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Brief Article
In vivo pulmonary delivery and magnetictargeting of dry powder nano-in-microparticles Dominique N. Price, Loreen R. Stromberg, Nitesh K Kunda, and Pavan Muttil Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00532 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017
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Molecular Pharmaceutics
In vivo pulmonary delivery and magnetictargeting of dry powder nano-inmicroparticles
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Dominique N. Price1, Loreen R. Stromberg1,2, Nitesh K. Kunda1, and Pavan Muttil1,3*
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1
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Health Sciences Center, Albuquerque, New Mexico
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2
Department of Mechanical Engineering, Iowa State University, Ames, Iowa
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3
The University of New Mexico Cancer Center, Albuquerque, New Mexico
Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico
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Conflicts of Interest: The authors have no conflicts of interest to declare.
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Abstract
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This brief communication evaluates the cytotoxicity and targeting capability of a dry powder
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chemotherapeutic. Nano-in-microparticles (NIMs) are a dry powder drug delivery vehicle
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containing superparamagnetic iron oxide nanoparticles (SPIONs) and either doxorubicin (w/w
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solids) or fluorescent nanospheres (w/v during formulation; as a drug surrogate) in a lactose
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matrix. In vitro cytotoxicity was evaluated in A549 adenocarcinoma cells using MTS and LDH
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assays to assess viability and toxicity after 48 hours of NIMs exposure. In vivo magnetic-field-
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dependent targeting of inhaled NIMs was evaluated in a healthy mouse model. Mice were
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endotracheally administered fluorescently-labeled NIMs either as a dry powder or a liquid
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aerosol in the presence of an external magnet placed over the left lung. Quantification of
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fluorescence and iron showed a significant increase in both fluorescence intensity and iron
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content to the left magnetized lung. In comparison, we observed decreased targeting of
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fluorescent nanospheres to the left lung from an aerosolized liquid suspension, due to the
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dissociation of SPIONs and nanoparticles during pulmonary administration. We conclude that
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dry powder NIMs maintain the therapeutic cytotoxicity of doxorubicin and can be better
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targeted to specific regions of the lung, in the presence of a magnetic field, compared to a liquid
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suspension.
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Keywords: Targeted pulmonary delivery; nano-in-microparticles (NIMs); pulmonary
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chemotherapeutic; aerosolized drug delivery; non-small cell lung cancer; superparamagnetic
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iron oxide nanoparticles (SPIONs)
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Introduction
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Lung cancer is the second most commonly diagnosed cancer in both men and women, and it has
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the highest mortality rate compared to all other types of cancer accounting for 26.5% of all
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cancer deaths1–3. Conventional chemotherapy for lung cancer is administered intravenously and
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does not distinguish between cancerous and healthy cells. The high concentrations of
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chemotherapeutic agents that are required for systemic administration often lead to nonspecific
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adverse effects, while lung tumor microenvironments that are distantly located from capillaries
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receive sub-therapeutic drug concentrations.
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Pulmonary drug delivery has been used to treat respiratory diseases such as asthma and
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microbial infections, as well as systemic diseases4–9. The lungs are well-suited for drug delivery
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due to their large surface area, thin alveolar epithelium, easily permeable membrane, and
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extensive vasculature, which allow substantial and rapid drug absorption for increased local and
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systemic efficacy9. Pulmonary delivery of chemotherapy has been evaluated in human trials for
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the treatment of lung cancer10–15. A phase I/II study of inhaled doxorubicin combined with
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systemic platinum-based therapy was administered to twenty-eight patients with advanced non-
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small cell lung cancer11. However, while the results of this localized delivery were promising, a
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few patients experienced dose-limiting pulmonary-toxicity. In a preclinical study, Dames and
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colleagues attempted to minimize toxicity to the whole lung by targeting a therapeutic surrogate
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to a specific lung lobe in mice using iron oxide nanoparticles in the presence of an external
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magnetic field16. However, targeting to the magnetized lobe was minimal due to the separation
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of the drug surrogate and iron oxide nanoparticles during pulmonary administration of the liquid ACS Paragon Plus Environment
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suspension.
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The aim of this study was two-fold, 1) to determine if dry powder nano-in-microparticles
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(NIMs)17 containing doxorubicin still display therapeutic cytotoxicity after the spray drying
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process and 2) to evaluate the magnetic-field-dependent targeting of dry powder NIMs
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administered endotracheally into the lungs of healthy mice (Fig. 1A). Our hypothesis was that
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the incorporation of a chemotherapeutic agent and iron-oxide nanoparticles in a powder matrix
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should prevent the separation of nanoparticles and therapeutic observed by Dames et al., while
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still targeting the drug payload to the magnetized lung region (Fig. 1A)16. This inhalable drug-
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delivery approach has the potential to reduce toxicity to healthy tissues by targeting drugs
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directly to specific regions of the lung, thus achieving therapeutic concentration near solid
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tumors (Fig. 1B)18,19.
