Label-free plasmonic nanostar probes to illuminate in vitro membrane receptor recognition

Peptide functionalized plasmonic nanostars evince Raman signals from targeted receptors in cells and modulate protein corona formation, improving targeting.

Cyclic(-Arg-Gly-Asp-D-Phe-Cys) purchased from Peptide international. Recombinant human αvβ3 integrin protein (αvβ3) was purchased from R&D systems. All cell culture reagents were purchased from Thermo Fischer Scientific (USA).

Instrumentation
Raman measurements were carried out using a Renishaw InVia instrument with 633 nm laser excitation. Extinction measurements were carried out using a VWR V-1600 Spectrophotometer. Scanning electron microscope (SEM) images were obtained using a Magellan XHR SEM. Size and zeta-potential measurements were carried out using a Malvern Zetasizer ZS and concentration calculated using a Malvern Nanosight 300NS.

Au NS Synthesis
Au multi-twined seeds were prepared by adding HAuCl4 (50 µl, 10 mM) to CTAC (1 mL, 100 mM). This solution was mixed for 30 minutes whilst NaBH4 solution (10 mM) was cooled in a 4 o C fridge. After 30 minutes, 150 µL of cold NaBH4 was added to the solution and left to mix for an additional 3 minutes. Upon addition of the chilled NaBH4, the solution turned orange signifying the synthesis of Au multi-twined seeds and was left overnight in the fridge prior to use. To grow the NS, a CTAC solution (10 mL, 100 mM) was prepared and 1 mL was removed and added to 1 µL of seed. The growth solution was prepared by adding HAuCl4 (450µL, 10 mM), AgNO3 (90 µL, 10 mM), HCl (180 µL, 1 M) and C6H8O6 (90 µL, 100 mM) to the remaining CTAC solution and left to mix for 5 minutes. The growth solution was split into 9x1 mL vials and different volumes of seed solution were added to each vial (800, 600, 400, 200, 100, 50, 30, 10, 1 µL). The solutions were left to shake for 1 hour. Then solutions were centrifuged at 6000 RPM for 20 minutes, and then resuspended in 1 mL of HPLC grade water (H2O) to remove excess CTAC. The centrifugation and resuspension were repeated twice. The NS were characterized using extinction spectroscopy and SEM. The chosen NS that were incubated with cells were also characterized for size, zeta potential, and concentration using a NanoSight (Malvern, Zetasizer ZS (Malvern) and extinction by UV-Vis spectroscopy.

SERS of RGDFC
Cyclic RGDFC (200 µM) was added to 1 mL of Au NS with LSPRs of 720 nm and left to incubate overnight. The sample was then centrifuged and resuspended (as described before) to remove excess RGDFC. The extinction of the Au-RGDFC NS was taken and compared to Au NS. Au and Au-RGDFC NS were then concentrated to a volume of 100 µL, from which 2 µL was spotted onto a clean glass slide (washed three time with ethanol and H2O) and left to dry. Average SERS spectra were obtained by focusing on 10 different particle clusters using a 50x objective lens and interrogated with 633 nm laser excitation with a power of 0.8 mW, centered at 1000 cm -1.

SERS of αvβ3 integrin
αvβ3 integrin (2 µL of 200 µg/mL) was added to 10 µL of Au-RGDFC NS and left to incubate for 2 hours. 2 µL of Au-RGDFC-αvβ3 NS conjugates were spotted onto a clean glass slide and left to dry. Spectra were obtained by focusing on different particle clusters with a 50x objective lens and interrogated with 633 nm laser excitation, with power of 0.8 mW, scanning between Raman shifts of 800-2000 cm -1 . Raman analysis was also carried out for Au and Au-RGDFC NS.

Characterization of protein corona
10 µL of Au or Au-RGDFC NSs were added to 10 µL of RPMI cell media or 10 µL of RPMI media supplemented with 10% FBS and incubated for 2 hours. 2 µL of the conjugates were then spotted onto a clean glass slide and left to dry. Average spectra were obtained by focusing on different particle clusters with a 50x objective lens and interrogated with 633 nm laser excitation, with power of 0.8 mW, scanning between Raman shifts of 800-2000 cm -1 . Extinction and size measurements were carried out by adding 500 µL of Au or Au-RGDFC NS with 500 µL of RPMI cell media with and without the addition of 10% FBS and analyzed after incubating for 2 hours.

