This new research improves the prospects for success of systemically delivered and X-Ray activated PDT. There was a very similar project at the University of Alberta in 2022 using a porphyrin based photosensitizer. The new research notes the superiority of Dr. McFarland's Ruthenium based PS to the porhyrin based one used in the earlier U of Alberta research.
https://www.sciencedirect.com/science/article/abs/pii/S1742706120305523 Both projects were funded by a CUA-CUOG Astellas Research Grant.
Astellas Pharma Canada is the first Japanese pharmaceutical company in Canada and is located in Markham, Ontario. Globally, it is one of the top 20 pharmaceutical companies in sales, and employs 17,000 people. They recently received FDA accelerated approval for their PADCEV drug in a combo treatment with Keydruda for metastatic urothelial carcinoma By chance, Astellas is less than a 20 km drive from Theralase.
High quantum efficiency ruthenium coordination complex photosensitizer for improved radiation-activated Photodynamic Therapy ORIGINAL RESEARCH article
Front. Oncol., 28 August 2023
Abul Kalam Azad 1 Lothar Lilge 2 Nawaid H. Usmani 1 John D. Lewis 1 Houston D. Cole 3 Colin G. Cameron 3 Sherri A. McFarland 3 Deepak Dinakaran 1,4*† Ronald B. Moore 1 ,5†
1 Dept. of Oncology, University of Alberta, Edmonton, AB, Canada
2 Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada
3 Dept. of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, US
4 Radiation Oncology Branch, National Cancer Inst., National Institute of Health, Bethesda, MD, US
5 Department of Surgery, University of Alberta, Edmonton, AB, Canada Traditional external light-based Photodynamic Therapy (PDT)’s application is limited to the surface and minimal thickness tumors because of the inefficiency of light in penetrating deep-seated tumors. To address this, the emerging field of radiation-activated PDT (radioPDT) uses X-rays to trigger photosensitizer-containing nanoparticles (NPs). A key consideration in radioPDT is the energy transfer efficiency from X-rays to the photosensitizer for ultimately generating the phototoxic reactive oxygen species (ROS). In this study, we developed a new variant of pegylated poly-lactic-co-glycolic (PEG-PLGA) encapsulated nanoscintillators (NSCs) along with a new, highly efficient ruthenium-based photosensitizer (Ru/radioPDT). Characterization of this NP via transmission electron microscopy, dynamic light scattering, UV-Vis spectroscopy, and inductively coupled plasma mass-spectroscopy showed an NP size of 120 nm, polydispersity index (PDI) of less than 0.25, high NSCs loading efficiency over 90% and in vitro accumulation within the cytosolic structure of endoplasmic reticulum and lysosome. The therapeutic efficacy of Ru/radioPDT was determined using PC3 cell viability and clonogenic assays. Ru/radioPDT exhibited minimal cell toxicity until activated by radiation to induce significant cancer cell kill over radiation alone. Compared to protoporphyrin IX-mediated radioPDT (PPIX/radioPDT), Ru/radioPDT showed higher capacity for singlet oxygen generation, maintaining a comparable cytotoxic effect on PC3 cells.
Introduction
Cancer is a leading cause of death worldwide, second to cardiovascular diseases. In 2020, an estimated 18.1 million cases of cancer and nearly 10 million deaths were reported globally (1). Conventional cancer treatments such as chemo- and radiotherapies have clinically been proven as effective therapeutics for many cancer types; however, clinical scenarios with radiation or chemo-resistant cancers still exist where the therapeutic effect is not durable and leads to treatment-refractory disease over time (2, 3). Alternative cancer treatment modalities have been developed, including photodynamic therapy (PDT), given its high selectivity and non or minimal invasiveness (4, 5). In PDT, the photosensitizer (PS) is activated by light irradiation that generates reactive oxygen species (ROS), mediating the cell-killing mechanism. However, the tissue penetration depth of light in non-invasive clinical PDT systems is often less than 1 cm, thus limiting the application of PDT to tumors that are located superficially on the skin and endoscopically accessible subcutaneous tissues (5, 6). Deeper situate or thicker tumors can still be treated with PDT, but invasive interstitial light catheters are required to deliver the activating light effectively (7). In recent years, radiation-activated PDT (radioPDT), where the photosensitizer uses energy from X-ray photons rather than optical photons for activation, is gaining momentum to mitigate the limitation of visible/near-infrared (NIR) light sources in penetrating deep tissue structures (7, 8). X-ray photons have a much larger penetration depth and can be used to induce radioluminescence within nanoscintillators (NSCs), which in turn will activate the adjacent PSs via Forster resonance energy transfer (FRET) to generate ROS (9).
