Anticancer Activity from Gold-alpha-Lactalbumin Nanoconstructs?

Abstract

Human α-Lactalbumin made lethal to tumor cells (HAMLET), and its bovine analogue BAMLET, have in the recent years shown promising results in cancer therapy. HAMLET contains several oleic acids which in turn stabilize a partially unfolded conformational state of the protein, inducing a higher surface activity compared to the native protein, and likely play a role in its cancer-detrimental function as well. Herein, we report the formation of gold-bovine α-lactalbumin nanoconstructs (Au–LA NCs) displaying conformational changes in the native protein and its concomitant ability to reduce HeLa cell viability approaching that of BAMLET. Modification of LA with gold was achieved via two synthesis protocols; (i) utilizing the intrinsic reduction potential of LA (Au–LAint) or (ii) the addition of an extrinsic reducing agent (Au–LAext). The gold–protein nanostructures formed were investigated using a palette of analytical probes including AFM, TEM, ζ-potential, UV–vis, circular dichroism, and fluorescence (steady-state and time-resolved). Toxicity toward HeLa cells was studied using a trypan blue assay and benchmarked against BAMLET. Whereas constructs from both synthetic protocols employed result in conformational changes of the protein and altered surface activity compared to the native protein, Au–LAint was found to display lipid-specific interaction and severe disruption of lipid monolayers. Au–LAint also revealed toxicity toward HeLa-cells comparable to that of BAMLET. The results imply that formation of Au–LA nanoconstructs could be a new route to making HAMLET-like materials, while also imparting a built-in optical probe from fluorescent or plasmonic gold species.

Self-assembly and characterization of transferrin–gold nanoconstructs and their interaction with bio-interfaces

“…In samples where only AuNCs are present, the AuNCs can be detected with fluorescence spectroscopy. [15][16][17] While the LSPR feature is prominent in the Tf-AuXH system, the band observed for Tf-AuINT has lower absorbance and occur at shorter wavelengths, indicating smaller AuNPs. 16 When AuNPs and AuNCs are in close proximity, the AuNPs can quench the emitted fluorescence from AuNCs.…”

“…16 When AuNPs and AuNCs are in close proximity, the AuNPs can quench the emitted fluorescence from AuNCs. [15][16][17] Steady-state emission spectra of the Tf-AuNCs upon excitation at 370 nm are shown in Figure 1b. UV-visible spectra of the different Tf-AuNCs studied here, as well as the reference systems (Tf only and Tf treated with NaBH 4 ), are shown in Figure 1a.…”

“…From the results shown in Table 2, neither the lifetimes nor the associated populations are significantly affected by the addition of NaBH 4 . 15,16 The biggest change in the average lifetime was calculated for Tf-AuINT. For the Tf-AuXL system, there is a minor decrease in the average lifetime compared to the native protein.…”

“…For the Tf-AuXL system, there is a minor decrease in the average lifetime compared to the native protein. 15,16 In order to ascertain that the observed changes in CD profiles did not emanate from signal attenuation from scattering contribution or reduction in protein concentration due to aggregation and subsequent precipitation, changes in the CD line shape were estimated via the ratio of mean residual ellipticity at 200 nm and 222 nm (I200/I222, Table 2). We attribute this to quenching from increased solvent exposure due to partial unfolding, energy transfer (FRET) to the embedded AuNCs, or a combination of these effects.…”

“…The phosphate buffer was prepared by mixing dipotassium hydrogen phosphate (KH 2 PO 4 , 50mM, Merck) and potassium dihydrogen phosphate (K 2 HPO 4 , 50mM, Merck) to a final pH of 7.4 15 . In order to remove excess TCAA, samples were dialysed against de-ionized (MilliQ) water for two hours, before water exchange and dialysis over night.…”