pubs.acs.org/molecularpharmaceutics
`
`Article
`
`Preparation and Characterization of Novel Magnetic Nano-in-
`Microparticles for Site-Specific Pulmonary Drug Delivery
`†
`†,‡,§
`†,∥
`Amber A. McBride,
`Dominique N. Price,
`Loreen R. Lamoureux,
`Alaa A. Elmaoued,
`and Pavan Muttil*,†,‡,§,∥
`⊥
`#
`Jose M. Vargas,
`Natalie L. Adolphi,
`‡
`†
`Biomedical
`Department of Pharmaceutical Sciences, College of Pharmacy,
`Nanoscience and Microsystems Graduate Program,
`Sciences Graduate Program, §
`#
`The University of New Mexico Cancer Center, and
`Department of Biochemistry and Molecular
`Biology, The University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, United States
`⊥
`The University of New Orleans, New Orleans, Louisiana 70148, United States
`
`†,‡
`
`∥
`
`ABSTRACT: 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.
`KEYWORDS: pulmonary delivery, magnetic microparticles, inhalable dry powders, SPIONs, lung cancer therapy, spray drying
`
`1. INTRODUCTION
`Lung cancer is the leading cause of cancer mortality worldwide,
`with 1.4 million people dying from the disease each year, as of
`2008.1 Lung cancer accounts for more deaths than breast,
`liver and kidney cancers combined.2 In the
`prostate, colon,
`United States alone, 160,000 people died of lung cancer in
`2010.3 Despite the use of new chemotherapeutic agents for
`lung cancer, the average patient five-year survival rate is 5−15%
`and has remained largely unchanged for decades.1
`These statistics are due, in part, to conventional drug delivery
`systems that neither deliver nor maintain sufficient drug
`concentration near solid lung tumors,4 leading to adverse
`effects in healthy tissues. The lung offers a unique and
`challenging route for drug delivery with high absorption and
`surface area, ca. 100 m2.5 Inhaled drug delivery is widely used
`for diseases such as asthma, COPD and cystic fibrosis and has
`shown promise as an alternate delivery method for lung cancer
`
`chemotherapeutics. However, it has been associated with side
`effects.6
`A major unmet medical need in the field of cancer therapy is
`to selectively deliver chemotherapeutic agents to lung tumors.
`The objective is to minimize side effects observed in healthy
`lung tissues as well as to achieve effective therapy. A phase I/II
`study of inhaled doxorubicin combined with oral cisplatin and
`docetaxel-based therapy for advanced non-small cell
`lung
`cancer showed the efficacy of
`inhaled therapy. Although
`seven evaluable patients responded to the combined inhaled
`and oral therapy, dose-limiting pulmonary toxicity was observed
`in two patients due to a lack of drug-tumor targeting.7,8 This
`
`Received: December 22, 2012
`Revised:
`August 19, 2013
`Accepted: August 21, 2013
`Published: August 21, 2013
`
`© 2013 American Chemical Society
`
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`study showed the benefit of delivering agents directly to the
`lung with diminished systemic side effects.
`Researchers have used Fe3O4 superparamagnetic iron oxide
`nanoparticles (SPIONs) as a means to target drugs to specific
`regions of the lung in animal models to mitigate the toxicity
`observed in healthy lung tissues.9−11 If successful, this approach
`will lead to improved tumor targeting, minimize the side effects
`observed in healthy tissues due to the chemotherapeutic agent,
`and maximize the therapeutic outcome. Targeted pulmonary
`drug delivery using SPIONs will also significantly lower the
`total drug dose required to achieve therapeutic response at the
`tumor site, thus further alleviating the side effects observed in
`healthy tissues.
`Previously, Dames, et al.11 and Rudolph et al.12 targeted
`nebulized suspension containing SPIONs and pDNA, a
`therapeutic mimic,
`to specific regions in a mouse lung.
