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.

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.

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.

Since Heimstaedt and Lehmann developed the first fluorescence microscope in the early 20th century,^1,2 it has become a critical tool for biological research. The use of fluorescent probes has allowed scientists and physicians to increase contrast and signal from a region of interest, allowing for the study of biological systems that could not previously be imaged. One of the most common uses for fluorescence is in the diagnosis, planning, and treatment of cancer. For example, fluorescence imaging has been used for guiding the resection of brain tumors. Gliolan^® [5-aminolevulinic acid hydrochloride (5-ALA)] is an FDA-approved imaging agent used for fluorescence-guided surgery of malignant gliomas (e.g., glioblastoma multiforme, GBM). 5-ALA is a hemoglobin precursor, with protoporphyrin IX (PpIX) being a fluorescent intermediary molecule.^3,4 Changes in various metabolic pathways, such as the upregulation of coproporphyrinogen and downregulation of ferrochelatase, lead to the preferential accumulation of PpIX in glioma cells over healthy brain tissues.⁵ By exciting the PpIX in tumor tissues with light at wavelengths in the range of 375-nm to 420-nm during surgery, tumor margins are highlighted for better detection and resection,⁶ increasing patient mean overall survival by an average of 3 months when compared with white light techniques.⁷

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 where 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 disrpution. We transfected HEK-293 cells with both NanoLuc and human ABCB1 or ABCG2, or their zebrafish homologs Abcb4 and Abcg2a, which are functionally homologous to human P-gp and ABCG2, respectively, and expressed at the zebrafish BBB. We evaluated the brightness of ten NanoLuc substrates, then screened the eight brightest for their ability to be effluxed by the ABC transporters. We identified one ABCB1 substrate, two Abcb4 substrates, six ABCG2 substrates, and four Abcg2a substrates. 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. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

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. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.