Inhibitor-conjugated harmonic nanoparticles targeting fibroblast activation protein

Abstract

The recent progress in the engineering of nanosized inorganic materials presenting tailored physical properties and reactive surface for post-functionalization has opened promising avenues for the use of nanoparticles (NPs) in diagnosis and therapeutic intervention. Surface decoration of metal oxide NPs with ligands modulating circulation time, cellular uptake, affinity and extravasation through active targeting led to efficient cancer specific bioimaging probes. The most relevant cancer biomarkers studied so far include surface and transmembrane cancer cell receptors. More recently, tumor microenvironments and more specifically the fibroblastic element of the tumor stroma have emerged as a valuable target for diagnosis and treatment of several types of cancers. In this study, a low molecular weight ligand targeting fibroblast activation protein α (FAP), which is specifically expressed by activated fibroblasts of the tumor stroma, was synthesized. This ligand demonstrated nanomolar inhibition of FAP with high selectivity with respect to prolyl oligopeptidase (PREP) and dipeptidyl peptidase (DPP) IV, as well as good biocompatibility toward a human lung tissue model. Bismuth ferrite (BFO) harmonic nanoparticles (HNPs) conjugated to this ligand showed target-specific association to FAP as demonstrated by reverse ELISA-type assay using Human Fibroblast Activation Protein alpha/FAP Alexa Fluor® 594-conjugated Antibody and multiphoton multispectral microscopy experiments. These functionalized HNPs may provide new nanocarriers to explore the role of FAP in tumorigenesis and to target the fibroblastic component of the tumor microenvironment.

Scheme 1. Synthesis of DIBO-PEG₃-FAPi, targeting ligand for conjugation to the surface of imaging NPs. Synthesis of Biotin-PEG₃-FAPi for evaluation of FAP targeting ability.

Fig. 1. Evaluation of the cytocompatibility of the targeting ligand on the human lung tissue model MucilAir™-HF. Tissue was exposed to repeated doses (10 μM or 100 μM) of Biotin-PEG₃-FAPi (day 0, 2, 4, 7, 9, 11 and 14) and measurements were made on different days (day 2, 4, 6, 8, 10, 12, 14 and 16). (A) Cytotoxicity was evaluated by dosing LDH in the culture supernatant. (B) Trans-epithelial electrical resistance (TEER) measurement. (C) Cilia beat frequency (CBF) measurement. Triton X-100 was used as positive control; DMSO (1%) was used as negative control.

Fig. 2. (A) Synthesis scheme of functionalized BFO-PEG-FAPi NPs; (B) dynamic light scattering analyses of functionalized nanoparticles.

Fig. 3. Association of BFO-PEG-FAPi NPs to FAP through target-specific interactions. HrFAP (FAP, 100 ng) was incubated for 2 h at 37 °C with BFO-PEG-FAPi NPs (100 μg mL⁻¹) or BFO-PEG NPs (100 μg mL⁻¹) in the presence of Human Fibroblast Activation Protein alpha/FAP Alexa Fluor® 594-conjugated Antibody (anti-FAP) (dilution 1 : 50). The samples were centrifuged (5 min, 10 000 rpm) and the supernatants were transferred into a flat bottom 96-well plate. Fluorescence was measured in a multi-well plate spectrophotometer (λ_ex/λ_em = 590/645 nm). HrFAP incubated with anti-FAP and BFO-PEG-FAPi NPs incubated with anti-FAP were used as controls. Results were analyzed using a Student's t-test. Fluorescence intensity of BFO-PEG-FAPi NPs incubated with FAP and anti-FAP was significantly lower than BFO-PEG NPs incubated with FAP and anti-FAP and the fluorescence intensity of the controls (all comparisons ***p < 0.001).

Fig. 4. Association of BFO-PEG NPs and BFO-PEG-FAPi NPs to hrFAP quantified by multiphoton multispectral microscopy. HrFAP (100 ng) was incubated for 2 h at 37 °C with BFO-PEG NPs (100 μg mL⁻¹) (A) or BFO-PEG-FAPi NPs (100 μg mL⁻¹) (B) in the presence of Human Fibroblast Activation Protein alpha/FAP Alexa Fluor® 594-conjugated Antibody (dilution 1 : 5). SH and fluorescence signals were epi-collected upon focused excitation at 840 nm. The ratio of the area of conjugates emitting in both SH and fluorescence channels to the area of SH emitting structures was averaged from four distinct regions of each sample (635 × 635 μm). Error bars represent standard deviation from n = 4 calculations.