`Although significant deposition of SPIONs was shown in the
`magnetized lobe, separation of pDNA from SPIONs during the
`delivery process was observed. Nebulized formulations used in
`these studies lead to separation of the drug surrogate from the
`SPIONs before reaching the intended target site. We propose
`formulating and characterizing an inhalable dry powder vehicle
`containing SPIONs and a chemotherapeutic agent. This novel
`approach overcomes the natural deposition mechanism of
`inhaled aerosol liquid droplets in the lungs that is limited by
`targeting of aerosols to the central and peripheral airways but
`not to local regions of the lung.
`The objective of this study was to formulate and characterize
`nano-in-microparticles (NIMs) containing SPIONs and the
`chemotherapeutic agent doxorubicin in a lactose matrix. NIMs,
`the first of their kind formulated in a dry powder form, will
`prevent premature separation of the chemotherapeutic agent
`and SPIONs.13
`
`2. EXPERIMENTAL SECTION
`2.1. Materials. Alpha-D-(+)-lactose monohydrate Respitose
`ML-001 was a gracious gift from DMV-Fonterra Excipients
`GmbH & Co. KG (Goch, Germany). FluidMAG-UC SPIONs
`with a hydrodynamic diameter of 50 nm were gifted and
`purchased from Chemicell GmbH (Berlin, Germany). Fluoro-
`Max Green Fluorescent Polymer nanospheres (0.025 μm
`diameter) were purchased from Thermo Fisher Scientific.
`Adriamycin (doxorubicin) was purchased from Selleck
`Chemicals, LLC (Houston, TX). A Gemini-NX 5 μm C18
`110 A 150 × 4.6 mm HPLC column was purchased from
`Phenomenex (Torrance, CA). OmniSolv acetonitrile, anhy-
`drous, was obtained from EMD Chemicals (Gibbstown, NJ),
`and all other reagents were of analytical grade and used as
`received. Borosilicate glass tubes (20 mm × 120 mm) were
`fabricated by Scientific Glass Co. Ltd. (Albuquerque, NM). A
`commercially available neodymium−iron−boron (NdFeB)
`permanent cylindrical magnet (grade N52, 22 mm long × 20
`mm in diameter) was purchased from Applied Magnets (Plano,
`TX).
`2.2. Preparation of Inhalable Magnetic Microparticles.
`NIMs containing fluorescent nanospheres or a chemo-
`therapeutic agent (doxorubicin) were prepared by spray drying.
`A suspension containing 78.2% lactose, 20% SPIONs and 2.8%
`doxorubicin (w/w) in double distilled water (ddH2O) was
`spray dried using a mini-spray dryer B-290 with a standard two-
`fluid nozzle (0.7 mm diameter) (Büchi Corporation, Flawil,
`Switzerland). NIMs were also formulated with a fluorescent
`nanosphere drug-surrogate; the drug was replaced with 4.0%
`
`nanospheres (w/w). Nanospheres were used in the NIM
`formulation, rather than a soluble dye, to avoid redistribution of
`free dye following deposition in unmagnetized regions. Spray
`drying parameters were the same for nanospheres and
`doxorubicin-containing NIMs and were as follows:
`inlet
`temperature 170 ± 2 °C, outlet temperature 103 ± 2 °C,
`aspirator rate 100%, pump flow rate of 10% and air flow rate
`742 L/h. Control lactose particles were prepared by spraying a
`5% lactose solution (w/v) in ddH2O using the above
`parameters. When spray drying doxorubicin and SPIONs, the
`spray dryer was isolated in a walk-in chemical hood that was
`vented to outside air to prevent exposure to toxic vapors.
`Chemotherapeutic-resistant PPE was worn when handling
`doxorubicin and NIMs.
