Poster O18 - Understanding Protein Interactions with Two-Dimensional Nanosilicate Particles

Introduction: Two-dimensional nanosilcate particles (NS) have demonstrated their utility in prolonging the release of macromolecules such as proteins. These molecules are adsorbed onto NS particles via electrostatic interactions due to the dual-charged surfaces of NS, which exhibit negatively charged faces and positively charged edges. Because of the unique NS charge characteristics, positively charged proteins are bound more efficiently than negatively charged ones. Burst release is greatly reduced through the formation of NS-protein complexes, the size of which is governed by the NS and protein concentrations, as well as the protein net charge. NS particles naturally degrade into their constituent ions in approximately 30 days, releasing electrostatically adsorbed cargo. However, little is known about the effects of protein adsorption on protein structure and bioactivity. For applications as the delivery of therapeutic proteins, bioactivity must be retained following interaction with the NS to render the delivery effective. Here, we explore how protein charge affects formation of NS-protein complexes as well as how adsorption to NS affects protein structure and bioactivity.

Materials and

Methods: Protein affinity to NS was explored by spectrophotometric measurement of unbound protein at varying concentrations of NS (0.001-10 mg/mL), with initial protein concentration kept constant at 35 µM. Proteins with varying molecular weight and isoelectric point were tested here, including lysozyme (Lys), ribonuclease A (RNase), bovine serum albumin (BSA), horseradish peroxidase (HRP), and pepsin. Unbound protein was separated via centrifugation. Fluorescence correlation spectroscopy (FCS) was used to determine the stability of NS-protein complexes. Fluorescently-labelled RNase (1 µg/mL) was mixed with 10 µg/mL NS. Next, increasing concentrations of non-fluorescent RNase, BSA, or Lys were added to the fluorescent RNase/NS complexes. Diffusivity, molecular brightness, and bound fraction were recorded. Tryptophan fluorescence, circular dichroism, and 8-anilino-1-naphthalenesulfonic acid (ANS) assays were all used to determine the effect of NS binding on protein secondary structure. For tryptophan fluorescence, Lys (250 µg/mL) was mixed with 0-10 mg/mL NS and fluorescence measured at 315-390 nm using a spectrometer. For circular dichroism, proteins (20 µM) were mixed in varying NS concentrations (0-10 mg/mL) and spectra recorded from 200-260 nm. ANS assay was performed by mixing proteins (20 µM) with ANS (20 µM) and varying NS concentration (0-10 mg/mL). Fluorescence spectra was measured at 400-700 nm and changes in fluorescence maxima and blue shifting were recorded. Lastly, protein bioactivity was quantified for Lys and HRP according to manufacturer protocols with varying NS concentration (0-10 mg/mL), and following release from polyethylene glycol hydrogels with and without NS. Release samples were taken every 7 d and specific activity recorded.

Results, Conclusions, and Discussions: Protein affinity assays revealed a strong correlation between protein isoelectric point and binding affinity to NS. Lys (isoelectric point of 11.3) was 50% bound to NS at a NS concentration of 0.15 mg/mL, while BSA (isoelectric point = 4.7) was 50% bound at 4.2 mg/mL NS. Because of the strong correlation, a plug-and-play delivery device could be developed, where NS concentration could be tuned to achieve desired release profiles based on the protein isoelectric point. FCS revealed fluorescently labelled RNase (isoelectric point = 8.6) could be displaced by non-fluorescent Lys. whereas BSA did not displace RNase, even at 1000-fold excess. Non-fluorescent RNase also displaced fluorescent RNase at higher concentrations, though not as effectively as Lys. These results indicate that the electrostatic interactions between NS and proteins are not permanent, but rather reversible and based upon protein affinity. Tryptophan fluorescence assays, circular dichroism, and ANS assays all indicated changes in protein secondary structure following adsorption onto NS particles (Figure 1A-B). The extent of this presumed unfolding of proteins was dependent on NS concentration, with higher NS concentration leading to more substantial protein unfolding. Tryptophan showed increased fluorescence and a slight red-shift in samples with elevated NS concentrations. Circular dichroism further confirmed a loss in secondary structure of proteins at concentrations above 0.1 mg/ml NS. ANS assays revealed an increase in fluorescence as NS concentration increased, indicating higher exposure of protein hydrophobic regions. Additionally, a substantial blue shift of fluorescent peaks was observed and was contingent on NS concentrations. Such changes in ANS fluorescence and wavelength at maximum were again dependent on protein isoelectric point, with positively charged Lys and RNase exhibiting the most significant changes. Together, these results indicate that proteins exhibit some degree of unfolding when interacting with NS. Lastly, protein release assays revealed a loss of Lys and HRP activity as NS concentration increased (Figure 1C). However, this activity was recovered following dissociation and release from NS particles (Figure 1D). This seemingly indicates that proteins are not permanently denatured when bound to NS, but rather temporarily unfolded. Future work will elucidate the structure and stoichiometry of these NS-protein complexes.

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