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. 2020 Mar;96(2):301-309.
doi: 10.1111/php.13155. Epub 2019 Oct 13.

Predictors and Limitations of the Penetration Depth of Photodynamic Effects in the Rodent Brain

Collin T Inglut #  1 , Brandon Gaitan #  1 , Daniel Najafali  1 , Irati Abad Lopez  1 , Nina P Connolly  2   3 , Seppo Orsila  4 , Robert Perttilä  4 , Graeme F Woodworth  2   3 , Yu Chen  1   2 , Huang-Chiao Huang  1   2
Affiliations

Predictors and Limitations of the Penetration Depth of Photodynamic Effects in the Rodent Brain

Collin T Inglut et al. Photochem Photobiol. 2020 Mar.

Abstract

Fluorescence-guided surgery (FGS) is routinely utilized in clinical centers around the world, whereas the combination of FGS and photodynamic therapy (PDT) has yet to reach clinical implementation and remains an active area of translational investigations. Two significant challenges to the clinical translation of PDT for brain cancer are as follows: (1) Limited light penetration depth in brain tissues and (2) Poor selectivity and delivery of the appropriate photosensitizers. To address these shortcomings, we developed nanoliposomal protoporphyrin IX (Nal-PpIX) and nanoliposomal benzoporphyrin derivative (Nal-BPD) and then evaluated their photodynamic effects as a function of depth in tissue and light fluence using rat brains. Although red light penetration depth (defined as the depth at which the incident optical energy drops to 1/e, ~37%) is typically a few millimeters in tissues, we demonstrated that the remaining optical energy could induce PDT effects up to 2 cm within brain tissues. Photobleaching and singlet oxygen yield studies between Nal-BPD and Nal-PpIX suggest that deep-tissue PDT (>1 cm) is more effective when using Nal-BPD. These findings indicate that Nal-BPD-PDT is more likely to generate cytotoxic effects deep within the brain and allow for the treatment of brain invading tumor cells centimeters away from the main, resectable tumor mass.

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Figures

Figure 1.
Figure 1.. Absorbance (blue) and fluorescence (red) spectra of BPD, PpIX, Nal-BPD, and Nal-PpIX.
(a) Free form BPD fully dissolved in DMSO has a strong absorbance peak at 435 nm (Soret band) and 690 nm (Q band). Typically, 690 nm light is applied to activate BPD for PDT. Fluorescent BPD has a peak emission in the NIR range (λmax at ~700 nm). (b) Free form PpIX fully dissolved in DMSO shows a strong absorbance peak at 405 nm (Soret band), with multiple Q bands in the green to red range. Typically, PpIX is activated by 635 nm light or 375–440 nm light for PDT or FGS, respectively. The fluorescence signal emitted by PpIX is in the red/NIR range between 635 and 700 nm. (c) Free BPD is hydrophobic and readily aggregates in PBS, as demonstrated by the decrease in magnitude of the absorbance spectrum, resulting in fluorescence quenching of BPD with minimal photoactivity (< 2%). Entrapping BPD in a nanoliposomal formulation restores its fluorescence and photoactivity (35.8 ± 0.2%) in PBS. (d) Likewise, free form PpIX aggregates in PBS, leading to a quenched state that is no longer activatable at 635 nm. Similarly, the encapsulation of PpIX within a nanoliposomal formulation restores its fluorescence and photoactivity up to 82.8 ± 0.1%.
Figure 2.
Figure 2.. Singlet oxygen (1O2) production and photobleaching of Nal-BPD and Nal-PpIX upon light activation.
(a) SOSG fluorescence emission and (b) photobleaching percentage of Nal-BPD and Nal-PpIX with respect to increasing fluences of light at 690 nm and 635 nm, respectively.
Figure 3.
Figure 3.. Experimental setup for examining the limit of the photodynamic effect.
Glass capillaries filled with 5 μM of Nal-BPD or Nal-PpIX were inserted at various heights (Z, cm) in Sprague Dawley rat brains and exposed to surface illumination of 690 nm or 635 nm at various fluences.
Figure 4.
Figure 4.. Depth of photosensitizer activation within brain tissue determined via degree of photobleaching.
Glass capillaries (300 μm in diameter) containing 5 μM of Nal-BPD (or Nal-PpIX) were inserted at varying depths within ex vivo rat brains and irradiated with light (690 nm or 635 nm; 100 mW/cm2; 0, 10, 20, 40, 80 J/cm2). Photobleaching of (a) Nal-BPD and (b) Nal-PpIX were evaluated at different depths and fluences. (c) Fluorescence images of capillaries filled with Nal-BPD showing 90, 73, and 10% photobleaching in brain tissues at 0, 0.4, and 1.4 cm deep, respectively. (d) At 80 J/cm2, 27% Nal-BPD photobleaching was observed 1 cm deep in tissue, in contrast to only 0–2% photobleaching of Nal-PpIX.
Figure 5.
Figure 5.. Visualization of the fluorescent emission of Nal-BPD and Nal-PpIX when excited at 375 nm.
(a) Capillaries (300 μm in diameter) filled with the photosensitizers (Nal-BPD or Nal-PpIX, 5 μM) were excited with 375 nm and captured with a phone camera (OnePlus 5). PpIX’s fluorescent signal can be easily visualized by the naked eye. (b) Capillaries were inserted into the brain and were excited to examine if the fluorescent signal could be visualized through brain tissue. The emission signal from PpIX could be seen when the capillary was inserted ~0.04–0.05 cm deep in tissue (black arrow).
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