`2.3. Microparticle Characterization. 2.3.1. Yield and
`Encapsulation Efficiency of Doxorubicin. NIM yield was
`calculated as the ratio of the mass of solids collected after spray
`drying to the amount of solids in the feed suspension. The
`percentage encapsulation efficiency (EE) and percentage
`doxorubicin loading in NIMs were determined using eqs 1
`and 2, respectively:
`×
`100
`actual weight of doxorubicin in NIMs
`theoretical weight of doxorubicin in NIMs
`
`% EE
`
`=
`
`Article
`
`(1)
`
`% doxorubicin loading
`actual weight of doxorubicin in NIMs
`=
`NIMs weight
`
`×
`
`100
`
`(2)
`
`2.3.2. SPION Characterization: Hydrodynamic Size and
`Zeta Potential. Hydrodynamic size (D50) was determined
`using dynamic light scattering (Zetasizer Nano ZSP, Malvern
`Instruments Ltd.). The samples were prepared by dispersing 1
`μL [50 mg/mL] of SPIONs in 1 mL of ddH2O (n = 9). The
`zeta potential of SPIONs was characterized by dispersing 0.5
`μL [50 mg/mL] of SPIONs in 1 mL of ddH2O (n = 9),
`expressed as size distribution by average intensity.
`2.3.3. NIM Hydrodynamic Size. The volume median
`the NIMs was determined by laser
`diameter (Dv 0.5) of
`diffraction using the cuvette disperser (Helos/KF-OM,
`Sympatec GmbH, Germany). Briefly, 5.0 mg of NIMs was
`suspended in 1 mL of acetonitrile and gently vortex-mixed. A
`200 μL aliquot of this suspension was added dropwise to the 6
`mL cuvette containing 5 mL of acetonitrile. Measurements
`were taken for 10 s using the R3 lens in triplicate.
`2.3.4.
`In Vitro Aerosolization Studies (Aerodynamic
`Diameter). The aerodynamic size of the spray dried NIMs
`containing dye was determined using a Next Generation
`Impactor (model 170 NGI, MSP Corporation, Shoreview,
`MN). NIM samples (6 mg) were aerosolized using a model
`DP4 dry powder insufflator for rat (Penn Century, Inc., USA).
`A pump (Copley Scientific, Nottingham, U.K.) was operated at
`a flow rate of 30.0 L/min for 10 min. Following aerosolization,
`particle deposition was measured by gravimetric method from
`collection cups. The percent cumulative mass fractions were
`plotted versus log aerodynamic diameters. The mass median
`aerodynamic diameter (MMAD) was estimated by linear
`interpolation that links the curve points at 50% deposition.
`The fine particle fraction (FPF; stage 3 to stage 7, i.e. <6.4 μm)
`was calculated as a percentage of total emitted dose (n = 3).
`2.3.5. NIM Morphology and Cross Sectional Analysis. To
`determine particle size, surface morphology and elemental
`analysis, NIMs were visualized using a high-resolution scanning
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`Figure 1. Tracheal mimic tube study. A tracheal mimic tube was fitted with an inhaler spacer and connected to vacuum pump mimicking breathing
`inspiration, determined by a flow meter at 15 L/min. A neodymium magnet was placed in a region of interest, and formulations of either NIMs or
`liquid suspension were administered.
`
`electron microscope energy dispersive X-ray spectrometry
`(EDS-SEM) (JEOL 5800LV). Briefly, NIMs were deposited
`onto a small piece of silicon wafer using double-sided magnetic
`adhesive tape and examined in the SEM at 0.3−20 kV. Using
`the focused ion beam SEM (FIB-SEM), electron-transparent
`slices of the NIMs were cut to reveal a cross-section of the
`microparticle. For transmission electron microscopy (TEM,
`JEOL 200CX TEM operated at 200 kV), NIMs were uniformly
`suspended in acetonitrile and 10 μL droplets were mounted on
`copper grids covered with holey carbon film. Samples were also
`analyzed by energy dispersive X-ray spectroscopy (EDS) for
`SPION loading in NIMs.
`2.3.6.
