Structure and Conformation of Individual Molecules upon Adsorption of a Mixture of Benzoporphyrins on Ag(111), Cu(111), and Cu(110) Surfaces.

We investigated the adsorption behavior of a mixture of six 2H-tetrakis-(3, 5-di-tert-butylphenyl)(x)benzoporphyrins (2H-diTTBP(x)BPs, x=0, 1, 2-cis, 2-trans, 3, and 4) on Ag(111), Cu(111) and Cu(110) at room temperature by scanning tunneling microscopy (STM) under ultra-high vacuum conditions. On Ag(111), we observe an ordered two-dimensional square phase, which is stable up to 400 K. On Cu(111), the same square phase coexists with a stripe phase, which disappears at 400 K. In contrast, on Cu(110), 2H-diTTBP(x)BPs adsorb as immobile isolated molecules or dispersed short chains along the [1 1 ‾ ${\bar{1}}$ 0] substrate direction, which remain intact up to 450 K. The stabilization of the 2D supramolecular structures on Ag(111) and Cu(111), and of the 1D short chains on Cu(110) is attributed to van der Waals interactions between the tert-butyl and phenyl groups of neighboring molecules. From high-resolution STM, we can assign all six 2H-diTTBP(x)BPs within the ordered structures. Moreover, we deduce a crown shape quadratic conformation on Ag(111) and Cu(111), an additional saddle-shape on Cu(111), and an inverted structure and a quadratic appearance on Cu(110). The different conformations are attributed to the different degree of interaction of the iminic nitrogen atoms of the isoindole and pyrrole groups with the substrate atoms.

Photoimmunotherapy Retains Its Anti-Tumor Efficacy with Increasing Stromal Content in Heterotypic Pancreatic Cancer Spheroids.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by increased levels of desmoplasia that contribute to reduced drug delivery and poor treatment outcomes. In PDAC, the stromal content can account for up to 90% of the total tumor volume. The complex interplay between stromal components, including pancreatic cancer-associated fibroblasts (PCAFs), and PDAC cells in the tumor microenvironment has a significant impact on the prognoses and thus needs to be recapitulated in vitro when evaluating various treatment strategies. This study is a systematic evaluation of photodynamic therapy (PDT) in 3D heterotypic coculture models of PDAC with varying ratios of patient-derived PCAFs that simulate heterogeneous PDAC tumors with increasing stromal content. The efficacy of antibody-targeted PDT (photoimmunotherapy; PIT) using cetuximab (a clinically approved anti-EGFR antibody) photoimmunoconjugates (PICs) of a benzoporphyrin derivative (BPD) is contrasted with that of liposomal BPD (Visudyne), which is currently in clinical trials for PDT of PDAC. We demonstrate that both Visudyne-PDT and PIT were effective in heterotypic PDAC 3D spheroids with a low stromal content. However, as the stromal content increases above 50% in the 3D spheroids, the efficacy of Visudyne-PDT is reduced by up to 10-fold, while PIT retains its efficacy. PIT was found to be 10-, 19-, and 14-fold more phototoxic in spheroids with 50, 75, and 90% PCAFs, respectively, as compared to Visudyne-PDT. This marked difference in efficacy is attributed to the ability of PICs to penetrate and distribute homogeneously within spheroids with a higher stromal content and the mechanistically different modes of action of the two formulations. This study thus demonstrates how the stromal content in PDAC spheroids directly impacts their responsiveness to PDT and proposes PIT to be a highly suited treatment option for desmoplastic tumors with particularly high degrees of stromal content.

Photodynamic Priming Modulates Endothelial Cell-Cell Junction Phenotype for Light-activated Remote Control of Drug Delivery.

