Evaluating the impact of bioinspired counterion inclusion on silk nanoparticle physicochemical attributes and physical stability

Abstract

Silk fibroin is a promising material for nanocarrier-based drug delivery applications due to its biocompatibility, biodegradability, and mechanical properties, which can be fine-tuned through processing conditions. In this study, we explore the impact of Ca²⁺ and K⁺ inclusion on the morphology of silk nanoparticles and evaluate the short- and long-term stability of silk nanoparticles formed by antisolvent precipitation in deionized water and sodium phosphate buffer. Using advanced electric asymmetric flow field-flow fractionation multiplexed with online detectors (EAF4-UV-MALS-DLS) and orthogonal analytics (DLS, ELS, NTA, FE-SEM), we analyze the physicochemical attributes of silk nanoparticles. We find significant differences in nanoparticle architecture and stability in different buffers, with notable differences in particle size (R _g and R _h), charge, and shape measured over 56 days. Notably, nanoparticles formulated with 0.7 mg Ca²⁺ and 1.1 mg K⁺ maintained superior physicochemical stability, making them promising candidates for future nanocarrier-based applications.

Fig. 1. Schematic of silk nanoparticle storage stability study illustrating measurement workflow and analytical techniques, performed (a) after 2 h incubation at ambient temperature, (b) over 56 days, and (c) the electrical asymmetric flow field-flow fractionation (EAF4) method used for nanoparticle characterization at day 0 and day 56. Abbreviations: deionized water (DI water); sodium phosphate buffer (NaPi buffer).

Fig. 2. Colloidal stability of silk nanoparticles. Diffusion coefficients, particle size, and polydispersity index (PDI) were determined by dynamic light scattering (DLS) as a function of nanoparticle concentration. (a) The impact of continuous phase, which was either deionized water (DI water), 10 mM sodium phosphate (NaPi) buffer (pH 7.4), 1× phosphate-buffered saline (PBS) (pH 7.0–7.2), or 0.9% w/v NaCl, on nanoparticle agglomeration behavior (n = 3). (b) Agglomeration behavior of silk nanoparticles as a function of nanoparticle concentration and pH (5.8–8.0) was assessed in 10 mM NaPi buffer (n = 3). All conditions were measured after 2 hour incubation at ambient temperature. Two-way ANOVA and Dunnett's multiple comparisons test were used in the statistical analysis, using 0.0625 mg/mL as a control baseline: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Colored asterisks represent statistical analyses for each sample and condition; 0 mg cation (control, black), 0.7 mg Ca²⁺ (light blue), 11.5 mg Ca²⁺ (blue), 1.1 mg K⁺ (light pink), 17.3 mg K⁺ (pink), pH 5.8 (orange), pH 7.4 (green), pH 8.0 (purple).

Fig. 3. Storage stability of silk nanoparticles in deionized water at 4 °C. (a) Particle size, concentration, polydispersity index (PDI), and zeta potential (ZP) measured over 56 storage days in deionized (DI) water at 4 °C (n = 3). (b) SEM images (10 000× and 40 000× magnifications) of silk nanoparticles dispersed in deionized water at 4 °C on day 0 and day 56 (n = 1). (c) The fractograms of silk nanoparticles obtained from the conventional asymmetric flow field-flow fractionation (AF4) (no applied current; 0 mA) (n = 3). Two-way ANOVA and Dunnett's multiple comparisons test were used in statistical analysis, comparing the impact of storage day to day 0; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Different colored asterisks represent statistical analysis for each sample; 0 mg (control, black trace), 0.7 mg Ca²⁺ (light blue trace), 11.5 mg Ca²⁺ (blue trace), 1.1 mg K⁺ (light pink trace), 17.3 mg K⁺ (pink trace). Abbreviations: dynamic light scatter (DLS); nanoparticle tracking analysis (NTA); electrophoresis light scattering (ELS); field-emission scanning electron microscopy (FE-SEM); multiangle light scatter (MALS); radius of gyration (R_g); crossflow (X-flow).

Fig. 4. Storage stability of silk nanoparticles in sodium phosphate (NaPi) buffer (pH 7.4) at 4 °C. (a) Particle size, concentration, polydispersity index (PDI), and zeta potential (ZP) measured over 56 storage days in 10 mM NaPi buffer (pH 7.4) at 4 °C (n = 3). (b) FE-SEM micrographs (10 000× and 40 000× magnifications) of silk nanoparticles dispersed in 10 mM NaPi buffer (pH 7.4) at 4 °C on day 0 and day 56 (n = 1). (c) Corresponding fractograms of nanoparticles obtained from conventional asymmetric flow field-flow fractionation (AF4) (n = 3). Two-way ANOVA and Dunnett's multiple comparisons test were used in statistical analysis, comparing the impact of storage duration; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Different color of an asterisk represents statistical analysis for each sample; black: 0 mg cation, light blue: 0.7 mg Ca²⁺, blue: 11.5 mg Ca²⁺, light pink: 1.1 mg K⁺, pink: 17.3 mg K⁺. Abbreviations: dynamic light scattering (DLS); nanoparticle tracking analysis (NTA); electrophoretic light scattering (ELS); field-emission scanning electron microscopy (FE-SEM); multiangle light scattering (MALS); radius of gyration (R_g); crossflow (X-flow).

Fig. 5. Electrical asymmetric flow field-flow fractionation (EAF4) fractograms of silk nanoparticles dispersed in deionized water and 10 mM sodium phosphate (NaPi) buffer (pH 7.4). Silk nanoparticles (0.1 mg/mL) were separated in 0.5 mM sodium carbonate under electrical fields with applied positive (+0.2 mA), neutral (0 mA), and negative (−0.2 mA) currents. (a) Nanoparticles EAF4 fractograms for day 0 and day 56 storage were presented as a function of normalized MALS signal and shape factor (R_g/R_h): (a) deionized water storage and (b) NaPi buffer (pH 7.4) storage (n = 3). Abbreviations: hydrodynamic radius (R_h); radius of gyration (R_g); multiangle light scattering (MALS).