‘Let there be light.’
That is the introductory phrase of the Hebrew Bible – as it should be! It’s not just the Bible: most cultures have a sun deity usually associated with health, joy, and success. The ubiquity of a sun deity in multiple cultures around the world stems from the fact that light has played a major role in the establishment of the universe. It is vital in making our planet’s temperature habitable, allowing crops to grow. It also aids human beings to synthesize Vitamin D for stronger bones, better immune function, and improved cardiac health. Sunlight has been known to improve health, but light can also be used for more specific purposes. Research suggesting that light has antimicrobial effects dates back to the late 1800s (Downes & Thos, 1877), with more recent research, such as preferential inactivation of microbes for cleaner rooms (Fahimipour, 2018), currently in the works.
However, light has been seen to have more potential than just its antimicrobial activity. It was in the laboratory of Hermann von Tappeiner, an Austrian pharmacologist, that the first strides towards photodynamic therapy were made (Tappeiner & Jesionek, 1903). Von Tappeiner and his colleagues carried out a clinical trial using eosin as a photosensitizer to treat basal cell carcinoma (a type of skin cancer). They found that injecting eosin solution followed by long-term exposure to light yielded total tumour resolution and a 12-month relapse-free period in 4 out of 6 patients. This showed that light, in combination with a photosensitive agent and oxygen, could be used to treat cancer. This is essentially the foundation of photodynamic therapy. Further research was performed to synthesize and characterize photosensitive agents and to understand their efficacy in the diagnosis and treatment of malignant tumours.
But what is the basis of this antitumor effect of light? And why is this therapy better (or worse) than chemotherapy?
Let’s examine the basic components required to carry out photodynamic therapy: a photosensitizer, a light source (of a particular wavelength that can excite the photosensitizer), and the presence of oxygen in the target tissue.
In 1948, research on laboratory animals showed that porphyrin (a photosensitive agent) and its derivatives accumulate in neoplastic (tumour-forming) tissue, as well as in embryonic and regenerating tissues (Figge, Weiland, & Manganiello, 1948). The affinity of porphyrin to neoplastic tissue was striking. The tumour fluoresced red under UV light of a specific wavelength, a stark contrast from the non-neoplastic tissue that showed no fluorescence, owing to a minimal accumulation of porphyrin (as compared to the neoplastic tissue).
Here, porphyrin acts as a photosensitizer. A photosensitizer is a chemical agent that absorbs light of a specific wavelength and generates ROS (reactive oxygen species) that are toxic to the cell, causing cellular death. It is important to note that this definition is concerning photodynamic therapy. If the term porphyrin sounds familiar, it’s because you’ve read about it in photosynthesis – chlorophyll, the light-sensitive agent required for photosynthesis, is a porphyrin derivative.
Structure of Chlorophyll (Puntener & Schlesinger, 2000)
These porphyrin and porphyrin derivatives react with the light of specific wavelengths (traditionally supplied using optical surface applicators (Chamberlain, et al., 2020)) to give rise to short-lived ROS, such as singlet oxygen radicals, which react with the DNA present in the cells and DNA strand breakage or DNA mutations.
Oxidation of guanine base can induce mispairing and lead to mutations (Wylie, 2006)
This causes the cell to undergo apoptosis (i.e., programmed cell death). If the cell can’t repair the DNA damage, it commits suicide to stop itself from dividing and passing on this damaged DNA to its daughter cells.
This method of cancer therapy provides an edge over systemically administered chemotherapy; it is an easy way to select tumour cells as the site of action of the therapeutic drug, as opposed to chemotherapeutic drugs which are administered intravenously and possess lesser selectivity, thus damaging normal, healthy cells as well. Iridium-associated albumin (human serum albumin, a protein found in the plasma of blood, was used) as a photosensitized molecule showed ample penetration in cancer cells (Zhang, et al., 2019).