The blood-brain barrier (BBB) remains a major obstacle for drug delivery to the central nervous system. In particular, the tight and adherens junctions that join the brain capillary endothelial cells limit the diffusion of various molecules from the bloodstream into the brain. Photodynamic priming (PDP) is a non-cytotoxic modality that involves light activation of photosensitizers to photochemically modulate nearby molecules without killing the cells. Here we investigate the effects of sub-lethal photochemistry on junction phenotype (i.e., continuous, punctate, or perpendicular), as well as the BBB permeability in a transwell model of human brain microvascular endothelial cells (HBMECs). We showed that PDP decreases the continuous junction architecture by ~20%, increases the perpendicular junction architecture by ~40%, and has minimal impact on cell morphology in HBMECs. Furthermore, transwell permeability assay revealed that PDP improves the HBMEC permeability to dextran or nanoliposomes by up to 30-fold for 6-9 days. These results suggest that PDP could safely reverse the mature brain endothelial junctions without killing the HBMECs. This study not only emphasizes the critical roles of PDP in the modulation junction phenotype, but also highlights the opportunity to further develop PDP-based combinations that opens the cerebrum endothelium for enhanced drug transporter across the BBB.

Unraveling the Photodynamic Activity of Cationic Benzoporphyrin-Based Photosensitizers against Bladder Cancer Cells.

In this study, we report the preparation of new mono-charged benzoporphyrin complexes by reaction of the appropriate neutral benzoporphyrin with (2,2'-bipyridine)dichloroplatinum(II) and of the analogs' derivatives synthesized through alkylation of the neutral scaffold with iodomethane. All derivatives were incorporated into polyvinylpyrrolidone (PVP) micelles. The ability of the resultant formulations to generate reactive oxygen species was evaluated, mainly the singlet oxygen formation. Then, the capability of the PVP formulations to act as photosensitizers against bladder cancer cells was assessed. Some of the studied formulations were the most active photosensitizers causing a decrease in HT-1376 cells' viability. This creates an avenue to further studies related to bladder cancer cells.

Breaking the selectivity-uptake trade-off of photoimmunoconjugates with nanoliposomal irinotecan for synergistic multi-tier cancer targeting.

BACKGROUND: Photoimmunotherapy involves targeted delivery of photosensitizers via an antibody conjugate (i.e., photoimmunoconjugate, PIC) followed by light activation for selective tumor killing. The trade-off between PIC selectivity and PIC uptake is a major drawback limiting the efficacy of photoimmunotherapy. Despite ample evidence showing that photoimmunotherapy is most effective when combined with chemotherapy, the design of nanocarriers to co-deliver PICs and chemotherapy drugs remains an unmet need. To overcome these challenges, we developed a novel photoimmunoconjugate-nanoliposome (PIC-Nal) comprising of three clinically used agents: anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody cetuximab (Cet), benzoporphyrin derivative (BPD) photosensitizer, and irinotecan (IRI) chemotherapy. RESULTS: The BPD photosensitizers were first tethered to Cet at a molar ratio of 6:1 using carbodiimide chemistry to form PICs. Conjugation of PICs onto nanoliposome irinotecan (Nal-IRI) was facilitated by copper-free click chemistry, which resulted in monodispersed PIC-Nal-IRI with an average size of 158.8 ± 15.6 nm. PIC-Nal-IRI is highly selective against EGFR-overexpressing epithelial ovarian cancer cells with 2- to 6-fold less accumulation in low EGFR expressing cells. Successful coupling of PIC onto Nal-IRI enhanced PIC uptake and photoimmunotherapy efficacy by up to 30% in OVCAR-5 cells. Furthermore, PIC-Nal-IRI synergistically reduced cancer viability via a unique three-way mechanism (i.e., EGFR downregulation, mitochondrial depolarization, and DNA damage). CONCLUSION: It is increasingly evident that the most effective therapies for cancer will involve combination treatments that target multiple non-overlapping pathways while minimizing side effects. Nanotechnology combined with photochemistry provides a unique opportunity to simultaneously deliver and activate multiple drugs that target all major regions of a cancer cell-plasma membrane, cytoplasm, and nucleus. PIC-Nal-IRI offers a promising strategy to overcome the selectivity-uptake trade-off, improve photoimmunotherapy efficacy, and enable multi-tier cancer targeting. Controllable drug compartmentalization, easy surface modification, and high clinical relevance collectively make PIC-Nal-IRI extremely valuable and merits further investigations in living animals.

