schultz & Furst, 2012). This single-point microrheology technique is also capable of extracting the relevant rheological information near the gel point, including the critical extent of gelation/transition concentration (by the identification of the gel point), the critical relaxation exponent, and other critical dynamic scaling exponents. Although not replacing bulk rheological methods entirely, microrheology complements macrorheological characterisation by providing information for samples that would otherwise be difficult (or impossible) to study using bulk rheology—e.g. microrheology’s small sample volume requirements compared with bulk rheology makes it possible to investigate the rheological properties of scarce and/or expensive biomaterials (Dodero et al., 2019; Larsen & Furst, 2008; Rodrigues, Mota, Gales & Campo-Deaño, 2022; Schultz & Furst, 2012).
However, as with any form of measurement, particle tracking experiments suffer from limitations inherent to the technique. Microrheological measurements can only be used to infer bulk moduli when certain conditions are met, including that the native length scale of the material is much smaller than the probe particle (Mason, 2000). It follows that local structure can have an adverse effect. If the particles are small compared to any structure, micron-scale measurements will probe the local viscoelastic properties of the material rather than its bulk properties, resulting in viscoelastic moduli measured on the micron scale lower than the corresponding bulk-scale moduli (Rich et al., 2011). This is particularly relevant for polymeric systems undergoing gelation with time (i.e. evolving materials) via a percolation transition, where the material is expected to be heterogeneous across different length scales. Conversely, MPT microrheology excels at providing insight into the local structures and rheology of the surrounding medium by using statistical techniques that consider the movements of individual particles exploring different local environments (Savin & Doyle, 2007), as opposed to the cross-correlated motion of a large ensemble of particles—e.g. inherent in light scattering methods, where probe aggregation may also be more difficult to discern (Dodero et al., 2019). Ultimately, researchers should be conscious that the local rheology and gelation properties may generally depend on the length scale that is probed.
Our aim here is to examine the effect of mixing a small amount of Cyanoflan into blood plasma on the final viscoelastic properties and state of the biomaterial, envisioning its use in biomedical applications—e.g. as a scaffold. Drawing on recent developments in understanding human blood plasma viscoelasticity (Rodrigues et al., 2022), we report an experimental study of its rheological evolution as progressively more biopolymer is incorporated into the biofluid. We characterise the dynamical and structural properties of these polysaccharide solutions using MPT microrheology and cryogenic scanning electron microscopy (cryo-SEM), respectively. Finally, we lay out key observations on how network structure, power-law exponent and complex modulus (from which solution zero-shear viscosity and longest relaxation time were inferred) evolve and depend on biopolymer concentration.
