Carrier-Free, Amorphous Verteporfin Nanodrug for Enhanced Photodynamic Cancer Therapy and Brain Drug Delivery.

Glioblastoma (GBM) is hard to treat due to cellular invasion into functioning brain tissues, limited drug delivery, and evolved treatment resistance. Recurrence is nearly universal even after surgery, chemotherapy, and radiation. Photodynamic therapy (PDT) involves photosensitizer administration followed by light activation to generate reactive oxygen species at tumor sites, thereby killing cells or inducing biological changes. PDT can ablate unresectable GBM and sensitize tumors to chemotherapy. Verteporfin (VP) is a promising photosensitizer that relies on liposomal carriers for clinical use. While lipids increase VP's solubility, they also reduce intracellular photosensitizer accumulation. Here, a pure-drug nanoformulation of VP, termed "NanoVP", eliminating the need for lipids, excipients, or stabilizers is reported. NanoVP has a tunable size (65-150 nm) and 1500-fold higher photosensitizer loading capacity than liposomal VP. NanoVP shows a 2-fold increase in photosensitizer uptake and superior PDT efficacy in GBM cells compared to liposomal VP. In mouse models, NanoVP-PDT improved tumor control and extended animal survival, outperforming liposomal VP and 5-aminolevulinic acid (5-ALA). Moreover, low-dose NanoVP-PDT can safely open the blood-brain barrier, increasing drug accumulation in rat brains by 5.5-fold compared to 5-ALA. NanoVP is a new photosensitizer formulation that has the potential to facilitate PDT for the treatment of GBM.

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, and expressed at the zebrafish BBB. We evaluated the luminescence of ten NanoLuc substrates, then screened the eight brightest to determine which are most efficiently effluxed by the ABC transporters. We identified one substrate efficiently pumped out by ABCB1, two by Abcb4, six by ABCG2, and four by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter.

Development of an Endoscopic Auto-Fluorescent Sensing Device to Aid in the Detection of Breast Cancer and Inform Photodynamic Therapy.

Breast cancer is the most diagnosed cancer type in women, with it being the second most deadly cancer in terms of total yearly mortality. Due to the prevalence of this disease, better methods are needed for both detection and treatment. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent biomarkers that lend insight into cell and tissue metabolism. As such, we developed an endoscopic device to measure these metabolites in tissue to differentiate between malignant tumors and normal tissue. We performed initial validations in liquid phantoms as well as compared to a previously validated redox imaging system. We also imaged ex vivo tissue samples after modulation with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and a combination of rotenone and antimycin A. We then imaged the rim and the core of MDA-MB-231 breast cancer tumors, with our results showing that the core of a cancerous lesion has a significantly higher optical redox ratio ([FAD]/([FAD] + [NADH])) than the rim, which agrees with previously published results. The mouse muscle tissues exhibited a significantly lower FAD, higher NADH, and lower redox ratio compared to the tumor core or rim. We also used the endoscope to measure NADH and FAD after photodynamic therapy treatment, a light-activated treatment methodology. Our results found that the NADH signal increases in the malignancy rim and core, while the core of cancers demonstrated a significant increase in the FAD signal.

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.

Depth-resolved imaging of photosensitizer in the rodent brain using fluorescence laminar optical tomography.

SIGNIFICANCE: Previous studies have been performed to image photosensitizers in certain organs and tumors using fluorescence laminar optical tomography. Currently, no work has yet been published to quantitatively compare the signal compensation of fluorescence laminar optical tomography with two-dimensional (2-D) imaging in tissues. AIM: The purpose of this study is to quantify the benefit that fluorescence laminar optical tomography holds over 2-D imaging. We compared fluorescence laminar optical tomography with maximum intensity projection imaging to simulate 2-D imaging, as this would be the most similar and stringent comparison. APPROACH: A capillary filled with a photosensitizer was placed in a phantom and ex vivo rodent brains, with fluorescence laminar optical tomography and maximum intensity projection images obtained. The signal loss in the Z direction was quantified and compared to see which methodology could compensate better for signal loss caused by tissue attenuation. RESULTS: The results demonstrated that we can reconstruct a capillary filled with benzoporphyrin derivative photosensitizers faithfully in phantoms and in ex vivo rodent brain tissues using fluorescence laminar optical tomography. We further demonstrated that we can better compensate for signal loss when compared with maximum intensity projection imaging. CONCLUSIONS: Using fluorescence laminar optical tomography (FLOT), one can compensate for signal loss in deeper parts of tissue when imaging in ex vivo rodent brain tissue compared with maximum intensity projection imaging.

Quantitatively relating brain endothelial cell-cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program.