Photodynamic therapy: Promoting in vitro efficacy of photodynamic therapy by liposomal formulations of a photosensitizing agent.

OBJECTIVE: A relatively low level of lysosomal photodamage has been shown capable of promoting the efficacy of photodamage simultaneously or subsequently directed to mitochondrial/ER sites. The procedure has hitherto involved the use of two photosensitizing agents that require irradiation at two different wavelengths and different formulation techniques. This, together with different pharmacokinetic profiles of the photosensitizers, adds a layer of complexity to a protocol that we have sought to circumvent. In this study, liposomal formulations were used to direct photodamage created by benzoporphyrin derivative (BPD, Verteporfin) to lysosomes, mitochondria and the ER. This resulted in the development of an optimal targeting profile using a single agent and a single wavelength of activating irradiation. MATERIALS/METHODS: These studies were carried out in monolayer cultures of OVCAR5 tumor cells. BPD localization was modified by lipid anchoring and formulation in liposomes, and was assessed by fluorescence microscopy. Irradiation was carried out at 690 ± 10 nm with photodamage assessed also using fluorescent probes and microscopy. RESULTS: BPD normally localizes in a wide variety of sub-cellular loci that include both mitochondria and the ER, but lysosomes are spared from photodamage. Using a liposomal formulation containing BPD anchored to a lipid resulted in the targeting of lysosomes. A mixture of liposomes containing "free" and "anchored" BPD was shown to significantly promote photokilling. Eliminating cholesterol from the formulation of the anchored product enhanced lysosomal photodamage; prior studies had revealed that excess cholesterol can have a cytoprotective effect when lysosomes are the PDT target. DISCUSSION: The ability of a liposomal formulation to change localization patterns permits directing photodynamic therapy toward specific sub-cellular loci, thereby promoting photokilling. Incorporating chemotherapeutic agents into such formulations could represent a logical next step in assessing the ability of directed photodamage to enhance tumor eradication. Lasers Surg. Med. 50:499-505, 2018. © 2018 Wiley Periodicals, Inc.

Simultaneous delivery of cytotoxic and biologic therapeutics using nanophotoactivatable liposomes enhances treatment efficacy in a mouse model of pancreatic cancer.

A lack of intracellular delivery systems has limited the use of biologics such as monoclonal antibodies (mAb) that abrogate molecular signaling pathways activated to promote escape from cancer treatment. We hypothesized that intracellular co-delivery of the photocytotoxic chromophore benzoporphyrin derivative monoacid A (BPD) and the anti-VEGF mAb bevacizumab in a nanophotoactivatable liposome (nanoPAL) might enhance the efficacy of photodynamic therapy (PDT) combined with suppression of VEGF-mediated signaling pathways. As a proof-of-concept we found that nanoPAL-PDT induced enhanced extra- and intracellular bevacizumab delivery and enhanced acute cytotoxicity in vitro. In an in vivo subcutaneous mouse model of pancreatic ductal adenocarcinoma, nanoPAL-PDT achieved significantly enhanced tumor reduction. We attribute this to the optimal incorporation of insoluble BPD into the lipid bilayer, enhancing photocytotoxicity, and the simultaneous spatiotemporal delivery of bevacizumab, ensuring efficient neutralization of the rapid but transient burst of VEGF following PDT. From the Clinical Editor: Most patients with pancreatic ductal adenocarcinoma (PDAC) by the time present the disease it is very advanced, which unavoidably translates to poor survival. For these patients, use of traditional chemotherapy often becomes ineffective due to tumor resistance to drugs. Photodynamic therapy (PDT) can be an effective modality against chemo-resistant cancers. In this article, the authors investigated the co-delivery of a photocytotoxic agent and anti-VEGF mAb using liposomes. This combination was shown to results in enhanced tumor killing. This method should be applicable to other combination of treatments.

A Physiochemical, In Vitro, and In Vivo Comparative Analysis of Verteporfin-Lipid Conjugate Formulations: Solid Lipid Nanoparticles and Liposomes.

