Investigation of cellular uptake mechanism of functionalised gold nanoparticles into breast cancer using SERS

Gold nanoparticles (AuNPs) are widely used in various applications such as cancer imaging and drug delivery. The functionalisation of AuNPs has been shown to affect their cellular internalisation, accumulation and targeting efficiency. The mechanism of cellular uptake of functionalised AuNPs by different cancer cells is not well understood. Therefore, a detailed understanding of the molecular processes is necessary to improve AuNPs for their selective uptake and fate in specific cellular systems. This knowledge can greatly help in designing nanotags with higher cellular uptake for more selective and specific targeting capabilities with less off-target effects. Here, we demonstrate for the first time a straightforward and non-destructive 3D surface enhanced Raman spectroscopy (SERS) imaging approach to track the cellular uptake and localisation of AuNPs functionalised with an anti-ERα (estrogen receptor alpha) antibody in MCF-7 ERα-positive human breast cancer cells under different conditions including temperature and dynamin inhibition. 3D SERS enabled information rich monitoring of the intracellular internalisation of the SERS nanotags. It was found that ERα-AuNPs were internalised by MCF-7 cells in a temperature-dependent manner suggesting an active endocytosis-dependent mechanism. 3D SERS cell mapping also indicated that the nanotags entered MCF-7 cells using dynamin dependent endocytosis, since dynamin inhibition resulted in the SERS signal being obtained from, or close to, the cell surface rather than inside the cells. Finally, ERα-AuNPs were found to enter MCF-7 cells using an ERα receptor-mediated endocytosis process. This study addresses the role of functionalisation of SERS nanotags in biological environments and highlights the benefits of using 3D SERS for the investigation of cellular uptake processes.

. Characterisation of ERα-AuNPs after their functionalisation (A) Extinction spectra of BPE-AuNPs (blue), PEG5000-AuNPs (grey) and ERα-AuNPs (orange) nanotags showing that there was a shift in the wavelength when the antibody was added to the surface of AuNPs (from 529 to 533 nm). (B) Differential light scanning analysis (DLS) of PEG5000-AuNPs (grey) and ERα-AuNPs (orange) nanotags confirmed the successful functionalisation of the anti-ERα antibody as the hydrodynamic diameter of AuNPs increased and became more positive as each layer was added. The ERα-AuNPs were 80  1.6 d.nm in comparison to the PEG5000-AuNPs (73  1.0 d.nm) at pH 7.0. (C) Z-potential of PEG5000-AuNPs (grey) and ERα-AuNPs (orange) nanotags showing the increase of the zeta potential values (from -56  0.7 mV to -52  1.1 mV). This was a further verification of the anti-ERα antibody attachment to the AuNPs surface since the antibody carried a slightly positive charge at pH 7.0 (isoelectric point of anti-ERα antibody: 8.3) that increased the charge of the AuNPs. (D) Agarose gel after electrophoresis showing the distance travelled by PEG5000-AuNPs and ERα-AuNP nanotags. Gel electrophoresis is a method of separation and analysis, based on the size and charge of the samples being analysed. Here, gel electrophoresis confirmed the PEG5000-AuNPs travelled further than the ERα-AuNPs suggesting that the nanotags were of different size and/or charge and successful antibody functionalisation. (E) Lateral flow immunosorbent assay strips showing the spot from ERα-AuNPs onto the detection zone of the nitrocellulose strip. The spot was present only for samples with the matching secondary IgG antibody for ERα applied (anti-rabbit). There was no detected spot when the nanotags were applied to a lateral flow that contained a non-specific secondary IgG antibody (antimouse) or when PEG5000-AuNPs was tested with the antirabbit IgG confirming the successful binding of the anti-ERα antibody on the AuNPs surface.
SI Figure S5. Schematic of lateral flow immunosorbent assay (LFA). For the lateral flow immunosorbent assay, the strips contained a sample pad dipped in the eluent (HEPES buffer), a conjugate pad to which the nanotags were applied, a nitrocellulose strip, where the antigen is immobilised and, finally, an absorbent pad which collects the excess eluent and nanotags Afterwards, the strip is placed in HEPES buffer which flows through the conjugate pad allowing the conjugate to join the flow on the strip. When the AuNP nanotags flow over the secondary antibody, they bind to it which immobilise the nanotags on the detection spot.

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This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins SI Figure S6. Raw Data and calculations to determine the ERα antibody loading onto AuNPs using the bicinchoninic acid assay (BCA). *Concentration of remaining antibody in the supernatant calculated from the calibration curve. Absorbance was corrected by subtracting the absorbance of the PEG5000-AuNPs control sample (0 μg/mL antibody) per Bio-Rad protocol. The red dots in the BCA assay represent ERα-AuNPs sample. **Amount of antibody absorbed onto AuNPs presented as the concentration and calculated as the difference in the antibody added and antibody remaining in the supernatant. *** The average number of antibody molecules adsorbed onto each AuNPs was calculated by dividing the concentration of adsorbed antibody (converted to 178.65 nM using antibody MW of 160,000 g/mL) by the concentration of AuNPs (Initial concentration was 0.028 nM and AuNPs were centrifuged and concentrated to 2.8 nM). The SERS analysis was carried out using a Snowy Range CBEx 2.0 handheld Raman spectrometer equipped with a 638 nm laser with a maximum laser power of 40 mW. The spectrum was collected using 100% laser power with 0.05 s accumulation time. The software used to acquire spectra was Peak 1.1.112.

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SI Figure S8. SERS intensity of ERα-AuNPs prepared in H20 or RPMI media. SERS analysis was carried out on Snowy Range CBEx 2.0 handheld Raman spectrometer (Snowy Range Instruments, Laramie WY USA equipped with a 638 nm laser with a maximum laser power of 40 mW).Samples were deposited in glass vials for interrogation. Spectra were collected using 10% laser power at the sample with a 1.0 s accumulation time. The software used to acquire spectra was Peak 1.1.112. Resulting spectra were baseline corrected in Matlab 2014b.

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This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins SI Figure S9. ERα expression in breast cancer cell lines. Cell lysates were prepared from breast cancer lines and western blot analysis carried out using an antibody to ERα, b-actin was used as a loading control.
SI Figure S10. Top view of False colour SERS map for MCF-7 cells incubated with only ERα-AuNP nanotags (60 pM, 2 h). The images were generated using a Renishaw InVia Raman microscope with 50× magnification NIR APO Nikon water immersion objective with a 0.75 NA and 1.2 mW laser power (10% power) from a HeNe 633 nm excitation source with step size y,x 1.0 μm, 0.1s acquisition time and a 1200 L mm -1 grating in high confocality mode. Scale bar= 20 μm. Free anti-ERα antibodies compete with anti-ERα antibodies attached on nanotags, resulting in blocking of ERα-AuNPs internalization via mERα-mediated endocytosis. CCV is, therefore, not formed. In normal conditions, for their internalisation, ERα-AuNPs first bind to mERα, forming a mERα-ERα-AuNPs complex. The complex binds the coat proteins, and CCV assembly begins. The CCV either grows to form a vesicle and the ERα-AuNP is then internaliz ed.