BACKGROUND: The endothelial cell-cell junctions of the blood-brain barrier (BBB) play a pivotal role in the barrier's function. Altered cell-cell junctions can lead to barrier dysfunction and have been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell-cell adhesion. Here, we used our novel Junction Analyzer Program (JAnaP) to quantify junction phenotype (i.e., continuous, punctate, or perpendicular) in response to various substrate compositions, cell culture times, and cAMP treatments in human brain microvascular endothelial cells (HBMECs). We then quantitatively correlated junction presentation with barrier permeability on both a "global" and "local" scale. METHODS: We cultured HBMECs on collagen I, fibronectin, collagen IV, laminin, fibronectin/collagen IV/laminin, or hyaluronic acid/gelatin for 2, 4, and 7 days with varying cAMP treatment schedules. Images of immunostained ZO-1, VE-cadherin, and claudin-5 were analyzed using the JAnaP to calculate the percent of the cell perimeter presenting continuous, punctate, or perpendicular junctions. Transwell permeability assays and resistance measurements were used to measure bulk ("global") barrier properties, and a "local" permeability assay was used to correlate junction presentation proximal to permeable monolayer regions. RESULTS: Substrate composition was found to play little role in junction presentation, while cAMP supplements significantly increased the continuous junction architecture. Increased culture time required increased cAMP treatment time to reach similar ZO-1 and VE-cadherin coverage observed with shorter culture, though longer cultures were required for claudin-5 presentation. Prolonged cAMP treatment (6 days) disrupted junction integrity for all three junction proteins. Transwell permeability and TEER assays showed no correlation with junction phenotype, but a local permeability assay revealed a correlation between the number of discontinuous and no junction regions with barrier penetration. CONCLUSIONS: These results suggest that cAMP signaling influences HBMEC junction architecture more than matrix composition. Our studies emphasized the need for local barrier measurement to mechanistically understand the role of junction phenotype and supported previous results that continuous junctions are indicative of a more mature/stable endothelial barrier. Understanding what conditions influence junction presentations, and how they, in turn, affect barrier integrity, could lead to the development of therapeutics for diseases associated with BBB dysfunction.

Immunological and Toxicological Considerations for the Design of Liposomes.

Liposomes hold great potential as gene and drug delivery vehicles due to their biocompatibility and modular properties, coupled with the major advantage of attenuating the risk of systemic toxicity from the encapsulated therapeutic agent. Decades of research have been dedicated to studying and optimizing liposomal formulations for a variety of medical applications, ranging from cancer therapeutics to analgesics. Some effort has also been made to elucidate the toxicities and immune responses that these drug formulations may elicit. Notably, intravenously injected liposomes can interact with plasma proteins, leading to opsonization, thereby altering the healthy cells they come into contact with during circulation and removal. Additionally, due to the pharmacokinetics of liposomes in circulation, drugs can end up sequestered in organs of the mononuclear phagocyte system, affecting liver and spleen function. Importantly, liposomal agents can also stimulate or suppress the immune system depending on their physiochemical properties, such as size, lipid composition, pegylation, and surface charge. Despite the surge in the clinical use of liposomal agents since 1995, there are still several drawbacks that limit their range of applications. This review presents a focused analysis of these limitations, with an emphasis on toxicity to healthy tissues and unfavorable immune responses, to shed light on key considerations that should be factored into the design and clinical use of liposomal formulations.

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

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.

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.

Estimation of Achievable Oxygen Consumption Following Transfusion With Rejuvenated Red Blood Cells.

Erythrocyte storage induces a nonphysiological increase in hemoglobin-oxygen affinity (quantified by low p50, the oxygen tension at 50% hemoglobin saturation), which can be restored through biochemical rejuvenation. The objective was to mathematically model the impact of transfusing up to 3 standard allogeneic units or rejuvenated units on oxygen delivery (DO2) and oxygen consumption (VO2). Oxygen dissociation curves were generated from additive solution-1 red blood cell (RBC) leukoreduced units (n = 7) before and after rejuvenation following manufacturer's instructions. Two of these units were used to prepare standard or rejuvenated donor RBC and added to samples of fresh whole blood. These admixtures were used to construct an in vitro transfusion model of postoperative anemia and determine a linear equation for calculating the sample p50, which was subsequently used to calculate DO2 and VO2 after simulated transfusions. Whole blood-packed red blood cell unit admixture p50s could be predicted from a linear model including the p50 of its components, the mass fraction of the transfused component, and interaction terms (R2 = .99, P < 0.001). Transfusion with standard units slightly, but significantly, increased projected DO2 compared with rejuvenated units (P = 0.03), but rejuvenated units markedly increased projected VO2 (P = 0.03). Standard units did not significantly change VO2 relative to pre-transfusion levels (P > 0.1). Using high-p50, rejuvenated RBC in simulated transfusions greatly improved projected VO2, indicating the potential for increased end-organ oxygen availability compared with standard transfusion. Patient capacity to increase cardiac output after cardiac surgery may be limited. Transfusing high-p50 RBC in this setting may improve the perioperative care of these patients.