EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

Abstract

The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm) showed a plasmon absorption band located within the near-infrared range (650-900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.

Fig 1. Molecular structure of EGF (chain B) according to (1JL9.pdb).

Aromatic residues are represented by different colors: Trp (red), Tyr (blue), Cys (green).

Fig 2. Temperature effect on EGF photochemistry: A) EGF fluorescence excitation and emission wavelengths were fixed at 295 nm and 330 nm, respectively, at 10°C, 15°C, 20°C, 25°C and 30°C; B) SYPRO^® Orange fluorescence excitation and emission wavelengths were fixed at 470 nm and 580 nm, at the same temperatures.

Continuous illumination was conducted during 2 hours and the excitation slit size was set at 0.8 mm (1.67 μW) for all experiments.

Fig 3. Arrhenius plot showing the linear correlation between the logarithm of the kinetic rate constant (ln k) and the inverse of temperature 1/T (ln k = ln A₀—E_a/RT), R² = 0.994).

The activation energy (E_a) and the pre-exponential factor (A₀) were 19.9±0.9 kJ.mol^-1 and 0.44±0.37 M^-1.s^-1, respectively. Uncertainty errors for ln k values are represented as error bars (percent of data: 1%).

Fig 4. UV-light power effect (different excitation slit openings) on EGF photochemistry: A) EGF fluorescence excitation and emission wavelengths were fixed at 295 nm and 330 nm, respectively, for 0.1 mm (0.12 μW), 0.5 mm (0.30 μW), 0.8 mm (1.67 μW), 1.2 mm (2.34 μW) and 2.0 mm (4.40 μW); B) SYPRO^® Orange fluorescence excitation and emission wavelengths were fixed at 470 nm and 580 nm, for the same power levels.

Continuous illumination was conducted during 2 hours and the temperature of each solution was kept at 20°C for all experiments.

Fig 5. EGF conjugated HAOA-coated gold nanoparticles represented as an illustration (upper corner) and as the TEM image at scale bar of 250 nm.

Fig 6. EGF fluorescence emission intensity at 330 nm for free EGF (2 hours 295 nm excitation), EGF-conjugated HAOA-coated gold nanoparticles (2 hours 295 nm excitation), and empty HAOA-coated gold nanoparticles and non-coated plain gold nanoparticles (1 hour 295 nm excitation).

All samples were analyzed at 20°C and excitation slit size fixed at 2.0 mm (4.40 μW).

Fig 7. A) Conjugation effect: EGF in supernatant (after conjugation) compared with free EGF and EGF-conjugated HAOA-coated gold nanoparticles; B) EGF-conjugated HAOA-coated gold nanoparticles, at different scale bar.

Fluorescence excitation spectra was fixed at 330 nm and fluorescence emission spectra was fixed at 295 nm. Experiments were conducted at 20°C and excitation slit size fixed at 2.0 mm (4.40 μW). No continuous excitation of EGF was conducted, beside the necessary for obtaining the represented spectra.

Fig 8. EGF fluorescence excitation and emission spectra acquired before and after 295 nm illumination for 2 hours.

Trp fluorescence excitation and emission wavelengths were fixed at 295 m and 330 nm, respectively. Excitation slit size was set at 2.0 mm (4.40 μW) and the temperature of each solution was kept at 20°C for all experiments.

Fig 9. SYPRO^® fluorescence excitation and emission spectra acquired before and after EGF 295 nm illumination for 2 hours.

SYPRO^® Orange fluorescence excitation and emission wavelengths were fixed at 470 nm and 580 nm, respectively. Excitation slit size was set at 2.0 mm (4.40 μW) and the temperature of each solution was kept at 20°C for all experiments.

Fig 10

A) Fluorescence emission spectra for NFK + Kyn, before and after excitation of free EGF and EGF-conjugated HAOA-coated gold nanoparticles, at a fixed wavelength of 320 nm. Experiments were conducted at 20°C and excitation slit size fixed at 2.0 mm (4.40 μW); B) Fluorescence emission spectra for Kyn + NFK, before and after excitation of free EGF and EGF-conjugated HAOA-coated gold nanoparticles, at a fixed wavelength of 360 nm. Experiments were conducted at 20°C and excitation slit size fixed at 2.0 mm (4.40 μW).

Fig 11. Colocalization of HAOA-coated gold nanoparticles conjugated with EGF.

EGF was dyed with Alexa Fluor 647 (red color) and HAOA-coated gold nanoparticles were dyed with Coumarin-6 (green color). The parts were the HAOA-coated gold nanoparticles are associated with EGF, in the same localization, are visible in yellow (scale bar at 5 μm).

Fig 12. The CD spectra of: free EGF (0.3 mg/mL), HAOA coated gold nanoparticles (without EGF), EGF-conjugated HAOA coated gold nanoparticles (16.5 μg/mL), non- conjugated EGF in supernatant, extracted by centrifugation, and EGF extracted after incubation of EGF-conjugated HAOA coated gold nanoparticles in phosphate buffer pH 5.5, at 37°C, for 72 hours.

Fig 13. Viability (%) of HaCaT cells exposed to EGF-conjugated HAOA-coated gold nanoparticles for 24 hours, assessed by MTT assay (n = 3–4; mean ± SD).

Fig 14. EGFR binding assay in A549 cell model, for 1.5 hours in contact with treatment (100X).

CN1 corresponds to the non-treated cells, while CN2 shows the exposure to HAOA-coated gold nanoparticles (without any dye). As for the treatment groups: A1) free EGF with Alexa Fluor 647, B1) EGF-conjugated HAOA-coated gold nanoparticles (only EGF is marked with Alexa Fluor 647), and C1) EGF-conjugated HAOA-coated gold nanoparticles (both EGF and HAOA-coated gold nanoparticles are marked with Alexa Fluor 647 and Coumarin-6, respectively). For A2, B2 and C2, anti-EGFR antibodies were added 1 hour before the addition of the tested samples.