VisudyneⓇ, a liposomal formulation of verteporfin (benzoporphyrin derivative; BPD), is the only nanomedicine approved to date for photodynamic therapy (PDT). We have previously demonstrated that BPD conjugated to the lysophospholipid 1-arachidoyl-2-hydroxy-sn-glycero-3-phosphocholine (BPD-PC) exhibits the greatest physical stability in liposomes, while maintaining cancer cell phototoxicity, from a panel of BPD lipid conjugates evaluated. In this study, we prepared 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-based solid lipid nanoparticles (LNPs) that stably entrap BPD-PC, which resemble the composition of the SpikevaxⓇ Moderna COVID-19 vaccine, and compared them to a DPPC based liposomal formulation (Lipo BPD-PC). We evaluated the photochemical, optical, and phototherapeutic properties of both formulations. We also investigated the in vivo distribution and tumor microdistribution of both formulations. Our results demonstrated that Lipo BPD-PC is able to generate 17% more singlet oxygen than LNP BPD-PC, while interestingly, LNP BPD-PC is able to produce 76% more hydroxyl radicals and/or peroxynitrite anion. Importantly, only 28% of BPD-PC leaches out of the LNP BPD-PC formulation during 7 days of incubation in serum at 37 °C, while 100% of BPD-PC leaches out of the Lipo BPD-PC formulation under the same conditions. Despite these differences, there was no significant difference in cellular uptake of BPD-PC or phototoxicity in CT1BA5 murine pancreatic cancer cells (derived from a genetically engineered mouse model). Interestingly, PDT using LNP BPD-PC was more efficient at inducing immunogenic cell death (calreticulin membrane translocation) than Lipo BPD-PC when using IC25 and IC50 PDT doses. In vivo studies revealed that CT1BA5 tumor fluorescence signals from BPD-PC were 2.41-fold higher with Lipo BPD-PC than with LNP BPD-PC; however, no significant difference was observed in tumor tissue selectivity or tumor penetration. As such, we present LNP BPD-PC as a unique and more stable nanoplatform to carry BPD lipid conjugates, such as BPD-PC, with a potential for future photodynamic immune priming studies and multiagent drug delivery.

Overcoming the effects of fluid shear stress in ovarian cancer cell lines: Doxorubicin alone or photodynamic priming to target platinum resistance.

Resistance to platinum-based chemotherapies remains a significant challenge in advanced-stage high-grade serous ovarian carcinoma, and patients with malignant ascites face the poorest outcomes. It is, therefore, important to understand the effects of ascites, including the associated fluid shear stress (FSS), on phenotypic changes and therapy response, specifically FSS-induced chemotherapy resistance and the underlying mechanisms in ovarian cancer. This study investigated the effects of FSS on response to cisplatin, a platinum-based chemotherapy, and doxorubicin, an anthracycline, both of which are commonly used to manage advanced-stage ovarian cancer. Consistent with prior research, OVCAR-3 and Caov-3 cells cultivated under FSS demonstrated significant resistance to cisplatin. Examination of the role of mitochondria revealed an increase in mitochondrial DNA copy number and intracellular ATP content in cultures grown under FSS, suggesting that changes in mitochondria number and metabolic activity may contribute to platinum resistance. Interestingly, no resistance to doxorubicin was observed under FSS, the first such observation of a lack of resistance under these conditions. Finally, this study demonstrated the potential of photodynamic priming using benzoporphyrin derivative, a clinically approved photosensitizer that localizes in part to mitochondria and endoplasmic reticula, to enhance the efficacy of cisplatin, but not doxorubicin, thereby overcoming FSS-induced platinum resistance.

Oxygen saturation and blood volume analysis by photoacoustic imaging to identify pre and post-PDT vascular changes.