In PDT, finding an appropriate PS is challenging. For clinical application, the ideal PS factors to be considered include chemical purity, stability in physiologic conditions, low dark toxicity, higher tumor selectivity, localization to critical intracellular structure to trigger cell death, quick clearance from the body, and high ROS generation under activating light for efficient therapeutic yield (10). The latter is important, particularly with low-energy input fluence treatments (11). Protoporphyrin IX (PPIX) is one of the most commonly studied PSs in PDT. PPIX and its prodrug 5-aminolevulinic acid (5-ALA), a naturally occurring amino acid, are precursors in the biosynthetic heme pathway. Upregulation of this pathway leads to PPIX accumulating in tumors, which is favorable for treating tumors with PDT (12–15). Nevertheless, the clinical application of PPIX, 5-ALA, and other porphyrin derivatives like Photofrin in PDT remain limited to superficial cancers, including non-melanoma skin cancers, bladder, esophageal, lung and head and neck cancers (5, 16, 17).
In contrast, radiotherapy is widely used to treat approximately 50% of all cancer patients (18). Modern radiotherapy can be highly focused anywhere in the body with intensity-modulated radiotherapy and image-guided radiotherapy (2). By doing this, the toxicity to efficacy ratio of radiotherapy has been greatly optimized. Nevertheless, the short and long-term toxicities of radiotherapy is a major limiting factor to expanding its application in cancer care, and causes significant permeant side-effects to patients (19).
To address this, a new field has emerged at the crossroads between radiation and PDT, where radiation energy excites the PS for subsequent photochemical activity in a phenomenon termed radiation-activated PDT (radioPDT). Doing so allows the therapeutic effect of radiotherapy to be augmented by radioPDT without additional radiation dose, which can lead to additional radiotoxicity. Achieving radioPDT through nanoparticles also allows for the opportunity for multimodal strategies such as therapy and diagnostic (theranostic) agents (20, 21). We previously demonstrated one such radioPDT agent consisting of polyethylene glycol conjugated to poly-lactic-co-glycolic acid (PEG-PLGA) encapsulating LaF3:Ce3+ NSC and PPIX PS nanoparticle (PPIX/radioPDT NP), with an impressive performance in vitro and in vivo (9). Given the emergence of ruthenium (Ru) coordination complexes as PSs with attractive properties for PDT, particularly with much higher phototherapeutic index and quantum yield (11, 22–24), we hypothesized a more efficient radioPDT system could be generated by substituting PPIX for a Ru PS (Figure 1). Herein, we tested ML19H02 (Ru) within our radioPDT NP (Ru/radioPDT) construct in vitro using the PC3 cell line, an aggressive prostate cancer cell line derived from grade IV metastatic prostatic adenocarcinoma (25). The characteristics and effectiveness of Ru/radioPDT were evaluated by light irradiation and under X-ray irradiation and compared to the previously reported PPIX/radioPDT (9).
