Chlorin e6 - polyvinylpyrrolidone mediated photosensitization is effective against human non-small cell lung carcinoma compared to small cell lung carcinoma xenografts

Abstract

Background: Photodynamic therapy (PDT) is an effective local cancer treatment that involves light activation of a photosensitizer, resulting in oxygen-dependent, free radical-mediated cell death. Little is known about the comparative efficacy of PDT in treating non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC), despite ongoing clinical trials treating lung cancers. The present study evaluated the potential use of chlorin e6 - polyvinylpyrrolidone (Ce6-PVP) as a multimodality photosensitizer for fluorescence detection and photodynamic therapy (PDT) on NSCLC and SCLC xenografts.

Results: Human NSCLC (NCI-H460) and SCLC (NCI-H526) tumor cell lines were used to establish tumor xenografts in the chick chorioallantoic membrane (CAM) model as well as in the Balb/c nude mice. In the CAM model, Ce6-PVP was applied topically (1.0 mg/kg) and fluorescence intensity was charted at various time points. Tumor-bearing mice were given intravenous administration of Ce6-PVP (2.0 mg/kg) and laser irradiation at 665 nm (fluence of 150 J/cm2 and fluence rate of 125 mW/cm2). Tumor response was evaluated at 48 h post PDT. Studies of temporal fluorescence pharmacokinetics in CAM tumor xenografts showed that Ce6-PVP has a selective localization and a good accuracy in demarcating NSCLC compared to SCLC from normal surrounding CAM after 3 h post drug administration. Irradiation at 3 h drug-light interval showed greater tumor necrosis against human NSCLC xenografts in nude mice. SCLC xenografts were observed to express resistance to photosensitization with Ce6-PVP.

Conclusion: The formulation of Ce6-PVP is distinctly advantageous as a diagnostic and therapeutic agent for fluorescence diagnosis and PDT of NSCLC.

Figure 1

Molecular structure of Ce6, PVP and the absorption spectra of Ce6-PVP in PBS measured from 400 to 800 nm. Ce6-PVP has a prominent absorption at 400 nm and 665 nm.

Figure 2

Fluorescence imaging of lung cancers xenografted on the CAM model. Representative of white light images of NSCLC and SCLC grafted on CAM before administration of photosensitizer (Fig 2A, B). Before incubation of Ce6-PVP, the CAM tumor xenografts were imaged under blue light illumination, to confirm that there was no autofluorescence. Tumor fluorescence images at 3 h post-topical administration of 1 mg/kg of Ce6-PVP under blue light illumination (Fig 2C, D).

Figure 3

Fluorescence kinetics of Ce6-PVP on NSCLC (▲) and SCLC (■) xenografted on CAM examined up to 24 h post topical drug administration. Values are expressed as red-to-blue intensity ratio of fluorescence images post administration of drug normalized with images before drug administration. For tumor, each point represents a mean of 5 eggs whereas for normal (●), each point represents a mean of 10 eggs. Bars = standard error of the mean. Non-linear regression analysis demonstrated that all the curves were statistically different with each other. The elimination rate constant for NSCLC, SCLC and normal CAM was in the following order: NSCLC < SCLC < normal CAM.

Figure 4

Receiver operating characteristic curves illustrating the ability of Ce6-PVP to separate NSCLC (solid line) and SCLC (dotted line) from normal chorioallantoic membrane in the CAM model. The ROC curve of two indistinguishable populations (i.e. abnormal versus normal region), represented by the 45-degree line (area under the ROC curve = 0.5), is included for comparison. Area under the ROC curve was 082 ± 0.04 (p < 0.0001) and 0.70 ± 0.05 (p = 0.0009118) for NSCLC and SCLC respectively.

Figure 5

A morphologic study of NSCLC and SCLC tumor damage efficiency using the method of vital staining with Evans blue at 48 h post Ce6-PVP mediated PDT. Strong homogeneous staining was observed in the untreated controls (Fig. 5A, B), whereas in the treated tumor at 3 h drug-light interval (DLI) (Fig 5C, D) and at 6 h DLI (Fig 5E, F). Tissues damage was clearly distinguishable as an unstained area in the the tumor. Drug dose: 2.0 mg/kg; light dose: 150 J/cm²; 125 mW/cm². Each data point is an average of at least 5 animals, Bars = standard error of the mean. *The mean difference is significant at the 0.05 level compared to the NSCLC group.