A study of concentration changes of Protoporphyrin IX and Coproporphyrin III in mixed samples mimicking conditions inside cancer cells for Photodynamic Therapy

Abstract

Photodynamic Therapy (PDT) using Aminolevulinic acid (ALA) could be an effective and minimally invasively applicable way to treat many different types of tumors without radiation and large incisions by just applying a light pulse. However the PDT process is difficult to observe, control and optimize and the dynamical relationships between the variables involved in the process is complex and still hardly understood. One of the main variables affecting the outcome of the process is the determination of the interval of time between ALA inoculation and starting of light delivery. This interval, better known as drug-light interval, should ensure that enough Protoporphyrin IX (PPIX) is located in the vicinity of functional structures inside the cells for the greatest damage during the PDT procedure. One route to better estimate this time interval would be by predicting PPIX from the dynamical changes of its precursors. For that purpose, in this work a novel optical setup (OS) is proposed for differentiating fluorescence emitted by Coproporphyrin III (CPIII) and PPIX itself in samples composed of mixed solutions. The OS is tested using samples with different concentrations in mixed solutions of PPIX and the precursor CPIII as well as with a Polymethyl methacrylate test sample as additional reference. Results show that emitted fluorescence of the whole process can be measured independently for PPIX and its precursor, which can enable future developments on PPIX prediction from the dynamical changes of its precursor for subject-dependent drug-light interval assessment.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Scheme with the main steps of the heme synthesis for PPIX formation from ALA.

Fig 2. Schematic of the OS showing the light path after each filter and the change of the spectral range.

blue: excitation beam, red: emission stemming from the excited samples, (description of abbreviations are shown in Table 1).

Fig 3. Protocol for the sample mixtures.

For each type of sample one concentration was kept constant while the other was gradually doubled. Dots represent the changing constituent while the background colour represents the constant constituent.

Fig 4. Position of the main peaks of the two porphyrin stem solutions.

Measured at pH 7.4 and a concentration of 247.56 μMol/l.

Fig 5. General setup for fluorescence measurements from the samples.

Fig 6. Raw data acquired with our filter-fluorometer: Exemplary for one measurement of a mixture with changing CPIII concentration.

The measurement begins with an offset (A), PMMA sample (B1 and B2) and the 4 different mixtures (C–F).

Fig 7. Changing CPIII concentration (A), Changing PPIX concentration (B).

First measurement line, second measurements dotted line and third measurement fine dotted line.

Fig 8. Differences changing CPIII concentration (A), differences changing PPIX concentration (B).

(CPIII A) 1: 0.12V ± 0.052V, 2: 0.38V ± 0.11V, 3: 0.67V ± 0.063V, 4: 0.93V ± 0.102V, (PPIX B) 1: 1.4V ± 0.032V, 2: 1.32V ± 0.03V, 3: 1.27V ± 0.097V, 4: 0.97V ± 0.082V.

Fig 9. Control of the spectra of the samples as measured with the Cary spectro-fluorometer.