Dominique N. Price

Preparation and Characterization of Novel Magnetic Nano-in-Microparticles for Site-Specific Pulmonary Drug Delivery

We propose the use of novel inhalable nano-in-microparticles (NIMs) for site-specific pulmonary drug delivery. Conventional lung cancer therapy has failed to achieve therapeutic drug concentrations at tumor sites without causing adverse effects in healthy tissue. To increase targeted drug delivery near lung tumors, we have prepared and characterized a magnetically responsive dry powder vehicle containing doxorubicin. A suspension of lactose, doxorubicin and Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) were spray dried. NIMs were characterized for their size and morphological properties by various techniques: dynamic light scattering (DLS) and laser diffraction (LS) to determine hydrodynamic size of the SPIONs and the NIMs, respectively; next generation cascade impactor (NGI) to determine the aerodynamic diameter and fine particle fraction (FPF); scanning (SEM) and transmission (TEM) electron microscopy to analyze particle surface morphology; electron dispersive X-ray spectroscopy (EDS) to determine iron loading in NIMs; inductively coupled plasma atomic emission spectroscopy (ICP-AES) and superconducting quantum interference device (SQUID) to determine Fe3O4 content in the microparticles; and high performance liquid chromatography (HPLC) to determine doxorubicin loading in the vehicle. NIMs deposition and retention near a magnetic field was performed using a proof-of-concept cylindrical tube to mimic the conducting airway deposition. The hydrodynamic size and zeta potential of SPIONs were 56 nm and -49 mV, respectively. The hydrodynamic and aerodynamic NIM diameters were 1.6 μm and 3.27±1.69 μm, respectively. SEM micrographs reveal spherical particles with rough surface morphology. TEM and focused ion beam-SEM micrographs corroborate the porous nature of NIMs, and surface localization of SPIONs. An in vitro tracheal mimic study demonstrates more than twice the spatial deposition and retention of NIMs, compared to a liquid suspension, in regions under the influence of a strong magnetic gradient. We report the novel formulation of an inhaled and magnetically responsive NIM drug delivery vehicle. This vehicle is capable of being loaded with one or more chemotherapeutic agents, with future translational ability to be targeted to lung tumors using an external magnetic field.

Oral Tolerance to Environmental Mycobacteria Interferes with Intradermal, but Not Pulmonary, Immunization against Tuberculosis

Bacille Calmette–Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB) and is administered in over 150 countries worldwide. Despite its widespread use, the vaccine has a variable protective efficacy of 0–80%, with the lowest efficacy rates in tropical regions where TB is most prevalent. This variability is partially due to ubiquitous environmental mycobacteria (EM) found in soil and water sources, with high EM prevalence coinciding with areas of poor vaccine efficacy. In an effort to elucidate the mechanisms underlying EM interference with BCG vaccine efficacy, we exposed mice chronically to Mycobacterium avium (M. avium), a specific EM, by two different routes, the oral and intradermal route, to mimic human exposure. After intradermal BCG immunization in mice exposed to oral M. avium, we saw a significant decrease in the pro-inflammatory cytokine IFN-γ, and an increase in T regulatory cells and the immunosuppressive cytokine IL-10 compared to naïve BCG-vaccinated animals. To circumvent the immunosuppressive effect of oral M. avium exposure, we vaccinated mice by the pulmonary route with BCG. Inhaled BCG immunization rescued IFN-γ levels and increased CD4 and CD8 T cell recruitment into airways in M. avium-presensitized mice. In contrast, intradermal BCG vaccination was ineffective at T cell recruitment into the airway. Pulmonary BCG vaccination proved protective against Mtb infection regardless of previous oral M. avium exposure, compared to intradermal BCG immunization. In conclusion, our data indicate that vaccination against TB by the pulmonary route increases BCG vaccine efficacy by avoiding the immunosuppressive interference generated by chronic oral exposure to EM. This has implications in TB-burdened countries where drug resistance is on the rise and health care options are limited due to economic considerations. A successful vaccine against TB is necessary in these areas as it is both effective and economical.

In Vivo Pulmonary Delivery and Magnetic-Targeting of Dry Powder Nano-in-Microparticles

This brief communication evaluates the cytotoxicity and targeting capability of a dry powder chemotherapeutic. Nano-in-microparticles (NIMs) are a dry powder drug delivery vehicle containing superparamagnetic iron oxide nanoparticles (SPIONs) and either doxorubicin (w/w solids) or fluorescent nanospheres (w/v during formulation; as a drug surrogate) in a lactose matrix. In vitro cytotoxicity was evaluated in A549 adenocarcinoma cells using MTS and LDH assays to assess viability and toxicity after 48 h of NIMs exposure. In vivo magnetic-field-dependent targeting of inhaled NIMs was evaluated in a healthy mouse model. Mice were endotracheally administered fluorescently labeled NIMs either as a dry powder or a liquid aerosol in the presence of an external magnet placed over the left lung. Quantification of fluorescence and iron showed a significant increase in both fluorescence intensity and iron content to the left magnetized lung. In comparison, we observed decreased targeting of fluorescent nanospheres to the left lung from an aerosolized liquid suspension, due to the dissociation of SPIONs and nanoparticles during pulmonary administration. We conclude that dry powder NIMs maintain the therapeutic cytotoxicity of doxorubicin and can be better targeted to specific regions of the lung in the presence of a magnetic field, compared to a liquid suspension.