`Iron Content Determination. To evaluate concen-
`trations of Fe in NIMs, samples were analyzed using inductively
`coupled plasma atomic emission spectroscopy (ICP-AES,
`Perkin-Elmer Elan II, Waltham, MA). The internal standard
`was iron (Fe) at mass 55.845. The analysis was performed
`comparably to method US EPA 200.8. Exactly 10 mL of the
`standards, samples, and QC samples were spiked with 1 mL of
`100 mg/L Fe as internal standard to correct for drifts in the
`signal that may be caused by sample matrix, viscosity of the
`solution, and/or peristaltic pump (sample) pulsing. The system
`was calibrated using NIST traceable calibration standards
`(stock solution) and QC solutions (stock solution). The system
`is sensitive down to the parts per billion (ppb) level. Three
`milligram NIM samples were taken for measurement and
`digested, using 2 mL of nitric acid, at 90 °C. After digestion was
`completed, digests were brought up to 10 mL final volume and
`transferred into ICP plastic tubes. Results were then calculated
`using the starting weight and the final volume after digestion.
`Results were expressed as μg/g of Fe and then converted to
`ppb based on the standard curve. Fe was calculated based on
`the basis of the molecular ratio of Fe3O4. Samples were run in
`duplicate.
`2.3.7. Doxorubicin Content Determination Using HPLC.
`We adapted an HPLC method reported by Mikan et al.14 and
`Urva et al.15 A stock standard of 65 μg/mL was prepared by
`placing 0.65 mg of doxorubicin into a 10 mL volumetric flask
`and diluted in pH 3.0 ddH2O to a total volume of 10 mL. A
`working standard of 20 μg/mL was prepared by transferring
`appropriate amounts of stock doxorubicin in a 5 mL volumetric
`flask and diluted in pH 3.0 water to a total volume of 5 mL.
`Both stock and working doxorubicin solutions were stored at 4
`°C. To create a standard curve, doxorubicin standards with
`concentrations of 0.078, 0.156, 0.31, 0.63, 1.25, 2.5, 5, 10, and
`20 μg/mL were prepared by accurately transferring appropriate
`volumes of working doxorubicin solution to HPLC vials. NIM
`
`concentrations of 2 and 14 μg/mL were prepared by dispersing
`NIMs into a suspension, separating the SPION component
`using a magnetic gradient, and transferring supernatant to
`HPLC vials. The HPLC system consisted of a 1260 Infinity
`Agilent LC (Agilent Technologies, Santa Clara, CA).
`Integrations, calculations, and plotting of chromatograms
`were performed with a Chemstation computing integrator
`(Agilent Technologies, Houston, TX). A C18 HPLC column
`was used, and the mobile phase was prepared by mixing
`acetonitrile and water, adjusted to pH 3.0 with phosphoric acid,
`at 72/28 (v/v) proportions. Doxorubicin eluted at 7.2 min with
`a flow rate of 1 mL/min. The UV detector was set at a
`wavelength of 254 nm. The HPLC apparatus was operated at
`room temperature.
`2.3.8. SPION Magnetization Measurement. Magnetic
`properties were measured with an MPMS-7XL SQUID
`Susceptometer (Quantum Design, San Diego, CA) integrated
`to a Physical Properties Measurement
`system (PPMS,
`Quantum Design, San Diego, CA). The ac susceptibility
`measurements were conducted in the temperature range from 4
`to 300 K with nominal magnetic field of 20 kOe. These
`measurements allowed us to calculate the magnetic moment of
`the SPIONs as well as the concentration of SPIONs in the
`NIMs.
