Multiplexed in vivo fluorescence optical imaging of the therapeutic efficacy of photodynamic therapy

Abstract

In our study we wanted to elucidate a time frame for in vivo optical imaging of the therapeutic efficacy of photodynamic therapy (PDT) by using a multiplexed imaging approach for detecting apoptosis and vascularization. The internalization of the photosensitizer Foslip(®) into tongue-squamous epithelium carcinoma cells (CAL-27) was examined in vitro and in vivo. For detecting apoptosis, annexin V was covalently coupled to the near-infrared dye DY-734 and the spectroscopic properties and binding affinity to apoptotic CAL-27 cells were elucidated. CAL-27 tumor bearing mice were treated with PDT and injected 2 days and 2 weeks thereafter with DY-734-annexin V. PDT-induced changes in tumor vascularization were detected with the contrast agent IRDye(®) 800CW RGD up to 3 weeks after PDT. A perinuclear enrichment of Foslip(®) could be seen in vitro which was reflected in an accumulation in CAL-27 tumors in vivo. The DY-734-annexin V (coupling efficiency 30-50%) revealed a high binding affinity to apoptotic compared to non-apoptotic cells (17.2% vs. 1.2%) with a KD-value of 20 nm. After PDT-treatment, the probe showed a significantly higher (p <0.05) contrast in tumors at 2 days compared to 2 weeks after therapy (2-8 h post injection). A reduction of the vascularization could be detected after PDT especially in the central tumor areas. To detect the therapeutic efficacy of PDT, a multiplexed imaging approach is necessary. A detection of apoptotic cells is possible just shortly after therapy, whereas at later time points the efficacy can be verified by investigating the vascularization.

Multifunctional calcium phosphate nanoparticles for combining near-infrared fluorescence imaging and photodynamic therapy

“…The same localization was seen after incubating CAL-27 cells with the photosensitizer formulation Foslip Ò [40], implying that a comparable PDT effect with a destruction of tumor cells is possible by photosensitizerloaded nanoparticles. The proven uptake of the nanoparticles into CAL-27 cells in vitro supports a real enrichment of the nanoparticles in tumor cells in vivo.…”

“…Apoptosis after nanoparticle-PDT was imaged with DY-734-annexin V, a fluorescent probe with high affinity to phosphatidylserine on apoptotic cells, which was designed and characterized previously [40]. In short, annexin V (Abcam, Cambridge, UK) was coupled to the NIRF dye DY-734-NHS (Dyomics GmbH, Jena, Germany) by mixing both components for 2 h in coupling buffer and subsequent extraction of the probe by ultracentrifugation.…”

Lowering photosensitizer doses and increasing fluences induce apoptosis in tumor bearing mice

“…A clear signal was only visible using the highest dose of PDT (40 µg Foslip per kg weight, 100 J/cm 2 ), indicating that the probe is specifically retained only in these tumors due to the presence of phosphatidylserine on the surface of apoptotic tumor cells. Indeed the occurrence of apoptosis due to PDT was already confirmed by the nuclei staining of tumor cells after PDT and the occurrence of condensed chromatin (equivalent drug doses) [7].…”

“…Fluorescence intensity of the mice was monitored before injection of the probe (−1 h) as well as between 4 to 24 h thereafter with the Maestro in vivo fluorescence imaging system (excitation/emission filter: 670 -710 nm/750 nm longpass, Cri Inc). Fluorescence intensity was analyzed semiquantitatively by spectral unmixing as described previously [7]. The fluorescence contrast from tumor to muscle was calculated by subtracting the average fluorescence signal of the muscle from that of the tumor.…”

“…Hereto, it was shown that it is possible to detect the efficacy of PDT at 2 days after therapy via multiplexed near-infrared fluorescence (NIRF) optical imaging of apoptotic tumor cells with a fluorescent annexin V probe and the tumor vascularization using an integrin targeting probe [7]. Nevertheless, the underlying molecular alterations after reduced photosensitizer concentrations and fluences of the laser light have never been investigated up to now.…”