DLS analysis of AuNPs modified with oligoglycines

DLS measurements revealed that the mean hydrodynamic diameter of AuNPs (measured by intensity in the backscattering mode) increased from ~127 to ~146 nm upon the addition of T₂C₈Gly₄ (Figure 1; Table 1). This positive shift, corresponding to an increase of ~19 nm, is consistent with the formation of a stable oligoglycine coating on the gold surface. The particle size distribution (PSD) of the T₂C₈Gly₄-alone suspension was bimodal, exhibiting peaks at ~81 and ~318 nm, representing distinct populations of oligoglycine self-assemblies. This bimodal distribution is typical for this peptide amphiphile and corroborates prior literature (Arabadzhieva et al. 2019). The coexistence of these two populations can be linked to morphologically divergent tectomer aggregates, wherein the smaller peak (~81 nm) likely represents spherical aggregates and the larger peak (~318 nm) corresponds to discotic structures. Notably, the presence of AuNPs transformed this inherent bimodal distribution into a single, uniform PSD centered at ~146 nm. This transition indicates that the initial spherical and discotic oligoglycine assemblies undergo a complete structural reorganization upon association with the AuNPs. The high-affinity peptide–nanoparticle interactions caused the redistribution of the peptide molecules from these heterogeneous bulk aggregates to the nanoparticle surface, thereby establishing a single population of complex structures dominated by a uniform oligoglycine–nanoparticle interface.

Figure 1

Mean size distribution by intensity for gold nanoparticles (AuNPs), two-antennary oligoglycine (T₂C₈Gly₄), and their mixture (AuNPs, 1 ml + T₂C₈Gly₄, 2 mg/ml)

The polydispersity index values of the T₂C₈Gly₄ aqueous solutions indicate a relatively broad size distribution of tectomers, whereas the addition of AuNPs narrowed the PSD, closely resembling the profile of the bare nanoparticle suspension.

Furthermore, zeta-potential measurements confirmed effective modification of the AuNPs and a complete reversal of their surface charge. While the bare AuNPs exhibited a negative zeta-potential of –41 mV, T₂C₈Gly₄–nanoparticle complexes reached a value of +52 mV, indicating a dense coating of positively charged oligoglycine assemblies.

AFM analysis of AuNPs modified with oligoglycines

AFM imaging examined the morphology of AuNPs, T₂C₈Gly₄, and their corresponding complexes (Figure 2). Bare AuNPs deposited on glass appeared nearly spherical, well-dispersed, and uniform in size, as confirmed by the associated height profile (Figure 2A–C). In AFM height images, the x–y plane represents lateral dimensions, while the z-axis corresponds to surface height, with brighter regions indicating higher topography.

Figure 2

Representative 2D and 3D atomic force microscopy (AFM) images showing surface morphology of (A–C) bare gold nanoparticles (AuNPs) deposited on a glass substrate, (D–F) T₂C₈Gly₄ oligoglycines deposited on a glass substrate, and (G–I) AuNPs functionalized with T₂C₈Gly₄. For non-expert readers, brighter (higher) regions in the images correspond to elevated surface features, while darker (lower) regions indicate valleys or exposed substrate. Panels (C), (F), and (I) show height profiles corresponding to the red-line cross-sections in (A), (D), and (G), respectively. Surface root-mean-square roughness (Rrms) values are 13.8 ± 0.9 nm for T₂C₈Gly₄ films and 17.5 ± 1.4 nm for AuNPs–T₂C₈Gly₄ complexes; bare AuNPs exhibit a more uniform height distribution consistent with low surface roughness

Oligoglycines alone formed a rough, heterogeneous film with pronounced surface irregularities (root-mean square roughness, R_rms = 13.8 ± 0.9 nm), which reflect uneven deposition (Figure 2D–F) and align with non-uniform supramolecular assembly on the substrate. Upon functionalization, AuNPs became partially embedded within the oligoglycine layer (Figure 2G–I), leading to a reduction in particle definition and an increase in roughness (R_rms = 17.5 ± 1.4 nm). This indicates an irregular peptide coating with variable thickness and incomplete surface coverage. These observations support a robust association between AuNPs and oligoglycines and highlight the intrinsic structural variability of the peptide layer along with the limitations of AFM in resolving fully embedded nanoparticles. Future work could address these shortcomings by optimizing nanoparticle-to-peptide ratios and deposition conditions.

DSC analysis of BS interactions with AuNPs and oligoglycines

DSC assessed the interactions of BS with AuNPs and T₂C₈Gly₄ (Figure 3). All thermograms were recorded using a TA Instruments DSC, with endothermic transitions plotted upward and exothermic transitions downward.