`2.3.9. Permanent Magnet Characterization. The cylin-
`drical permanent magnet (2.5 cm length × 2 cm diameter) was
`characterized using a Bell-5180 series Hall effect gauss/
`teslameter and an STD18-0404 axial gaussmeter probe (Sypris
`Test and Measurement, F.W. Bell, Orlando, FL). The Hall
`effect gaussmeter was used to measure the flux density (B) at
`increasing distances from the surface of the magnet. A ruler was
`taped parallel to the magnet, and the flux density was measured
`with the axial gaussmeter probe in 1 mm increments from a
`distance of zero to 30 mm from the magnet. Because magnetic
`force is proportional to the magnetic field gradient (ΔB/Δx, in
`units of G/mm), the gradient was determined by dividing ΔB,
`the change in the flux density between each successive
`measurement, by Δx, the change in distance between each
`successive measurement. The magnetic field gradient measure-
`ments were used to indicate the relative strength of
`the
`magnetic attractive force on the NIMs at different positions for
`the subsequent tracheal mimic tube study.
`2.4. Proof of Concept Tracheal Mimic Tube Study.
`Cylindrical borosilicate glass tubes were designed and fabricated
`to mimic the conducting airways of the human respiratory tract.
`The dimensions of the tube were similar to those of a human
`male adult (20 mm diameter × 200 mm length × 1 mm
`
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`Figure 2. Hydrodynamic size and stability characterization of SPIONs. SPIONs are a component of the NIM drug delivery vehicle. (A) The
`transmission electron microscopy (TEM) micrograph shows monodisperse 50 nm diameter single-domain Fe3O4 nanoparticles. (B) Dynamic light
`scattering (DLS) shows a D50 of 56 nm. (C) Zeta-potential shows nanoparticle charge of −49 mV.
`
`Figure 3. SEM images of (A) lactose particles. (B, C) NIMs containing SPIONs, doxorubicin and lactose. (D) A cross-section of a single NIM
`ablated with a focused ion beam. (E) TEM images of electron-dense SPIONs concentrated toward the outer surface of NIMs. (F) A magnified
`version of E showing the distribution of SPIONs.
`
`thickness). Two different formulations were examined: NIMs
`and a nebulized liquid suspension. Both formulations contained
`the green fluorescent nanospheres as a surrogate for
`doxorubicin. Briefly, 5.0 mg of NIMs was weighed for each
`run; for the preparation of the liquid suspension 5.0 mg of the
`NIMs were suspended in 3 mL of water to have equal
`proportions of the nanospheres and SPIONs. The permanent
`magnet was placed on the external vertical axis of the tube to
`achieve retention of the NIMs near the magnet (Figure 1).
`The magnetic retention of
`the NIMs was examined by
`placing the magnet at 0 and 4 mm from the tube based on the
`magnetic strength characterized earlier. These two distances
`were measured from the outer surface of
`the glass tube
`(thickness 1 mm). Aerosol generators (model DP-4 dry powder
`insufflator and model IA-1C liquid suspension microsprayer
`(Penn Century, Inc., Philadelphia, PA, USA)) were used to
`aerosolize the NIMs and the liquid suspension, respectively (n
`= 3) (Figure 1). The glass tube was connected to a vacuum
`source that was adjusted to a flow rate of 15 L/min. Relative
`fluorescence intensity was quantified using the Carestream
`Molecular Image Station 4000 MM Pro using excitation and
`emission wavelengths of 488 and 508 nm, respectively. Student
`t tests were used to quantify statistical significance (Graphpad
`prism, La Jolla, CA).
`
`3. RESULTS
`3.1. NIM Formulation. NIMs were formulated by spray
`drying doxorubicin and SPIONs in a lactose matrix. For the
`purpose of drug delivery and bioavailability to tumors, NIMs
`were characterized on the basis of size, surface morphology and
`magnetic properties. SEM and TEM micrographs indicate
`in shape with a diameter of 1.6 μm
`NIMs to be spherical
`(Figure 3C and Figure 3E, respectively). TEM micrographs
`revealed increased electron dense (black) areas indicating the
`presence of iron atoms. Cross-sectional analysis by TEM shows
`that SPIONs are preferentially distributed on the outer surface
`of the NIMs (Figure 3E). SEM micrographs revealed a rough
`outer surface due to SPIONs protruding from the lactose
`matrix (Figure 3C). The surface area of NIMs may be
`influenced by the extent of the surface roughness. This is an
`important characteristic of dry powers when mitigating drug−
`drug particle agglomeration formation.16
`3.2. Characterization of SPIONs: Hydrodynamic Size,
`Zeta Potential, and TEM. SPIONs had an average radius of
`56 ± 6 nm (Figure 2B) and a density of 2.5 g/cm3. SPIONs
`had an average zeta potential of −49 mV (Figure 2C) and
`confer colloidal stability against aggregation. TEM micrographs
`of SPIONs showed magnetite crystals on the order of 6 nm that
`are combined to form a single-domain 50 nm core (Figure 2A).
