Exploring Morphology of Thermoplasmonic Nanoparticles to Synergize Immunotherapeutic Fibroblast Activation Protein-Positive Cell Sensitization and Photothermal Therapy

Abstract

The precision of photothermal therapy (PTT) is often hindered by the challenge of achieving selective delivery of thermoplasmonic nanostructures to tumors. Active targeting, which leverages synthetic molecular complexes to address receptors overexpressed by malignant cells, enables such specificity and facilitates the combination of the PTT with other anticancer therapies. In this study, we developed thermoplasmonic nanoconjugates consisting of (i) 20 nm spherical gold nanoparticles (AuNPs) or gold nanostars (AuNSs) as nanocarriers, and (ii) surface-passivated antibody-based fibroblast activation protein (FAP)-targeting modules, used in adaptive chimeric antigen receptor T-cells immunotherapy. The nanoconjugates demonstrated excellent stability and specific binding to FAP-expressing fibrosarcoma HT1080 genetically modified to express human FAP, as confirmed by fluorescence activated cell sorting, immunofluorescence, and surface plasmon resonance scattering imaging. Moreover, the nanocarriers showed significant photothermal conversion after visible and near-infrared irradiation. Quantitative thermal lens spectroscopy demonstrated the superior photothermal capability of AuNSs, achieving up to 1.5-fold greater thermal enhancement than AuNPs under identical conditions. This synergistic approach, combining targeted immunotherapy with the thermoplasmonic nanocarriers, not only streamlines nanoparticle delivery, increasing photothermal yield and therapeutic efficacy but also offers a comprehensive and potent strategy for cancer treatment with the potential for superior outcomes across multiple modalities.

Keywords: fibroblast activation protein; gold nanoparticles; immunotherapeutic target modules; photothermal therapy; specific cell targeting; thermal lens spectroscopy.

Scheme 1

a) Schematic illustration of the Au‐nanoplatforms using both spherical and branched AuNPs as nanocarriers for anti‐FAP TMs. The blue TM component is the ScFv that specifically binds to FAP and is directed toward the peptide epitope E5B9, which interacts with AuNP surfaces. The red TM component consists of human IgG (IgG4). This versatile approach aims to enhance cell labeling of FAP‐overexpressing cancer cells, enabling their subsequent ablation via photothermal conversion. b) Surface biofunctionalization of AuNPs was achieved through the Au‐S covalent bond between the cysteine‐terminated E5B9 peptide epitope (shown in orange with sulfur atom acts as the anchor point as shown in pink), and Au surfaces (I–III), facilitating the formation of a protein monolayer.

Figure 1

a) Schematic illustration depicts the synthetic methodologies employed in the fabrication of spherical and star‐shaped AuNPs. b) TEM micrographs displaying the morphology of the prepared particles: I) spherical AuNPs and II) AuNSs). c) digital micrographs showing the stability test for spherical AuNPs screened at varying concentrations of TM formats. The results suggest that the optimal concentrations for fully coating the AuNPs while maintaining their original red color, indicative of high stability, are 5 μg mL⁻¹ for ScFv TMs and 10 μg mL⁻¹ for IgG4 TMs. d) UV‐Vis absorption spectra of bare spherical AuNPs with λ_LSPR at 518 nm and bare AuNSs with λ_LSPR at 775 nm. Spherical AuNPs and AuNSs conjugated with 5 μg mL⁻¹ of ScFv TMs showed redshifts of 5 nm and 19 nm in their SPR bands, respectively. When conjugated with 10 μg mL⁻¹ of IgG4 TMs, the SPR bands of the spherical AuNPs and AuNSs exhibited additional redshifts of 1 nm and 5 nm, respectively. e) 1% Agarose gel electrophoresis analysis demonstrating the migration bands of coated and uncoated AuNPs. It is noticeable that the bare spherical AuNPs aggregated within the well, whereas bare AuNSs predominantly remained in the well with some smearing, indicating minimal migration and the absence of distinct bands.

Figure 2

a) Digital photograph of the photothermal experimental setup showing the 808 nm laser source directed toward the AuNP sample, which is secured in place using a 3D‐printed holder, and an IR camera positioned at an optimal angle for recording the heat conversion process. b) Schematic representation of the thermoplasmonic heating of AuNPs under irradiation from a NIR laser source; notice the generation of a spatial distribution of temperature at distance from the NP surface. c,d) IR images and corresponding temperature profiles as a function of time for the AuNP and AuNSs samples (OD = 2), recorded over 25 min. e,f) TL spectroscopic profile of AuNPs at the onset of irradiation (50 ms); (e) laser on–off cycle; (f) Tris‐HCl buffer excited at 784 nm; g) spherical AuNPs at varying concentrations excited at 784 nm; and h) as a function of particle size, excited at 520 nm, i) AuNSs at different concentrations excited at 784 nm.

Figure 3

a) Viability assessment of the cells following treatment with nanoconjugates (OD = 0.1). b) Surface expression of hFAP on HT1080 hFAP cells assessed by FACS using the commercial anti‐FAP 5b9 mAb (n = 1). c) Schematic illustration of the fluorescent‐based assay used for the FACS experiment. d) Gel electrophoresis analysis demonstrating the migration bands of bare AuNPs, E5B9, and 5B9‐coated AuNPs. e) Volume‐weighted DLS readouts showing the size evolution of AuNPs and AuNSs coated with E5B9 and 5B9. f) FAP‐specific binding of the nanoconjugates to hFAP cells evaluated by FACSs (n = 1). g) Fluorescence microscopy images of hFAP cells at different conditions. Scale bars: 50 μm.

Figure 4

Light‐scattered images of hFAP cells following two hours of incubation with OD 0.75 of: a) AuNPs/IgG4 with an excess IgG4, b) AuNPs/ScFv with an excess of ScFv, c) AuNP/IgG4, d) AuNP/ScFv, e) AuNSs/IgG4 with an excess IgG4, f) AuNS/ScFv with an excess of ScFv, g) AuNS/IgG4, and h) AuNS/ScFv. The color scale at the bottom indicates the spectral scattering profiles: Blue represents cellular structures and organelles. Reddish hues correspond to the scattering of spherical AuNPs. Yellowish hues indicate the scattering of AuNSs. Yellow and white arrows highlight regions of increased nanoparticle accumulation. Scale bars: 50 μm.