In this study, the blood volume and oxygen saturation of tumors were measured after photoacoustic imaging (PAI) under conditions of pre-photodynamic therapy (PDT), post-PDT, and 4 hrs, and 24 hrs post-PDT. PDTs with aminolevulinic acid (ALA) and low and high doses of benzoporphyrin derivative (BPD) were conducted to observe oxygen saturation changes, and the rapid oxygen consumption in the blood detected due to the action of BPD at the vascular level resulted in the recovery of PDT completion. Likewise, blood volume changes followed by ALA-PDT and BPD-PDT at low and high doses depicted a fast expansion of the blood volume after treatment. The tumor subjected to a high dose of ALA-PDT showed a partial alteration of Hb-pO2 in the first 24 hrs, as did the tumors treated with two ALA- and BPD-mediated PDTs. The Hb-pO2 started reducing immediately post-PDT and was less than 30% after 4 hrs until 24 hrs post-PDT. Reduced vascular demand was possibly due to tumor necrosis, as shown by the permanent damage in the cancer cells' bioluminescence signal. The ALA-mediated PDT-subjected tumor showed a 50% drop in BV at 24 hrs post-PDT, which is suggestive of vascular pruning. The studied data of blood volume against BLI showed the blood volume and oxygenation variations validating the cells' metabolic activity, including cell death.

In-Vitro Use of Verteporfin for Photodynamic Therapy in Glioblastoma.

BACKGROUND: Stummer et al. established fluorescence-guided surgery (FGS) for glioblastoma (GBM) using 5-aminolevulinic acid (5-ALA). Its metabolite, protoporphyrin IX (PPIX), is also a photosensitizer and can be used for photodynamic therapy (PDT) using a laser beam of 635 nm. The porphyrin derivate verteporfin (VP) was discovered to have properties to penetrate the brain, pharmacologically target glioma cells, and is approved for PDT of choroidal neovascularization in wet age-related macular degeneration at 689 nm. OBJECTIVE: To elucidate whether GBM cell lines are susceptible to PDT with second-generation photosensitizer VP. METHODS: Human glioma cell lines LN229, HSR-GBM1, and a low-passage patient-derived GBM cell line P1 were treated with variable concentrations of VP for 24 h, followed by PDT at 689 nm using a diode laser light. Cell viability was measured using the MTT assay and VP uptake was measured using a desktop cytometer. RESULTS: Significantly higher cell death following PDT with VP compared to VP treatment alone or no treatment was detected in all cell models (LN229, HSR-GBM1, P1). Flowcytometric measurements revealed a concentration-dependent cellular uptake of VP after 24 h incubation up to 99% at 10 µM (HSR-GBM1). CONCLUSION: This study demonstrates that PDT with VP causes cell death in GBM cells at marginal concentrations. Additionally, red spectrum fluorescence was detected at therapeutic concentrations in all cell lines, validating the cellular uptake of VP in GBM cells. VP, therefore, is not only a potential drug for targeting GBM pharmacologically but can be used as an optical imaging dye in surgery and photosensitizer to make GBM susceptible to PDT.

Aromatic Ring Fusion to Benzoporphyrin via γ-ortho Cyclodehydrogenation on a Ag(111) Surface.

Aromatic ring fusion to porphyrins and their derivatives represents an attractive route to tune the molecular conjugation and thus expand their functionalities. Here, we report the expansion of the aromatic π-system of palladium tetraphenyltetrabenzoporphyrins (Pd-TPTBP) via surface-assisted γ-ortho cyclodehydrogenation on Ag(111). The chemical transformation of Pd-TPTBP into different products at an elevated temperature of 600 K was revealed at the single-molecule level using bond-resolved scanning tunneling microscopy with a CO-functionalized tip. We captured a series of γ-ortho cyclodehydrogenation products, wherein the maximum extent to which the reaction can progress is associated with 7-fold C-C formation to afford nearly planar γ-ortho fused porphyrins with 66 conjugated π-electrons. In addition, a small number of molecules undergo C-C bond dissociation of meso-phenyl at elevated temperature, producing fully planar γ-ortho fused products lacking one or two phenyl moieties. Scanning tunneling spectroscopy measurements and DFT calculations suggest the electronic gap of the γ-ortho fused porphyrin decreases compared to that of the precursor. The HOMO and LUMO of the planar γ-ortho fused products are localized on the partially fused benzo moieties and the meso-position, respectively.