`3.3. Yield, Encapsulation Efficiency and Doxorubicin
`Loading. The theoretical powder yield was 60.9% based on a
`
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`Figure 4. Quantification of Fe3O4 in NIMs. (A) Schematic depicting cross-section NIMs and SPION localization. (B) ICP-AES chromatogram of
`Fe3O4 loading in NIMs. (C) EDS/SEM Fe depth analysis. (D) EDS/TEM Fe depth analysis. (E) Hysteresis curve shows superparamagnetism and
`demonstrates the total electromagnetic unit (emu), enabling the calculation of Fe3O4.
`
`Figure 5. A cylindrical glass tube was designed to mimic the conducting airway of the human pulmonary tract. (A) Magnetic gradient
`characterization of a permanent magnet used to direct and retain NIMs to a region of interest. The magnet was characterized over a distance of 30
`mm and fit with the function 1/x3. (B) The targeting and retention of a fluorescent dye with and without a magnet using NIMs. (C) The targeting
`and retention of a fluorescent dye with and without a magnet using a liquid suspension. The magnetic retention of the NIMs was examined by
`placing the magnet at 0 and 4 mm from the tube. These two distances were measured from the outer surface of the glass tube (thickness 1 mm). (D)
`The NIMs formulation is 2.4 times more fluorescent in a magnet region of interest (p = 0.0154).
`
`mass balance performed on the solids (SPIONs, doxorubicin,
`lactose) and NIMs before and after spray drying, respectively.
`Using HPLC, % EE was quantified in NIMs to be 57%.
`Doxorubicin eluted with a retention time of 7.2 min.
`Theoretically, NIMs contained 2.8% (w/w solids) doxorubicin,
`and actual doxorubicin loading was quantified to be 1.6%. The
`doxorubicin loss is attributed to the spray drying process; small
`particles of doxorubicin (which have a characteristic red color)
`were visually observed trapped in the filter of the machine.
`3.4. NIM Characterization. The average hydrodynamic
`NIM diameter was 1.6 μm using laser diffraction. This size
`correlates with the size assessed using SEM (Figure 3B). A
`MMAD of 3.27 μm was obtained using a NGI. The
`aerodynamic diameter takes the density of the NIMs into
`
`account and is fundamental to particle deposition in the lung
`and hence for inhaled drug delivery. The average fine particle
`fraction (FPF) (≤6.4 μm) of the NIMs was >90%, and the
`geometric standard deviation (GSD) was ±1.69.
`3.5. Permanent Magnet Characterization. Single and
`two combined permanent magnets were characterized for their
`magnetic field strength. The magnetic field strength on the
`surface of the single magnet was 0.58 T. Magnetic field strength
`did not increase when two permanent magnets were combined
`(data not shown). Magnetic field lines are parallel to the surface
`of the magnet and diverge with increasing distance, resulting in
`a weaker flux density at larger distances; the magnetic force acts
`on the NIMs when they enter this region of diverging magnetic
`field. The direction of the force moves magnetic objects from
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`regions of lower flux density to regions of greatest flux density
`(i.e., the surface of the magnet). This study also shows that the
`geometry and orientation of the magnet is critical for significant
`deposition of the NIMs near the magnet.9 Figure 5A shows that
`the magnet was characterized over a distance of 30 mm with
`decreasing magnetic gradient strength (G/mm).
