The self-associative properties of SSAs 1 -5, and co-formulations a -j, were studied using previously published methodologies. Initially, we quantified the strength of homogeneous SSA anion self-association events, to confirm the effect of the squaramide -(thio)urea substitution. Quantitative 1 H NMR spectroscopy experiments detected the presence of lower order self-association events only in DMSO-d6 1% DCM solutions (112 mM). Complimentary 1 H NMR DOSY experiments conducted in DMSO-d6 0.5 % H2O solutions at 298 K confirmed the SSA anion and cation components to diffuse at different rates, providing supporting evidence towards the lack of any strong ion pairing interactions between the SSA anionic and cationic components under these experimental conditions. Calculating the hydrodynamic diameter (dH) of the SSA anion from these data confirmed the dH < 1.7 nm for 1 -5 (Section S8, Figures ), supporting the formation of lower order self-associated species, such as dimers. The formation of SSA anionic dimers has also been shown in the solid state through single crystal X-ray diffraction experiments. As shown in Figure , the anionic component of squaramide incorporated SSA 5 was also shown to form a hydrogen bonded dimer in the solid state through single crystal X-ray diffraction studies. Here the SSA anion dimer is stabilised through the formation of four intermolecular urea-anion hydrogen bonds. Proton NMR spectroscopy dilution study data, obtained from a DMSO-d6 0.5% H2O solution, were then fitted to both the EK and CoEK self-association models using Bindfit v0.5. These data were found to fit the EK over the CoEK model, which alongside the additional NMR spectroscopy data caused us to hypothesise the presence of SSA anion hydrogen bonded dimers present in a DMSO-d6 0.5% H2O solution. Fitting these data to the EK binding isotherm enables the calculation of a dimerization constant (Kdim, Table ) for SSAs 1, 2, 3 and 5. The dimerization constants calculated confirm that substitution of the sulfonate ion (1 and 3) for the carboxylate ion (2) increase Kdim by approximately 50-fold. However, substituting the thiourea (2) for the squaramide (5) functionality increases the Kdim by a further 70-fold. Therefore, confirming our initial hypothesis, that incorporating the squaramide residue within the SSA anion structure would increase the strength of SSA anion self-association events. -82 e a -Evidence of higher order self-association events obtained, exact percentages could not be calculated. b -This represents the limit of solubility preventing determination of CAC. c -Surface tension at limit of solubility. d -Slow exchange processes prevented Kdim calculation. e -Multiple component system prevented data fitting. Moving into aqueous solutions, quantitative 1 H NMR spectroscopy experiments were undertaken in a D2O:EtOH 19:1 solution (2.78 mM), to confirm the presence of higher order selfassociation events, leading to the formation of aggregates which exhibit solid-like properties, and thus appear silent by 1 H NMR spectroscopy. All SSAs and co-formulations exhibited evidence of higher order self-association events under these experimental conditions, see Table . Interestingly, for homogeneous solutions of 1 -5 there was not much variation in the proportion of SSA to be taken up into these higher-order aggregates under these experimental conditions. The dH of these higher-order aggregates, subsequently determined by dynamic light scattering (DLS) for 1 -5 (Table ), showed those higher order aggregates produced to exhibit a similar size and size distribution profile (intensity peak maxima = 143-184 nm), with the exception of 3 (intensity peak maxima = 672 nm), which is the only SSA to contain the inclusion of a benzothiazole moiety.