Generating Large Numbers of Pancreatic Microtumors on Alginate-Gelatin Hydrogels for Quantitative Imaging of Tumor Growth and Photodynamic Therapy Optimization.

The emerging use of 3D culture models of cancer has provided novel insights into the therapeutic mechanisms of photodynamic therapy on a mesoscopic scale. Especially microscale tumors grown on scaffolds of extracellular matrix can provide statistically robust data on the effects of photosensitizers and photodynamic therapy by leveraging high-throughput imaging-based assays. Although highly informative, the use of such 3D cultures can be impractical due to the high costs and inter-batch variability of the extracellular matrix scaffolds that are necessary to establish such cultures. In this study, we therefore provide a protocol to generate inexpensive and defined hydrogels composed of sodium alginate and gelatin that can be used for culturing 3D microtumors in a manner that is compatible with state-of-the-art imaging assays. Our results reveal that the alginate-gelatin hydrogels can perform similarly to a commercially available ECM scaffold in terms of facilitating microtumor growth. We then applied these microtumor models to quantify the uptake and dark toxicity of benzoporphyrin derivative encapsulated in liposomes with either an anionic or a cationic surface charge. The results indicate that cationic liposomes achieve the highest level of uptake in the microtumors, yet also exert minor toxicity. Moreover, we reveal that there is typically a significant positive correlation between microtumor size and liposome uptake. In conclusion, alginate-based hydrogels are inexpensive and effective scaffolds for 3D culture models of cancer, with versatile applications in research toward photodynamic therapy.

Photoacoustic nanodroplets for oxygen enhanced photodynamic therapy of cancer.

Photodynamic therapy (PDT) is a well-known cancer therapy that utilizes light to excite a photosensitizer and generate cytotoxic reactive oxygen species (ROS). The efficacy of PDT primarily depends on the photosensitizer and oxygen concentration in the tumor. Hypoxia in solid tumors promotes treatment resistance, resulting in poor PDT outcomes. Hence, there is a need to combat hypoxia while delivering sufficient photosensitizer to the tumor for ROS generation. Here we showcase our unique theranostic perfluorocarbon nanodroplets as a triple agent carrier for oxygen, photosensitizer, and indocyanine green that enables light triggered spatiotemporal delivery of oxygen to the tumors. We evaluated the characteristics of the nanodroplets and validated their ability to deliver oxygen via photoacoustic monitoring of blood oxygen saturation and subsequent PDT efficacy in a murine subcutaneous tumor model. The imaging results were validated with an oxygen sensing probe, which showed a 9.1 fold increase in oxygen content inside the tumor, following systemic administration of the nanodroplets. These results were also confirmed with immunofluorescence. In vivo studies showed that nanodroplets held higher rates of treatment efficacy than a clinically available benzoporphyrin derivative formulation. Histological analysis showed higher necrotic area within the tumor with perfluoropentane nanodroplets. Overall, the photoacoustic nanodroplets can significantly enhance image-guided PDT and has demonstrated substantial potential as a valid theranostic option for patient-specific photodynamic therapy-based treatments.

Use of photoimmunoconjugates to characterize ABCB1 in cancer cells.

Accurate detection of ATP-binding cassette drug transporter ABCB1 expression is imperative for precise identification of drug-resistant tumors. Existing detection methods fail to provide the necessary molecular details regarding the functional state of the transporter. Photo-immunoconjugates are a unique class of antibody-dye conjugates for molecular diagnosis and therapeutic treatment. However, conjugating hydrophobic photosensitizers to hydrophilic antibodies is quite challenging. Here, we devise a photoimmunoconjugate that combines a clinically approved benzoporphyrin derivative (BPD) photosensitizer and the conformational-sensitive UIC2 monoclonal antibody to target functionally active human ABCB1 (i.e., ABCB1 in the inward-open conformation). We show that PEGylation of UIC2 enhances the BPD conjugation efficiency and reduces the amount of non-covalently conjugated BPD molecules by 17%. Size exclusion chromatography effectively separates the different molecular weight species found in the UIC2-BPD sample. The binding of UIC2-BPD to ABCB1 was demonstrated in lipidic nanodiscs and ABCB1-overexpressing triple negative breast cancer (TNBC) cells. UIC2-BPD was found to retain the conformation sensitivity of UIC2, as the addition of ABCB1 modulators increases the antibody reactivity in vitro. Thus, the inherent fluorescence capability of BPD can be used to label ABCB1-overexpressing TNBC cells using UIC2-BPD. Our findings provide insight into conjugation of hydrophobic photosensitizers to conformation-sensitive antibodies to target proteins expressed on the surface of cancer cells.