`3.6. Magnetization Measurement. A SQUID is a
`sensitive magnetometer that is used to measure extremely
`small magnetic fields, and with this device we measured the
`magnetic moment of NIMs. Susceptibility curves were obtained
`to provide the specific magnetization (s) in emu/g at a
`particular magnetic field strength. This is related to the
`susceptibility through the equation χ = (s*ρ)/H where ρ is the
`particle density. The zero coercivity and the reversible
`hysteresis behavior indicate the superparamagnetic nature of
`the SPIONs (Figure 4E). The specific saturation magnetization
`for the NIM formulation was 8 emu/g. This value is below the
`saturation magnetization known for bulk Fe3O4 (Ms = 92 emu/
`g) as expected given that lactose does not contribute to the
`magnetic moment. The magnetization measurement suggests
`that the NIMs are approximately 9% Fe3O4 by weight. The
`magnetization was corrected by the diamagnetic response of
`the sample-holder, and normalized by sample mass.
`Using ICP-AES, elemental Fe in NIMs was 7.67%. When
`converting this to magnetite, Fe3O4 was calculated to be 10.6%
`by weight, in good agreement with the Fe3O4 fraction obtained
`from the magnetization measurements (9%). EDS-SEM and
`EDS-TEM quantified Fe on the NIM surface, and Fe3O4 was
`calculated to be 16 ± 0.5% and 19 ± 0.5%, respectively (Figure
`4C,D). These numbers are further understood knowing that
`EDS-TEM has a depth resolution of 50 nm whereas EDS-SEM
`penetrates an average sample depth of ∼1 μm.17 TEM
`micrographs (Figure 3E) qualitatively supported this observa-
`tion that SPIONs are less densely distributed/localized in the
`center of
`the NIMs. Thus EDS-SEM quantitates a lower
`amount of Fe3O4 given this analytical tool penetrates the
`sample more deeply than EDS-TEM.
`3.7. Proof of Concept Tracheal Tube Study. Magnetic
`retention of NIMs containing fluorescent nanospheres was
`quantified in relation to its fluorescence intensity (Figure
`5B,C). Quantitative fluorescence analysis showed a 2-fold
`increased deposition of aerosolized NIMs at 0 mm (mean =
`534.97, SD = 131.17) near the magnet based on greater
`fluorescence intensity, compared to the aerosolized liquid
`formulation at 0 mm (mean = 221.67, SD = 26.70) (t = 4.054,
`p = 0.0154, df = 4, two-tailed Student t test) (Figure 5B,C,D).
`Control studies of untargeted NIMs (absence of magnetic
`targeting) qualitatively show no mean fluorescence over
`background in the region of
`interest (Figure 5B,C).
`Quantitative fluorescence analysis showed increased deposition
`of NIMs at 4 mm (mean = 385.65, SD 30.15), compared to the
`aerosolized liquid formulation (mean 277.30, SD = 88.16)
`(Figure 5D). A 28% decrease in the fluorescence intensity of
`NIMs was observed when the magnet was placed 4 mm away
`from the tube compared to 0 mm.
`
`4. DISCUSSION
`This research proposes the use of regional chemotherapy by the
`pulmonary route with the intent of increasing drug exposure
`near a solid tumor. Despite inhaled drug delivery being used for
`respiratory diseases for over 30 years,18 targeted drug delivery
`to specific regions of
`the lung has not been adequately
`explored. We show here, for the first time, the formulation of
`
`Article
`
`magnetic NIMs containing SPIONs and doxorubicin, by the
`process of spray drying. NIMs can be guided to a region of
`interest in a tracheal tube with strategic placement of an
`external magnet. Inhalable dry powders will allow higher doses
`of drug to be delivered to cancerous lung regions without
`increasing side effects observed in surrounding healthy tissues,
`compared to a liquid suspension. Using this novel delivery
`method, we do not expect
`to overcome oropharyngeal
`deposition. This delivery method will only localize drug near
`the target region that does not impact the upper regions of the
`respiratory tract.
