RESUMEN
BACKGROUND: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and laser irradiation. The effectiveness of current PDT using red light for advanced cancers is limited, because red light can only reach depths within a few millimeters. To enhance the antitumor effect for lung cancers, we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin and a PS, mono-L-aspartyl chlorin e6 (NPe6). METHODS: We examined whether cetuximab encapsulated in endosomes was released into the cytosol by PS in PDT under light irradiation. A431 cells were treated with fluorescein isothiocyanate-labeled cetuximab, NPe6, and light irradiation and were observed with fluorescence microscopy. We analyzed the cytotoxicity of saporin-conjugated cetuximab (IT-cetuximab) in A431, A549, and MCF7 cells and the antitumor effect in model A549-bearing mice in vivo using the iTAP method. RESULTS: Fluorescent microscopy analysis showed that the photodynamic effect of NPe6 (20 µM) and light irradiation (37.6 J/cm2 ) caused the release of cetuximab from the endosome into the cytosol. In vitro analysis demonstrated that the iTAP method enhanced the cytotoxicity of IT-cetuximab by the photodynamic effect. In in vivo experiments, compared with IT-cetuximab alone or PDT alone, the iTAP method using a low dose of IT-cetuximab showed the greatest enhancement of the antitumor effect. CONCLUSIONS: Our study is the first report of the iTAP method using NPe6 for lung cancer cells. The iTAP method may become a new, minimally invasive treatment superior to current PDT methods.
Asunto(s)
Inmunotoxinas , Neoplasias Pulmonares , Fotoquimioterapia , Humanos , Animales , Ratones , Fotoquimioterapia/métodos , Inmunotoxinas/farmacología , Inmunotoxinas/uso terapéutico , Cetuximab/farmacología , Cetuximab/uso terapéutico , Fototerapia , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
RATIONALE: Drug discovery studies invariably require qualitative and quantitative analyses of target compounds at every stage of drug discovery. We have developed a system combining molecular interaction analysis and mass spectrometry (LC-MS) using the principle of nanopore optical interferometry (nPOI) called molecular interaction kinetics-mass spectrometry (MIK-MS). Since nPOI has high binding capacity, the bond-dissociated compound can be directly detected using LC-MS. In this study, we use carbonic anhydrase II (CAII) as a ligand and apply six small compounds as analytes and report the affinity analysis using MIK-MS. METHODS: CAII was immobilized onto a COOH sensor chip using standard amine coupling. A reference surface was prepared by activating and subsequently blocking the surface under identical conditions. An amount of 50 µL of mix solution was injected over the reference channel and sample channel for CAII immobilization. The solutions eluting from the sensor chip were collected from the waste-line of the SKi Pro system every 30 s. Reconstructed elution samples were then injected into the LC-MS/MS system. RESULTS: A mixture containing furosemide, acetazolamide, 4-sulfamoylbenzoic acid, 5-(dimethylamino)-1-naphthalene sulfonamide (DNSA), sulfanilamide and sulpiride (15 µM each) was injected into the CAII-immobilized sensor chip, and the fractions eluted from the SKi Pro system were collected and subjected to selected reaction monitoring LC-MS characterization. Specific results were obtained for acetazolamide, DNSA, furosemide and sulpiride. The results suggest that the association-dissociation curve of a mixed sample can be obtained by one-time MIK-MS analysis. CONCLUSIONS: Six small-molecule binders of CAII were analyzed quantitatively using nPOI and MIK-MS, and the results were compared to published surface plasmon resonance (SPR) results. The nPOI and SPR results show good agreement, confirming the reliability of the analysis. Time-dependent binding results may be obtained by our MS sensorgram approach. Drugs that meet medical needs in a short period are required; this nPOI-LC-MS system is considered an important tool for rapid drug discovery.