In vivo biodistribution and ototoxicity assessment of cationic liposomal-ceftriaxone via noninvasive trans-tympanic delivery in chinchilla models: Implications for otitis media therapy.

OBJECTIVES: We report the in vivo biodistribution and ototoxicity of cationic liposomal-ceftriaxone (CFX) delivered via ear drop formulation in adult chinchilla. METHODS: CFX was encapsulated in liposomes with size of ∼100 nm and surface charge of +20 mV. 100 µl liposomes or free drug was applied twice daily in both external ear canals of adult chinchillas for either 3 or 10 days. Study groups included free ceftriaxone (CFX, Day 3: n = 4, Day 10: n = 8), liposomal ceftriaxone (CFX-Lipo, Day 3: n = 4, Day 10: n = 8), and a systemic control group (Day 3: n = 4, Day 10: n = 4). Ceftriaxone delivery to the middle ear and systemic circulation was quantified by HPLC assays. Liposome transport was visualized via confocal microscopy. Auditory brainstem response (ABR) tests and cochlear histology were used to assess ototoxicity. RESULTS: Liposomal ceftriaxone (CFX-Lipo) displayed a ∼658-fold increase in drug delivery efficiency in the middle ear relative to the free CFX (8.548 ± 0.4638% vs. 0.013 ± 0.0009%, %Injected dose, Mean ± SEM). CFX measured in blood serum (48.2 ± 7.78 ng/ml) following CFX-Lipo treatment in ear was 41-fold lower compared to systemic free-CFX treatment (1990.7 ± 617.34 ng/ml). ABR tests and histological analysis indicated no ototoxicity due to the treatment. CONCLUSION: Cationic liposomal encapsulation results in potent drug delivery across the tympanic membrane to the middle ear with minimal systemic exposure and no ototoxicity.

2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature.

PURPOSE: Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100 nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. PROCEDURES: Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12 weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13 Gy partial body irradiation at 42- and 90 days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. RESULTS: We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. CONCLUSIONS: Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

Photoactivated HPPH-Liposomal therapy for the treatment of HPV-Negative head and neck cancer.

OBJECTIVES: Human Papillomavirus (HPV)-negative head and neck cancer (HNC) is an aggressive malignancy with a poor prognosis. To improve outcomes, we developed a novel liposomal targeting system embedded with 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), a chlorin-based photosensitizer. Upon exposure to 660 nm light, HPPH phototriggering generates reactive oxygen species. The objective of this study was to evaluate biodistribution and test efficacy of HPPH-liposomal therapy in a patient-derived xenograft (PDX) model of chemoradioresistant HNC. MATERIALS AND METHODS: PDX models were developed from two surgically resected HNCs (P033 and P038) recurrent after chemoradiation. HPPH-liposomes were created including trace amounts of DiR (Ex/Em 785/830 nm), a near infrared lipid probe. Liposomes were injected via tail vein into PDX models. Biodistribution was assessed at serial timepoints in tumor and end-organs through in vivo DiR fluorescence. To evaluate efficacy, tumors were treated with a cw-diode 660 nm laser (90 mW/cm2, 5 min). This experimental arm was compared to appropriate controls, including HPPH-liposomes without laser or vehicle with laser alone. RESULTS: HPPH-liposomes delivered via tail vein exhibited selective tumor penetration, with a peak concentration at 4 h. No systemic toxicity was observed. Treatment with combined HPPH-liposomes and laser resulted in improved tumor control relative to either vehicle or laser alone. Histologically, this manifested as both increased cellular necrosis and decreased Ki-67 staining in the tumors treated with combined therapy. CONCLUSIONS: These data demonstrate tumor-specific anti-neoplastic efficacy of HPPH-liposomal treatment for HNC. Importantly, this platform can be leveraged in future studies for targeted delivery of immunotherapies which can be packaged within HPPH-liposomes.

X-ray and MR Contrast Bearing Nanoparticles Enhance the Therapeutic Response of Image-Guided Radiation Therapy for Oral Cancer.