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Experimental Methods
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Materials
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Alpha-D-(+)-lactose monohydrate Respitose ML-001 was a gift from DMV-Fonterra
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Excipients GmbH & Co. KG (Goch, Germany). FluidMAG-UC SPIONs with a hydrodynamic
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diameter of 50 nm was purchased from Chemicell GmbH (Berlin, Germany). Fluorescent dye-
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containing NIMs were formulated with Molecular Probes® FluoSpheres® Carboxylate-
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Modified Nanospheres (F8783, 0.02µm, λex = 660nm and λem = 680nm, Molecular Probes®,
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Life Technologies, Thermo Fisher Scientific, Inc. Waltham, MA, USA).
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Formulation and characterization of the NIMs delivery vehicle
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Spray drying of the NIMs delivery vehicle was performed as described previously17.
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Briefly, a suspension containing approximately 20% (w/w) SPIONs and either 2.8% (w/w)
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doxorubicin (Selleck Chemicals, LLC, Houston, TX, USA) (w/w) (for in vitro studies) or 10%
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(w/w) fluorescent nanospheres (fluorescent drug surrogate for in vivo studies) was spray-dried
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in MilliQ water and a lactose matrix (2.5% w/v total feed concentration) (Fig. 1A). A B-290 ACS Paragon Plus Environment
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mini-spray dryer with a standard two-fluid nozzle (0.7mm diameter) (Büchi Corporation,
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Flawil, Switzerland), was used to spray dry the suspension with the following parameters: inlet
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temperature 170 ± 2°C, outlet temperature 103 ± 2°C, aspirator rate 100%, and an atomization
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air flow rate of 742NL/h. Spray-dried control microparticles were formulated containing a
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higher concentration of 5% lactose only (w/v) in MilliQ water to keep particle sizing similar.
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Figure 1: Magnetic field-dependent lung targeting. A) Pictorial representation of NIMs dry
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powder showing lactose (gray), doxorubicin (red), and SPIONs (black)17. B) Scanning electron
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microscope image of NIMs dry powder. C) Schematic of endotracheal delivery of magnetically-
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targeted NIMs. D) Orientation of the permanent magnet in thoracotomized mice prior to
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pulmonary delivery of NIMs. E) Orientation of the trachea, right lung, and left lung prior to
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imaging. SPIONs are visible in the trachea and left lung (shown by arrows) demonstrating
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magnetic targeting.
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Dry powder NIMs particle sizing
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NIMs geometric particle size was measured using a Mastersizer 3000 analyzer (Malvern
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Instruments Ltd, Worcestershire, UK) attached to a dry-dispersion accessory device (Aero S,
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Malvern Instruments). A pressure of 4 bars was used for the venturi dispenser with a feed rate of
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90%. The refractive index of the sample was taken to be the average of the NIMs components
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multiplied by the percent (w/w) contained in the formulation: 70% w/w lactose (1.35), 20% w/w
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SPIONs (2.42), and 10% w/w fluorescent nanospheres (1.52) for a weighted average of 1.58.
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The results were expressed in terms of Dv50 (volumetric median diameter). All samples were
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analyzed in triplicate and are expressed as mean ± standard deviation. The aerodynamic
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diameter of the NIMs was determined using a Next Generation Impactor (NGI) (model 170,
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MSP Corporation, Shoreview, MN). NIMs were insufflated into the NGI using the DP4 dry
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powder insufflator™ for rat (Penn Century, Inc., USA). The NGI was operated with a flow rate
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of 30L/min for 10min. Particle deposition was determined by the gravimetric method.
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Cell culture
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Human lung adenocarcinoma A549 cells (ATCC® CCL-185™) (American Type Tissue
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Culture, Manassas, Virginia, USA) were grown in Ham’s F12K (Kaighn’s) medium and
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supplemented with fetal bovine serum (FBS), 5% L-glutamine, 3% antimycotic, and 3%
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antibiotic (Life Technologies, Grand Island, NY, USA).