High-power light-emitting diode array design and assembly for practical photodynamic therapy research.

SIGNIFICANCE: Commercial lasers, lamps, and light-emitting diode (LED) light sources have stimulated the clinical translation of photodynamic therapy (PDT). Yet, the continued exploration of new photosensitizers (PSs) for PDT often requires separate activation wavelengths for each agent being investigated. Customized light sources for such research frequently come at significant financial or technical cost, especially when compounded over many agents and wavelengths. AIM: LEDs offer potential as a cost-effective tool for new PS and multi-PS photodynamic research. A low-cost-per-wavelength tool leveraging high-power LEDs to facilitate efficient and versatile research is needed to further accelerate research in the field. APPROACH: We developed and validated a high-power LED array system for benchtop PDT with a modular design for efficient switching between wavelengths that overcome many challenges in light source design. We describe the assembly of a low-cost LED module plus the supporting infrastructure, software, and protocols to streamline typical in vitro PDT experimentation. RESULTS: The LED array system is stable at intensities in excess of 100 mW / cm2 with 2.3% variation across the illumination field, competitive with other custom and commercial devices. To demonstrate efficacy and versatility, a primary ovarian cancer cell line was treated with two widely used PSs, aminolevulinic acid and verteporfin, using the LED modules at a clinically relevant 50 J / cm2 light dose that induced over 90% cell death for each treatment. CONCLUSIONS: Our work provides the community with a tool for new PS and multi-PS benchtop photodynamic research that, unlike most commercial light sources, affords the user a low barrier to entry and low-cost-per-wavelength with the goal of illuminating new insights at the forefront of PDT.

Mutual impact of clinically translatable near-infrared dyes on photoacoustic image contrast and in vitro photodynamic therapy efficacy.

Photodynamic therapy (PDT), a spatially localized phototoxic therapy that involves irradiation of a photosensitizer (PS) with specific wavelengths of light, has shown exceptional promise in impacting cancer treatment outcomes, particularly oral cancer. To reduce PDT outcome variability, attempts toward image-guided personalized PDT are being pursued by monitoring PS uptake either via fluorescence or photoacoustic imaging (PAI), a nonionizing modality dependent on optical absorption properties of the tissue. PAI-guided PDT requires a near-infrared contrast agent for deep tissue imaging with minimal photobleaching effect. We evaluate the impact of PDT agent, benzoporphyrin derivative (BPD), on PAI agent indocyanine green (ICG) and vice versa, given that they have different optical absorption properties and singlet oxygen quantum yields for PDT. Specifically, we demonstrate in two oral squamous cell carcinoma lines (FaDu and SCC4) that ICG has minimal effect on BPD PDT efficacy when irradiated with either a continuous or pulsed laser. Furthermore, the impact of BPD on ICG photodegradation was monitored with PAI in tissue-mimicking phantoms. These studies inform us that the combination of BPD and ICG can be utilized for PAI-guided PDT. However, researchers need to consider the photodegradation effects of ICG in the presence of BPD when designing their drug delivery strategies for PAI-guided PDT.

Reactive oxygen species explicit dosimetry to predict tumor growth for benzoporphyrin derivative-mediated vascular photodynamic therapy.