But there is one drawback to this technique: using optical fibres or lasers as light irradiation sources does not allow for the treatment of deeply-seated tumours. If the tumour is in the neck or located in a cavity that opens to the external environment (say, your alimentary canal), the light treatment is done by inserting an endoscope (a device used to look inside your body) to locate the region where the tumour is present, followed by a fibre optic cable to irradiate it. However, if the tumour is present on, for example, the liver (as is the case with hepatic cancers), it is difficult to have light penetrate the tissue overlying the tumour; this tissue comprises skin, endothelial vessels, and muscles. To irradiate these tumours with fibre optic cables, they need to be implanted into the site of the tumour, which is an invasive procedure and may have high levels of risk associated with it.
Complications surrounding dark toxicity arise. ‘Dark toxicity’ of a photosensitizer refers to its potential to reduce the viability of a cell in the absence of irradiation, i.e., it occurs without photoexcitation. This is dangerous as it may damage the normal, healthy cells as well; remember, it is the location-specific irradiation that produces tumour-specific cytotoxic interactions in cancer cells. Furthermore, high-intensity light also does not provide light homogenously and does not cause the breakdown of non-identified malignant tissue.
Owing to these issues, it became increasingly necessary to come up with an alternative way to irradiate the neoplastic tissue of deeply-seated tumours.
A solution arrived in the form of chemiluminescence, which is the release of electromagnetic radiation as light generated as the energy released from a chemical reaction. Chemiluminescent agents can be transferred to the site of the tumour to generate light energy. This was done using luminol as the chemiluminescent source to target erythroleukemic tissue (cancerous red blood cells) with transferrin-hematoporphyrin as a photosensitive molecule (Laptev, 2006). In this method, however, it is important to find a chemiluminescent source that emits light of the wavelength required to activate the photosensitive compound used.
Bioluminescence is a special case of chemiluminescence, wherein the chemical reaction that generates light is enzyme-catalyzed, and usually involves oxidation of a specific substrate (enzyme-mediated oxidation). Bioluminescence occurs naturally in a wide variety of living organisms (you may be familiar with glowing jellyfish) and can be utilized for supplying light for photoexcitation of photosensitizers accumulated in deep-lying malignancies.
The bioluminescence-mediated photodynamic therapeutic system consists of a substrate, an enzyme, a photosensitive molecule, and, as always, the presence of tissue oxygen. In most cases, the mechanism involves the enzyme being conjugated to or immobilized upon quantum dots, which exhibit luminescence upon being treated with the appropriate substrate. Quantum dots are microscopic semiconductors of dimensions in nanometres. When these dots are luminesced with UV light, an electron gets excited to a higher energy level. When this excited electron returns to the ground state, it emits light, which excites the photosensitizer, further generating cytotoxic ROS, leading to cell death. This is known as bioluminescence resonance energy transfer (BRET) and involves the transfer of energy between a luminescent donor and an excitation-prone acceptor. BRET is commonly used for studying specific protein-protein interactions and conformational rearrangements in live cells (Kobayashi, 2019). The therapeutic applications of BRET are recently being realized, with BRET-mediated photodynamic therapy being a highly potent one. Quantum dots can also be used as bioluminescent probes, in addition to this application (Evanko, 2006).
The first use of BRET-mediated PDT was demonstrated by Theodossiou (T, et al., 2003), albeit, in this case, the gene encoding the enzyme necessary for luminescence was expressed by a line of cancer cells via genetic engineering, giving rise to a self-illuminated PDT system. The photosensitizer used was Rose Bengal. However, such use of genetic engineering is not widely feasible for cancer treatment, and thus other methods of bringing the enzyme in contact with its substrate at the site of the tumour had to be thought of.
A new coelenterazine-based system was developed, which involved immobilizing luciferase – the enzyme that catalyzes bioluminescent oxidation of the substrate coelenterazine – onto a quantum dot (Hsu CY, 2013). When luciferase encounters coelenterazine, it causes the quantum dot to fluoresce by BRET from coelenterazine to the quantum dot. This fluorescence activates the photosensitizer meta-tetra(hydroxyphenyl) chlorin to generate ROS, leading to cell death and tumour inhibition due to cytotoxicity. This system is known as QD-Rluc8 (QD = quantum dots, Rluc = luciferase from Renilla).