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Cytotoxicity studies
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Confluent cells were exposed to dry powder NIMs containing doxorubicin (D-NIMs)
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and controls (doxorubicin solution, lactose solution, and SPIONs suspension). A549 cell
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viability and toxicity were tested using the CytoTox96® Non-Radioactive (3-(4,5-
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dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
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cell proliferation assay and the CytoTox96® Non-Radioactive lactate dehydrogenase (LDH)
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cytotoxicity assay, respectively. Briefly, D-NIMs were uniformly dispersed in F12K non-
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supplemented media with FBS at three different doxorubicin concentrations: 0.16µg/mL (low
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concentration), 1.6µg/mL (medium concentration), and 16µg/mL (high concentration) (referred
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to henceforth as low, medium, and high dose). The same media was used to generate free
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doxorubicin solutions with concentrations of 0.3µg/mL (low), 3µg/mL (medium), and 30µg/mL
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(high). Controls consisting of lactose solution and SPIONs suspension were used at
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concentrations similar to those present in NIMs, as ratios from the spray dried feed solution
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were held constant relative to doxorubicin. Prior to exposure, A549 cells were washed with
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fresh media and 100µL of the suspensions/solutions (D-NIMs; doxorubicin only; lactose;
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SPIONs).
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CellTiter 96® Aqueous One Solution Reagent MTS assay (Promega, Madison,
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Wisconsin, USA) and CytoTox96® Non-Radioactive Assay LDH assay (Promega, Madison,
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Wisconsin, USA) were performed according to the manufacturer’s instructions. In brief, at time
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periods of 1, 8, 24, 48 hours post-exposure, 80µL of cell culture supernatant was aspirated from
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each well and centrifuged at 10,000 rpm for 10 minutes to settle any SPIONs or cells. MTS
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reagent was mixed with the cell culture supernatant at a ratio of 1:6 (MTS) or 1:2 (LDH),
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aliquoted into a black optical bottom 96-well plate and incubated at 37°C and 5% CO2 for 3
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hours (MTS) or 30 min (LDH). For LDH development, 50µL of stop solution was added to each
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well. Absorbance was measured at a wavelength of 490nm. Results were plotted as absorbance
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over time with respect to increasing amounts of doxorubicin present in the D-NIMs.
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In vivo study design
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Male Balb/c mice (6-8 weeks old; Jackson Laboratory, Sacramento, CA, USA) were
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used for the in vivo targeting studies. All rodents were housed in a temperature and light cycle
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controlled facility, and their care followed the guidelines of the National Institutes of Health and
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an approved Institutional Animal Care and Use Committee protocol (IACUC protocol number
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11-100747-HSC). Mice were randomly assigned to 5 different groups with n=3 per group: 1)
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Untreated control mice; 2) Mice administered NIMs dry powders magnetically-targeted to the
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left lung; 3) Mice administered NIMs dry powders in the absence of magnetic targeting; 4) Mice
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administered NIMs liquid suspensions magnetically-targeted to the left lung; 5) Mice
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administered NIMs liquid suspensions in the absence of magnetic targeting. For the in vivo
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studies, doxorubicin in the NIMs was replaced with fluorescent nanoparticles as a drug
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surrogate to facilitate imaging.
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Mice were anesthetized with a standard dose of xylazine/ketamine. A dry powder
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Insufflator™ for mouse (DP-4-M; Penn-Century Inc., Wyndmoor, PA, USA) was attached to a
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1mL disposable plastic syringe that was used to endotracheally administer the dry powder
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NIMs. Briefly, anesthetized mice were placed on their back on a intubation platform (Penn-
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Century Inc., Wyndmoor, PA, USA) and the tracheal opening was visualized by inserting a
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small animal laryngoscope (Model LS2; Penn-Century Inc.)20. The insufflator delivery tube was
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inserted gently into the trachea of the animal, proximal to the carina, until the bend of the
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delivery tube was positioned at the incisors. Skin over the left lung of the mouse was carefully
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dissected without exposing the thoracic cavity
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neodymium−iron−boron (NdFeB) permanent cylindrical magnet (grade N52, 22mm long ×
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20mm in diameter; Applied Magnets, Plano, TX) was centered 1mm above the left lung to avoid
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contact with the tissue, with the edge of the magnet perpendicular to the upper region of the left
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lung (Fig. 1C). Skin removal was performed to decrease the distance between the lung and the
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magnet, and maximize the magnetic targeting. The orientation of the magnet over the left lung
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was critical for the NIMs to be sufficiently attracted to the magnetic pole that was created by the
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externally-applied magnetic field. We had previously characterized the permanent magnet to
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have a magnetic field of 0.58 Tesla17.