Photodynamic therapy (PDT) is a well-established treatment modality for cancer and other malignant diseases; however, quantities such as light fluence and PDT dose do not fully account for all of the dynamic interactions between the key components involved. In particular, fluence rate (ϕ) effects, which impact the photochemical oxygen consumption rate, are not accounted for. In this preclinical study, reacted reactive oxygen species ([ROS]rx) was investigated as a dosimetric quantity for PDT outcome. The ability of [ROS]rx to predict the cure index (CI) of tumor growth, CI = 1 - k / kctr, where k and kctr are the growth rate of tumor under PDT study and the control tumor without PDT, respectively, for benzoporphyrin derivative (BPD)-mediated PDT, was examined. Mice bearing radiation-induced fibrosarcoma (RIF) tumors were treated with different in-air fluences (Φ = 22.5 to 166.7 J / cm2) and in-air fluence rates (ϕair = 75 to 250 mW / cm2) with a BPD dose of 1 mg / kg and a drug-light interval (DLI) of 15 min. Treatment was delivered with a collimated laser beam of 1-cm-diameter at 690 nm. Explicit measurements of in-air light fluence rate, tissue oxygen concentration, and BPD concentration were used to calculate for [ROS]rx. Light fluence rate at 3-mm depth (ϕ3 mm), determined based on Monte-Carlo simulations, was used in the calculation of [ROS]rx at the base of tumor. CI was used as an endpoint for three dose metrics: light fluence, PDT dose, and [ROS]rx. PDT dose was defined as the product of the time-integral of photosensitizer concentration and ϕ3 mm. Preliminary studies show that [ROS]rx best correlates with CI and is an effective dosimetric quantity that can predict treatment outcome. The threshold dose for [ROS]rx for vascular BPD-mediated PDT using DLI of 15 min is determined to be 0.26 mM and is about 3.8 times smaller than the corresponding value for conventional BPD-mediated PDT using DLI of 3 h.

Theoretical investigation of the structural and spectroscopic properties of expanded metalloporphyrin complexes.

The frontier molecular orbitals, UV-Vis absorption spectra, charge transfer (CT) and triplet excited states of 12 expanded D-A porphyrin/benzoporphyrin complexes were investigated using the density functional theory (DFT) method and time-dependent DFT in this work. The results showed that thiophene was an effective fragment for absorption of 'long wavelength', while the benzoporphyrin worked on the 'short wavelength', which was derived from its saddle-shaped structure; this expanded D-A conjugated system had a mild CT process with anthraquinone/isoindigo as acceptors and a strong CT process with naphtoquinone as acceptor. In addition, based on the simulation of the triplet state, the theoretical phosphorescence wavelength range of this series of derivatives was between 1000 and 1200 nm. This study is expected to assist the design of conjugated porphyrin for the field of porphyrin chemistry.

Systematic Evaluation of Light-Activatable Biohybrids for Anti-Glioma Photodynamic Therapy.

Photosensitizing biomolecules (PSBM) represent a new generation of light-absorbing compounds with improved optical and physicochemical properties for biomedical applications. Despite numerous advances in lipid-, polymer-, and protein-based PSBMs, their effective use requires a fundamental understanding of how macromolecular structure influences the physicochemical and biological properties of the photosensitizer. Here, we prepared and characterized three well-defined PSBMs based on a clinically used photosensitizer, benzoporphyrin derivative (BPD). The PSBMs include 16:0 lysophosphocholine-BPD (16:0 Lyso PC-BPD), distearoyl-phosphoethanolamine-polyethylene-glycol-BPD (DSPE-PEG-BPD), and anti-EGFR cetuximab-BPD (Cet-BPD). In two glioma cell lines, DSPE-PEG-BPD exhibited the highest singlet oxygen yield but was the least phototoxic due to low cellular uptake. The 16:0 Lyso PC-BPD was most efficient in promoting cellular uptake but redirected BPD's subcellular localization from mitochondria to lysosomes. At 24 h after incubation, proteolyzed Cet-BPD was localized to mitochondria and effectively disrupted the mitochondrial membrane potential upon light activation. Our results revealed the variable trafficking and end effects of PSBMs, providing valuable insights into methods of PSBM evaluation, as well as strategies to select PSBMs based on subcellular targets and cytotoxic mechanisms. We demonstrated that biologically informed combinations of PSBMs to target lysosomes and mitochondria, concurrently, may lead to enhanced therapeutic effects against gliomas.