SERS of αvβ3 integrin in complex matrix
10 µL of Au-RGDFC NSs, 10 µL of FBS supplemented RPMI media and 2 µL of αvβ3 integrin (200 µg/mL) were added together and incubated for 2 hours. FBS supplemented media was also added to Au and Au-RGDFC NS. 2 µL of all the conjugates were spotted onto a clean glass slide and left to dry. Spectra were obtained by focusing on different particle clusters with a 50x objective lens and interrogated with 633 nm laser excitation, with power of 0.8 mW, scanning between Raman shifts of 800-2000 cm -1 .

NS cell incubation and SERS analysis
SW620 cells were cultured in FBS supplemented RPMI media using previously reported published procedures 1 and cells and media transferred to a culture flask (2 mL) with coverslip. 200 µL of Au NS (acting as a control) and Au-RGDFC NS were added to the culture flask and incubated for 2 hours. The cell media was then removed and the cells which had adhered to the coverslip were washed with 2 mL of sterile PBS (three times) and fixed by immersing the coverslip in 4% formaldehyde for 15 minutes. The coverslip was then removed and washed again with PBS and H2O and left to dry. The cells were then imaged by focusing on the cell membrane with a 50x laser objective with a numerical aperture of 0.75 and Raman mapped using a 1x1 µm step size, 633 nm laser excitation, with a laser power of 4 mW, scanning between 800-2000 cm -1 .

RGD Blocking
2 mL of FBS supplemented RPMI media and SW620 cells were added to a culture flask with coverslip. 200 µL of RGD (stock 7.3 mM) was added to flask and left incubating for 2 hours. Au-RGDFC NS were the added to the flask for an additional 2 hours and the cells were washed, fixed and analyzed as described in the previous section.

Data analysis
Raw SERS spectra were preprocessed (baselined and normalized) prior to analysis using the PLS toolbox plugin. Multivariate curve resolution (MCR) was carried out to classify the SERS. Heat and score maps were reconstructed using MATLAB. False color images were created using WiRE software on baselined SERS maps (baselined with WiRE intelligent fit) using direct classical least squares (DCLS) with the MCR generated αvβ3 integrin spectrum as a reference. Figure S1 Normalized extinction spectra and SEM images of Au NS synthesized using different volumes of Au multi-twinned seeds.

Supplementary Figures
By decreasing the volume of gold multi-twinned seed added to growth solution, the LSPR of the synthesized NS are red shifted as seen in the extinction spectra and NS color change. The SEMs of 4 NS demonstrate the size and shape of synthesized particles associated with the LSPR. High volumes of seed (500 µL) yield triangular shaped particles, whilst decreasing the volume allows more branch growth to occur and the synthesized particles are star shaped (10 µL and 1 µL). Cyclic RGDFC was successfully synthesized to both NS evident in the blue shifting and peak broadening which occurred in the extinction spectra. The SERS spectra also indicate conjugation has occurred due to the presence of the 1000 cm -1 peak from the phenylalanine and 1030 cm -1 arginine residues of the RGDFC molecules.

Figure S4
False color images of SW620 cell incubated with Au-RGDFC NS created using the MCR generated spectrum of purified αvβ3 integrin as a reference. MCR reference spectrum and extracted spectra from within the blue box and green box for each cell. Cells were mapped using a 50x microscope objective, 1x1 µm step size, 633 nm laser excitation with a laser power of 4 mW, scanning between 800-2000 cm -1 . Figure S4 demonstrates the reproducibility of the signal obtained from cells incubated with Au-RGDFC NS. Of the 3 cells presented, the DCLS false color images indicate many areas where the spectra have similar spectral contributions as the MCR αvβ3 spectrum. The signal is also concentrated to small areas of the cell due to integrin clustering that occurs on the cell membrane.

Figure S5
Comparison of white light and DCLS false color images obtained from 5 cells that were incubated with a) Au NS and b) Au-RGDFC NS. Cells were mapped using a 50x microscope objective, 1x1 µm step size, 633 nm laser excitation with a laser power of 4 mW, scanning between 800-2000 cm -1 By comparing the DCLS false color images of cells incubated with Au and Au-RGDFC NS, there is a clear difference in the intensity and number of the pixels in each map. This is due to the Au-RGDFC NS correctly targeting the αvβ3 integrin and enhancing it and other cellular components, whereas the Au NS are not attracted to the αvβ3 and the small number of spectra with signal is due to NS electrostatically binding to the cell surface, while most are washed away or stuck to the glass surface, enhancing the protein corona.  Phenylalanine, tryptophan, C-C