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and a commercially available
Approximately 2.0mg of NIMs were loaded in the insufflator and administered using the 8 ACS Paragon Plus Environment
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plastic syringe with an air volume of 500µL for a total of 10 puffs (10 actuations). Of the 2.0mg
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loaded in the insufflator, 0.5mg was delivered into the mouse lung based on the gravimetric
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analysis of the insufflator before and after dosing. A NIMs liquid suspension was administered
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using the MicroSprayer® aerosolizer (1A-1C; Penn-Century) for mouse and was attached to a
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hand-operated, high-pressure syringe (FMJ-250; Penn-Century)21. Mice were endotracheally
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intubated and 50µL of saline containing 0.5mg of the NIMs dry powder (w/v solids; containing
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equivalent amounts of the fluorescent dye and SPIONs) was administered directly into the
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airways with the MicroSprayer®. Control mice were treated similarly using the dry powder
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NIMs and liquid suspension in the absence of magnetic-field-dependent targeting. Mice were
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sacrificed immediately after pulmonary delivery; lungs and trachea were removed en bloc,
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separated (Fig. 1D), and fluorescence and iron deposition were quantified in the respective
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tissues.
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Fluorescence quantification
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NIMs dry powder and NIMs suspension were administered endotracheally and targeted
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to the left lung in the presence of a permanent magnet. Individual lung lobes and trachea were
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excised and imaged immediately for fluorescence (λex = 660nm and λem = 680nm) with an
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exposure time of 10 seconds using a Caliper IVIS Lumina II imaging system (Caliper Life
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Sciences). A region of interest (ROI) was drawn around the trachea, left lung, right lung, and
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background. The ROI area was kept constant at 2.5 cm2 and fluorescence was expressed as units
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of average radiance efficiency (RE) (p/sec/cm2/sr)/(µW/cm2)22.
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Equation 1. Fluorescence calculations
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% Targeting =
23
Targeting Efficiency L:R Ratio = %
24 25
× 100 %
Percent targeting was calculated by determining ratios of the fluorescence intensity (FI individual)
of ROIs from individual tissue (e.g. left lung) in the numerator, divided by the sum of ACS Paragon Plus Environment
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fluorescence intensity (FI
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denominator (Equation 1), and multiplied by 100. Targeting efficiency L:R ratio was calculated
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using the percent targeting of the left lung in the numerator, divided by the percent targeting of
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the right lung in the denominator.
sum)
of the three ROI areas (trachea, left lung, and right lung) in the
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Iron quantification
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Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to
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measure ferrous iron content in the mouse lungs and trachea. Briefly, tissue was digested with
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0.5mL nitric acid (HNO3) at 105 °C for 1 hour and vortexed until completely dissolved
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(protocol modified from Niazi et al.23). After cooling, the samples were brought up to a known
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volume with MilliQ water. Samples were analyzed with a PerkinElmer Optima 4300 DV ICP-
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OES. The recommended wavelength for iron was used, Fe (II)-λ-259.939 nm. A five-point
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calibration standard range was used that ranged from 0.1 ppm to 25 ppm (mg/L). Endogenous
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iron was analyzed from the lungs of untreated mice (n = 3), and the average of these values was
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subtracted from the treated mice. Calibration and instrument verification samples were
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incorporated before and after analyzing the samples, as well as periodically throughout the
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measurements.
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Equation 2. Iron calculations
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% Targeting =
20
Targeting Efficiency L:R Ratio = %[]
[] []
× 100 %[]
21
Targeting efficiency ratio was calculated by determining ratios of the iron content from
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individual tissue (e.g. left lung) in the numerator, divided by the sum of the total iron content for
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all three tissues (trachea, left lung, and right lung) in the denominator (Equation 2), and
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multiplied by 100. Targeting efficiency L:R ratio was calculated using the percent targeting of
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the left lung in the numerator, divided by the percent targeting of the right lung in the
2
denominator.
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Dye and iron targeting corroboration
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Equation 3. Fluorescence and iron differentials
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Fluorescence differential =
6
Iron differential = [ )*+]
!" #$
!" #$
− &'$(" #$
− [ )*+]&'$(" #$
7
Targeting of dye and iron was corroborated by calculating the differential between the
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fluorescence (relative efficiency) or iron concentrations in left lung compared to the right
9
(Equation 3).