INTRODUCTION: Radiation therapy for head and neck squamous cell carcinoma is constrained by radiotoxicity to normal tissue. We demonstrate 100 nm theranostic nanoparticles for image-guided radiation therapy planning and enhancement in rat head and neck squamous cell carcinoma models. METHODS: PEG conjugated theranostic nanoparticles comprising of Au nanorods coated with Gadolinium oxide layers were tested for radiation therapy enhancement in 2D cultures of OSC-19-GFP-luc cells, and orthotopic tongue xenografts in male immunocompromised Salt sensitive or SS rats via both intratumoral and intravenous delivery. The radiation therapy enhancement mechanism was investigated. RESULTS: Theranostic nanoparticles demonstrated both X-ray/magnetic resonance contrast in a dose-dependent manner. Magnetic resonance images depicted optimal tumor-to-background uptake at 4 h post injection. Theranostic nanoparticle + Radiation treated rats experienced reduced tumor growth compared to controls, and reduction in lung metastasis. CONCLUSIONS: Theranostic nanoparticles enable preprocedure radiotherapy planning, as well as enhance radiation treatment efficacy for head and neck tumors.

Dynamic NIR Fluorescence Imaging and Machine Learning Framework for Stratifying High vs. Low Notch-Dll4 Expressing Host Microenvironment in Triple-Negative Breast Cancer.

Delta like canonical notch ligand 4 (Dll4) expression levels in tumors are known to affect the efficacy of cancer therapies. This study aimed to develop a model to predict Dll4 expression levels in tumors using dynamic enhanced near-infrared (NIR) imaging with indocyanine green (ICG). Two rat-based consomic xenograft (CXM) strains of breast cancer with different Dll4 expression levels and eight congenic xenograft strains were studied. Principal component analysis (PCA) was used to visualize and segment tumors, and modified PCA techniques identified and analyzed tumor and normal regions of interest (ROIs). The average NIR intensity for each ROI was calculated from pixel brightness at each time interval, yielding easily interpretable features including the slope of initial ICG uptake, time to peak perfusion, and rate of ICG intensity change after reaching half-maximum intensity. Machine learning algorithms were applied to select discriminative features for classification, and model performance was evaluated with a confusion matrix, receiver operating characteristic curve, and area under the curve. The selected machine learning methods accurately identified host Dll4 expression alterations with sensitivity and specificity above 90%. This may enable stratification of patients for Dll4 targeted therapies. NIR imaging with ICG can noninvasively assess Dll4 expression levels in tumors and aid in effective decision making for cancer therapy.

NIR-II window tracking of hyperglycemia induced intracerebral hemorrhage in cerebral cavernous malformation deficient mice.

Second near infrared (NIR-II) window fluorescence imaging between 1000 and 1700 nm with reduced scattering and autofluorescence and deep tissue light penetration allows early and non-invasive determination of vascular pathologies. Here, we demonstrate in vivo NIR-II imaging techniques for tracking hyperglycaemia-induced Intracerebral Hemorrhage (ICH) and Blood Brain Barrier (BBB) hyperpermeability in Cerebral Cavernous Malformation (CCM) deficient mice (CCM1+/-). We synthesised PEGylated Ag2S quantum dots (QDs) with a bright fluorescent emission peak centred at 1135 nm under an 808 nm NIR light for dynamic imaging of cerebral vasculature in mice and determined the development of ICH and BBB impairment in hyperglycaemic CCM1+/- mice. In vivo optical imaging was conducted with micro-CT (including k-mean cluster analysis) as well as in vivo permeability assays using FITC-dextran perfusion and IgG staining, respectively. The increased BBB permeability in CCM1+/- mice was further demonstrated to be associated with a high-glucose-caused decrease of CCM1 expressions. This study validates that deep-penetrating NIR-II QDs can be used for the tracking of ICH and BBB hyperpermeability in transgenic mice models of cerebral vascular anomalies.