`4.1. Formulation of a Novel Inhalable NIMs Delivery
`Vehicle. NIMs were formulated using pulmonary-compatible
`lactose rather than a biodegradable polymer. This is to allow for
`the immediate release of drug from the vehicle after deposition
`and to minimize the patient contact time with the magnetic
`field. After NIM deposition at the target site, the NIM will
`disintegrate quickly in the lung parenchyma to release drug that
`will diffuse into the target tumor mass. Neither doxorubicin nor
`SPIONs are conjugated to the delivery vehicle thus eliminating
`any limitations on the release of bound drug near the target site.
`4.2. Characterization of NIMs. NIMs exhibit a rough
`outer surface, as seen from the SEM images (Figure 3B,C).
`This facilitates a beneficial disaggregation of particles when
`administered as a dry powder.16 Currently, dry powder flow
`and dispersion are improved by incorporating larger particles
`(50−100 μm) as carriers19 to facilitate disaggregation of dry
`powders during inhalation. Should NIM flow or dispersion be a
`problem, the addition of carrier particles would be considered.
`The flow dynamics of the NIMs after inhalation can also affect
`the drug deposition and would require further studies. A logical
`next step, and our next pursuit, will involve the administration
`of NIMs
`in animal models
`to investigate the targeting
`capabilities in the upper and lower respiratory tract.
`NIMs can be designed to modulate drug release after the
`particles have been guided to the targeted lung regions in the
`future. If this concept is successful, NIMs can also be used to
`deliver doublet chemotherapeutic agents since doublet therapy
`is the cornerstone of treatment for lung cancer. Future studies
`also need to investigate the effect of SPIONs on cellular uptake
`and pulmonary toxicity in animal models,
`since surface
`properties of SPIONs are known to affect the cellular uptake
`and cytotoxicity. However, the biodegradability and biocompat-
`ibility of SPIONs have been proven for many years in the
`clinical setting as a contrast agent
`in magnetic resonance
`imaging.20,21
`4.3. Magnetic Properties of Aerosolized NIMs. The
`small magnetic moment of individual SPION in an aerosolized
`liquid droplet cannot be guided easily in the presence of an
`external magnet. However, when SPIONs are assembled
`together in NIMs,16 the net magnetic moment of aggregated
`SPIONs is large enough to be manipulated with a medically
`compatible external magnet. In addition, droplets containing
`nanosized SPIONs for inhaled drug delivery are easily exhaled
`due to their small size; therefore encapsulating the SPIONs into
`micrometer sized NIMs will help to resolve this problem. The
`combined magnetic moment of the SPIONs present in the
`NIMs may allow the particles to be retained at the target site
`until the NIMs disintegrate therefore releasing the drug. In
`addition, NIMs could also be retained at the target site by
`entrapment from lung mucus and cilia.
`A 28% decrease in the fluorescence intensity of NIMs was
`observed when the magnet was placed 4 mm away from the
`
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`Molecular Pharmaceutics
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`tube compared to 0 mm. This decrease in fluorescence, and
`therefore magnetic force,
`is a challenge that needs to be
`addressed since the magnetic field has to penetrate a larger
`distance if this targeting mechanism is to be applied in humans.
`Since the magnetic gradient decreases with the distance to the
`target tissue, the main limitation of this delivery mechanism
`could relate to the strength of the external field that can be
`applied to the patient
`to obtain the necessary magnetic
`gradient; however, high field gradient electromagnets are being
`used in animals for magnetic targeting22 and substantial field
`gradients are also used in MRI imaging. Further studies are
`needed to validate the safety of the strength of an external
`magnet as it relates to magnetic nanoparticles.
`The magnetization s-curve of NIMs loaded with 9% w/w
`Fe3O4 is shown (Figure 4E) and has negligible coercivity, and
`conseque