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Statistical analysis
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All statistical analysis was performed using GraphPad Prism statistical software
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(GraphPad Software, San Diego, CA). Paired t-tests with Bonferroni correction were used to
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compare two or more groups of independent data. The difference between variants was
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considered significant if p < .05. For comparisons of more than two groups an ordinary one-way
16
ANOVA was used to determine significance, with Tukey’s multiple comparisons and a Brown-
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Forsythe test used to compare variances. If more than two variables were taken into account, a
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two-way ANOVA was employed with Sidak’s multiple comparison. A post hoc power
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calculation was performed using the free program G*power (Dusseldorf, Germany)24,25. For in
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vivo animal experiments with equivalent sample sizes and an effect size of n = 3, the power was
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calculated to be > 0.95.
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Results
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Dry powder NIMs formulation and characterization
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Dry powder NIMs for pulmonary administration were prepared as described previously
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using lactose as the bulking agent17,26. NIMs were characterized for aerodynamic and geometric
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diameters, surface charge of the SPIONs, morphology, magnetic behavior, iron content, and
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drug loading (Table 1)17. For this study, doxorubicin, a common chemotherapeutic agent with
3
demonstrated activity against lung cancer, was used. NIMs with doxorubicin will be referred to
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as D-NIMs. Table 1. Characterization of SPIONs and NIMs* SPIONs average radius density zeta potential
NIMs
56 ± 6nm 2.5 g/cm3 -49 mV
Concentration (w/w) 1.6% Doxorubicin 10.6% Fe3O4 Laser Diffraction 1.6µm Next generation Impaction MMAD‡ 3.27µm FPF‡ > 90% GSD‡ ± 1.69
*
For more details on the NIMs powder preparation and characterization consult McBride et al [17] For NIMs dry powder ‡ Mass Median Aerodynamic Diameter (MMAD), Fine Particle Fraction (FPF), Geometric Standard Deviation (GSD) †
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In vitro evaluation of chemotherapeutic efficacy
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To assess whether the doxorubicin in D-NIMs retained its cytotoxicity (and therefore its
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therapeutic effect) after spray drying, a MTS assay was utilized to measure cell viability of lung
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adenocarcinoma cells after D-NIMs exposure. We hypothesized that D-NIMs would quickly
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release the doxorubicin and would have similar or increased cytotoxicity compared to free
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doxorubicin in A549 cells. The left panel of Figure 2 compares A549 cells treated with D-
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NIMs, as well as controls consisting of spray-dried lactose, SPIONs alone, and free doxorubicin
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solution. A549 cells were exposed to low, medium, and high doses of free doxorubicin solution
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or D-NIMs for a total of 48 hours (Fig. 2). It is important to note that the concentration of
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doxorubicin was approximately doubled in the free doxorubicin solution control group
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compared to the D-NIMs dry powder group at all three doses. The low concentration of free
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doxorubicin solution (0.03µg) or D-NIMs (0.016µg) showed minimal cytotoxicity, and cells
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were as viable as untreated A549 cells after 48 hours of exposure (Fig. 2A, MTS panel).
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However, medium and high doses of doxorubicin solution and D-NIMs were toxic to 12 ACS Paragon Plus Environment
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adenocarcinoma cells compared to SPION and lactose controls. Importantly, at both medium
2
and high doses, no significant differences were seen between the free doxorubicin and D-NIMs
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groups ((67±1.67)% and (58±0.51)% respectively), despite D-NIMs containing doxorubicin at
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nearly half the concentration of the free doxorubicin group. (Fig 2C, MTS panel).
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Figure 2. MTS viability and LDH cytotoxicity assay of A549 cells exposed to doxorubicin-
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loaded NIMs. MTS (left panel) and LDH (right panel) assay measuring viability and
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cytotoxicity, respectively, of A549 lung adenocarcinoma cells after 48 hours of exposure to A) 13 ACS Paragon Plus Environment
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low dose of D-NIMs compared to free doxorubicin (1µg NIMs containing 0.016µg doxorubicin
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compared to 0.03µg free doxorubicin), B) medium dose of D-NIMs compared to free
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doxorubicin (10µg NIMs containing 0.16µg doxorubicin compared to 0.3µg free doxorubicin),
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and C) high dose of D-NIMs compared to free doxorubicin (100µg NIMs containing 1.6µg
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doxorubicin compared to 3.0µg free doxorubicin). Free doxorubicin control (dark gray), Spray-
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dried lactose control (light gray), SPIONs control (dashed gray), D-NIMs (black); for both MTS
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and LDH panels. Lactose and SPIONs controls were used at the ratios from the spray dried feed
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solution and were held constant relative to doxorubicin. A two-way ANOVA with Sidak’s
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multiple comparison test was used to determine statistical significance. *p