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Discussion
RadioPDT is an emerging field in anticancer therapy for the non-invasive treatment of deep-seated tumors, otherwise not amenable to light-dependent PDT. The X-ray absorption and luminescence produced by the LaF3:Ce3+ NSC upon irradiation can, in turn, activate PSs to generate ROS. The level of activation and efficacy is far in excess of what is achievable by several organic PSs, such as PPIX derivatives under X-ray radiation (7, 9, 32). Incorporating an NSC with PPIX into a nanoparticle construct can significantly improve the radioPDT process. This has been reported in multiple other previous studies with similar nanocarriers (9, 33, 34). However, PPIX’s relatively low quantum yield and high photobleaching rate may still limit the overall system’s efficacy (9). Here, a new NP complex consisting of 3 components: polymer PEG-PLGA as a carrier for drug delivery, LaF3:Ce3+ NSC for X-ray energy capture and transfer to a Ru coordination complex PS demonstrates a potentially more efficient method of conducting radioPDT. This new Ru/radioPDT NP exhibits several favorable characteristics over PPIX/radioPDT NP. The synthesized Ru/radioPDT NP was comparable to previously reported PPIX/radioPDT with a uniform size distribution of 100-120 nm. This size range corresponds to the ideal size required for increasing the circulatory half-life and the subsequent bioavailability of NPs at the tumor site (35, 36). Ru/radioPDT NP also exhibited a moderately charged -17.4 mV zeta potential, as opposed to -27 mV for PPIX/radioPDT NP, which helps maintain stability in aqueous conditions and in circulation (37), but the more positive charge predicts for increased likelihood of Ru/radioPDT NP to interact with negatively charged cell membranes and be endocytosed and localize into critical organelle structures such as the endoplasmic reticulum (38). In addition, the measured stability of the NPs was well preserved for up to 48 hrs in physiologic media, which allows adequate circulation and cellular uptake time before it can be activated by radiation.
The singlet oxygen generation with Ru/radioPDT is higher than PPIX/radioPDT for the same light irradiation parameters. Based on this observation, one expects a higher PC3 cell killing efficiency under similar conditions, but cell death was exceedingly efficient in all the experimental conditions upon light irradiation. Radiation activation augmented the PC3 cytotoxicity in both radioPDT NP formulations, with an additional 16% improvement in efficacy for Ru/radioPDT over PPIX/radioPDT. Curiously, this did not manifest as a statistically significant difference despite better singlet oxygen yield, but this perhaps relates to the low input energy of 3 Gy in the clonogenic assay. In a multifractionated regimen, similar to clinical radiotherapy use, a statistically significant result could be likely. Mechanistically, Ru/radioPDT was taken up by the cells and entered into different cytoplasmic structures but without entering the nucleus, suggesting that the augmented effect of Ru/radioPDT in PC3 cell death, is related to the damage of cytoplasmic organelles, particularly the endoplasmic reticulum, which is known to be sensitive to ROS stress and affects cell survival (39). Further experiments are needed to understand the mechanisms of Ru/radioPDT induced cell death upon radiation activation in detail.
Conclusion
To improve radioPDT efficiency and increase its ability to provide an additional anticancer effect over radiation alone, a new radioPDT NP system consisting of PEG-PLGA encapsulated LaF3:Ce3+ NSC and Ru PS was developed. Ru/radioPDT showed higher singlet oxygen generation with moderately increased potential in PC3 cell killing efficiency over PPIX/radioPDT. Favorable characteristics of size, surface charge, stability, and cellular uptake were maintained. Together, this shows the suitability of Ru/radioPDT NPs in improving efficiency for anticancer radiation therapy in pre-clinical and eventual clinical scenarios.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.
Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.
Funding
This research was supported by funding from the Canadian Institute of Health Research (CIHR) NFRFE-2019-01265 grant and Canadian Urologic Oncology Group (CUOG) Astellas Research Grant Program funded by Astellas Pharma Canada, Inc. and jointly established by Astellas Pharma Canada, Inc., CUOG, and the CUA, Montreal, QC. JL held the Frank and Carla Sojonky Chair in Prostate Cancer Research funded by the Alberta Cancer Foundation from 2012 to 2021. RM holds the Mr. Lube Chair in Uro-Oncology, Edmonton, AB.
Acknowledgments
The authors gratefully acknowledge Dr. Xuejun Sun and the University of Alberta’s Cell Imaging Facility for his help with TEM and EFTEM studies.
Conflict of interest
The authors declare the following competing financial interest: SM has a potential research conflict of interest due to a financial interest with Theralase Technologies, Inc. and PhotoDynamic, Inc. A management plan has been created to preserve objectivity in research in accordance with UTA policy.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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