Longitudinal Assessment of Lung Cancer Progression in Mice Using the Sodium Iodide Symporter Reporter Gene and SPECT/CT Imaging

Lung cancer has the highest mortality rate of any tissue-specific cancer in both men and women. Research continues to investigate novel drugs and therapies to mitigate poor treatment efficacy, but the lack of a good descriptive lung cancer animal model for preclinical drug evaluation remains an obstacle. Here we describe the development of an orthotopic lung cancer animal model which utilizes the human sodium iodide symporter gene (hNIS; SLC5A5) as an imaging reporter gene for the purpose of non-invasive, longitudinal tumor quantification. hNIS is a glycoprotein that naturally transports iodide (I-) into thyroid cells and has the ability to symport the radiotracer 99mTc-pertechnetate (99mTcO4-). A549 lung adenocarcinoma cells were genetically modified with plasmid or lentiviral vectors to express hNIS. Modified cells were implanted into athymic nude mice to develop two tumor models: a subcutaneous and an orthotopic xenograft tumor model. Tumor progression was longitudinally imaged using SPECT/CT and quantified by SPECT voxel analysis. hNIS expression in lung tumors was analyzed by quantitative real-time PCR. Additionally, hematoxylin and eosin staining and visual inspection of pulmonary tumors was performed. We observed that lentiviral transduction provided enhanced and stable hNIS expression in A549 cells. Furthermore, 99mTcO4- uptake and accumulation was observed within lung tumors allowing for imaging and quantification of tumor mass at two-time points. This study illustrates the development of an orthotopic lung cancer model that can be longitudinally imaged throughout the experimental timeline thus avoiding inter-animal variability and leading to a reduction in total animal numbers. Furthermore, our orthotopic lung cancer animal model is clinically relevant and the genetic modification of cells for SPECT/CT imaging can be translated to other tissue-specific tumor animal models.

Respiratory Tract Deposition and Distribution Pattern of Microparticles in Mice Using Different Pulmonary Delivery Techniques

Pulmonary delivery of drugs and vaccines is an established route of administration, with particulate-based carriers becoming an attractive strategy to enhance the benefits of pulmonary therapeutic delivery. Despite the increasing number of publications using the pulmonary route of delivery, the lack of effective and uniform administration techniques in preclinical models generally results in poor translational success. In this study, we used the IVIS Spectrum small-animal in vivo imaging system to compare the respiratory tract deposition and distribution pattern of a microsphere suspension (5 µm) in mice after 1, 4, and 24 h when delivered by oropharyngeal aspiration, the Microsprayer® Aerosolizer, and the BioLite Intubation System, three-widely reported preclinical inhalation techniques. We saw no significant differences in microsphere deposition in whole body images and excised lungs (at 1, 4, and 24 h); however, the three-dimensional (3D) images showed more localized deposition in the lungs with the MicroSprayer® and BioLite delivery techniques. Further, oropharyngeal aspiration (at 1 h) showed microsphere deposition in the oral cavity, in contrast to the MicroSprayer® and BioLite systems. The studies shown here will allow researchers to choose the appropriate pulmonary delivery method in animal models based on their study requirements.

Delivery of Therapeutics to the Lung

Pulmonary delivery in animal models can be performed using either direct administration methods or by passive inhalation. Direct pulmonary delivery requires the animal to be endotracheally intubated, whereas passive delivery uses a nose-only or a whole-body chamber. Endotracheal delivery of therapeutics and vaccines allows investigators to deliver the payload directly into the lung without the limitations associated with passive pulmonary administration methods. Additionally, endotracheal delivery can achieve deep lung delivery without the involvement of other exposure routes and is more reproducible and quantitative than passive pulmonary delivery in terms of accurate dosing. Here we describe the endotracheal delivery of both liquids and dry powders for preclinical models of treatment and exposure.

Abstract 102: A longitudinal, orthotopic mouse imaging model using modified hNIS-A549 human lung adenocarcinoma cells and SPECT/CT imaging

The human sodium iodide symporter (hNIS; SLC5A5) is a membrane glycoprotein that is a transporter for iodide uptake and also concentrates the radiotracer technetium 99mTc-pertechnetate (99mTc). In this study, we investigated the use of hNIS as an imaging reporter gene expressed in lung cancer cells in nude mice that can be quantified using single-photon emission computed tomography (SPECT) imaging. The radionuclide 99mTc was selected for its rapid clearance from the body and its short physical half-life (i.e., 6 hours). These characteristics allowed for the development a highly sensitive spatial and temporal tumor-imaging model. Human lung adenocarcinoma A549 cells were genetically modified using plasmid and lentiviral vectors to stably express hNIS. Using these modified cells, we monitored xenograft and orthotopic tumor growth using SPECT/CT for a total of 47 days. hNIS expression was quantified using qPCR and tumor progression was monitored by SPECT voxel analysis and validated using HE 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 102. doi:10.1158/1538-7445.AM2014-102