Figure 3

Deconvoluted differential scanning calorimetry (DSC) thermograms of blood serum (BS). (A) Native BS with multiple thermal domains resolved by Gaussian deconvolution (albumin dominant; higher-temperature globulin contributions). (B) BS after interaction with gold nanoparticles, showing broadened transitions and the appearance of a high-temperature component (> 88°C), attributed to corona-associated protein stabilization. (C) Comparison of BS before (red) and after interaction with oligoglycine T₂C₈Gly₄ at 2 mg/ml (blue) and 4 mg/ml (green); oligoglycine (6 mg/ml in saline) is shown in grey. Measurements were performed using a TA Instruments DSC (endothermic up, exothermic down). High-temperature exothermic features (≈98–103°C) indicate peptide-induced protein aggregation

The serum thermograms exhibited multiple overlapping thermal domains corresponding to the denaturation of major protein fractions. To resolve these transitions, the profiles were analyzed by Gaussian deconvolution, enabling the separation and quantitative evaluation of individual components.

Native BS showed at least five partially overlapping thermal domains (Figure 3A), reflecting the complex composition of serum proteins. Albumin dominated the profile, while the higher-temperature transitions were due to globulins (primarily IgG), transferrin, and complement C proteins, consistent with literature reports (Garbett et al. 2009; Garbett and Chaires 2012). Deconvolution further allowed determining the relative contributions of each component to the total calorimetric enthalpy, confirming that albumin represented the major fraction and globulins contributed smaller but significant high-temperature components.

The thermal shifts in Figure 3B indicate the formation of a nanoparticle-associated protein corona modulating the structural stability of serum proteins. Specifically, the 2.5°C decrease in the albumin-associated transition suggests partial albumin destabilization upon adsorption to the AuNP surface, due to altered intramolecular hydrogen-bonding and hydrophobic interactions. This is consistent with previous studies reporting that AuNP–protein interactions can induce conformational rearrangements and partial unfolding of serum proteins, thereby altering their thermal denaturation behavior (Saptarshi et al. 2013). Concurrently, the calorimetric contribution of albumin decreased (from 29.4 to 23.8 mJ) while the globulin-associated region increased (from 13.8 to 16.6 mJ), further suggesting a redistribution of serum protein interactions following nanoparticle exposure. Such behavior aligns with the dynamic nature of protein corona formation and competitive adsorption processes at the nanoparticle interface (Vilanova et al. 2016). Nevertheless, owing to the complex, overlapping nature of serum DSC transitions, definitive attribution to specific protein populations remains challenging. A weak high-temperature transition emerged at ~88–100°C (1.7 mJ), which may additionally reflect thermally stable protein–nanoparticle assemblies, potentially corresponding to strongly bound corona structures (Lundqvist et al. 2017). Overall, AuNP exposure may have induced moderate but detectable reorganization of the serum protein thermal landscape, predominantly affecting albumin-associated transitions without causing extensive aggregation seen with T₂C₈Gly₄.

The interaction of T₂C₈Gly₄ with BS (Figure 3C) induced pronounced, concentration-dependent changes in the DSC profiles, upshifting the main transition temperature and triggering exothermic transitions at higher temperatures (98–103°C). Such exothermic events suggest protein aggregation and restructuring, implying new intermolecular interactions within protein–peptide complexes. The shift of the exothermic peak to lower temperatures at higher peptide concentrations indicates that aggregation is initiated more readily, consistent with an increased number of peptide–protein interaction sites.

These findings confirm that T₂C₈Gly₄ interacts more strongly with BS than with bare AuNPs, significantly altering protein thermal behavior and driving aggregation. This aligns with literature showing that DSC sensitively reports protein–ligand interactions through ligand-induced shifts in unfolding transitions, enabling the determination of binding affinities and associated thermodynamic parameters (Linkuvienė et al. 2022), as well as with reports that high-temperature exothermic effects can reflect protein aggregation or reorganization processes associated with nanoparticle–protein corona dynamics (Li et al. 2021).

SPE

SPE supported and complemented the DSC findings. Bare AuNPs induced only minor shifts in the protein profile, consistent with their weak effects on albumin stability and minimal influence on other serum proteins (Table 2).

In contrast, T₂C₈Gly₄ produced a pronounced, concentration-dependent redistribution of protein fractions, characterized by a marked increase in the α₁ fraction along with a reduction in albumin, β, and γ fractions (Table 2). These changes align with extensive peptide–protein binding and complex formation. Notably, the increase in the α₁ fraction did not reflect an actual increase in native α₁ proteins, but rather the formation of peptide–protein aggregates that migrated into this electrophoretic region. Thus, the α₁ zone effectively acts as a region of accumulation of aggregation products with altered electrophoretic mobility. This redistribution corresponds well with the DSC results, where the high-temperature exothermic transitions represent aggregation and restructuring processes, revealing the formation of new intermolecular interactions within these peptide–protein assemblies.

Together, the DSC and SPE data demonstrate that T₂C₈Gly₄ induces substantially stronger and qualitatively different interactions with serum proteins than bare AuNPs. While the peptide promotes protein reorganization, aggregation, and redistribution, AuNPs primarily induce moderate albumin destabilization and yield a limited population of thermally stabilized protein corona complexes.