The efficiency of inducing anti-tumour action in deeply-seated tumours tissue was evaluated by (Kim et al., 2015). The source of luminescence was BRET Luc-QD, as was seen earlier, with chlorin e6 (chlorins, like porphyrins, are a class of photosensitizers) as the photosensitizer. It was found that cytotoxic ROS accumulated on the cell surface instead of in the cytoplasm. The amount of photosensitizer activated was found to be higher than that activated by a laser light source, and tumour growth and metastasis of the tumour to distant organs was inhibited, indicating that BRET-mediated PDT is a viable method for tumour therapy.
Therefore, bioluminescence (arguably the coolest form of light generation!) looks to be a promising approach to PDT of cancer. It also is one of the most eco-friendly, as all chemicals used are of natural origin (or are analogous to those of natural origin). Light derived from enzyme-mediated reactions is used to kill cancer cells, encouraging them to ‘go towards the light’. There are still numerous research efforts being undertaken to make this technique more specific and potent and generate new photosensitizers. If these efforts are successful, bioluminescence-driven anti-tumour PDT could possibly be one of the mainstream methods to treat deep-tissue cancers.
References
[1] Chamberlain, S., Cole, H., Roque, J. 3., Bellnier, D., McFarland, S., & S. G. (2020). TLD1433-Mediated Photodynamic Therapy with an Optical Surface Applicator in the Treatment of Lung Cancer Cells In Vitro. Pharmaceuticals (Basel).
[2] Downes, A., & Thos, P. B. (1877). Researches on the Effect of Light upon Bacteria and other Organisms. Researches on the Effect of Light upon Bacteria and other Organisms. Proceedings of the Royal Society of London, 488-500.
[3] Evanko, D. (2006). Bioluminescent quantum dots. Nature Methods, 240.
[4] Fahimipour, A. H. (2018). Daylight exposure modulates bacterial communities associated with household dust. Microbiome, 275.
[5] Figge, F., Weiland, G., & Manganiello, L. O. (1948). Studies on cancer detection and therapy; the affinity of neoplastic, embryonic, and traumatized tissue for porphyrins, metalloporphyrins, and radioactive zinc hematoporphyrin. Studies on cancer detection and therapy; the affinity of neoplastic, embryonic, and traumatized tissue for porphyrins, metalloporphyrins, and radioactive zinc hematoporphyrin, 657.
[6] Hsu CY, C. C. (2013). Bioluminescence resonance energy transfer using luciferase-immobilized quantum dots for self-illuminated photodynamic therapy. Biomaterials, 1204-1212.
[7] Kim, Y., & al, e. (2015). Bioluminescence-Activated Deep-Tissue Photodynamic Therapy of Cancer. Theranostics, 805-817.
[8] Kobayashi, H. P. (2019). Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells. Nature Protocols, 1084–1107.
[9] Laptev, R. N. (2006). Intracellular chemiluminescence activates targeted photodynamic destruction of leukemic cells. British journal of cancer, 189-196.
[10] Puntener, A., & Schlesinger, U. (2000). Colorants for Non-Textile Applications, Natural Dyes. Elsevier Science.
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[12] Tappeiner, H. V., & Jesionek, H. (1903). Therapeutische Versuche mit fluoreszierenden Stoffen. Münchener Medizinische Wochenschrift, 223-226.
[13] Wylie, D. (2006). Evidence for DNA Oxidation in Single Molecule Fluorescence Studies (Doctoral dissertation, Ohio University).
[14] Zhang, P., Huang, H., Banerjee, S., Clarkson, G. J., Ge, C., Imberti, C., & & Sadler, P. J. (2019). Nucleus-Targeted Organoiridium-Albumin Conjugate for Photodynamic Cancer Therapy. Angewandte Chemie, 2350–2354.
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