preparation of spherical monodisperse ferrimagnetic iron-oxide microparticles between 1 and 5 μm...

*Corresponding author. Tel.:#49-89-893237-19; fax:#49-89-893237-11.E-mail address: [email protected] (W. MoK ller).

Journal of Magnetism and Magnetic Materials 225 (2001) 8}16

Preparation of spherical monodisperse ferrimagnetic iron-oxidemicroparticles between 1 and 5 �m diameter

Winfried MoK ller��*, Gerhard Scheuch�, Knut Sommerer�, Joachim Heyder�

�GSF * National Research Center for Environment and Health, Institute for Inhalation Biology, Robert Koch Allee 6,D-82131 Gauting, Germany

�InAMed * Institute for Aerosol Medicine GmbH, Robert Koch Allee 6, D-82131 Gauting, Germany

Abstract

The production of spherical monodisperse iron-oxide microparticles in the size range between 0.8 and 5�m isdescribed. The particles can be ferrimagnetic (Fe

�O

�) or non-magnetic (�-Fe

�O

�). The particles were radiolabeled with

��Tc or ��In, and the leakage of the radiolabel within 24 h was 0.1% in the human lung. The particles can be used tostudy the motility and the integrity of living cells. � 2001 Elsevier Science B.V. All rights reserved.

Keywords: Iron-oxide particles; Spherical; Monodisperse; Ferrimagnetic; Spinning top aerosol generator; Nebulization; Technetium-99m (Tc-99m); Radiolabeling; Magnetite; Preparation; Indium-111 (In-111); Lung imaging; Cell motility; Alveolar clearance

1. Introduction

Ferrimagnetic iron-oxide microparticles havebeen typically used for recording materials [1], butthese particles are also very useful as tracers forinvestigating the behavior of air-borne matter inthe human and animal respiratory tract [2}4], andthe mechanical properties of living cells [4,5]. Ironoxide is chemically stable and is not toxic andnon-carcinogenic [6]; therefore, it allows clearancestudies in the human lungs over time-periods up to1 year. The maximum working-place concentra-tion of respirable iron-oxide dust is 4 and1.5mg/m� for dust penetrating into the alveolarregion of the lungs [7]. Welders are exposed to

relatively high concentrations of particulateiron oxide, where burdens up to several thousandmilligrams could be detected [8,9]. This ma-terial is deposited and recovered in the lungs overmonths and years.Ferromagnetic and ferrimagnetic particles can bedetected in the human body by magnetopneumo-graphic (MPG) methods [4,10]. Ferrimagneticparticles were also used for measurements of mac-rophage functions and cellular integrity (visco-elas-ticity) in vivo and in vitro [4,5]. External magnetictwisting forces allow to study mechanisms that arerequired for the motility of animal cells [11].Ligands can be coupled to the magnetite particlesin order to attach them to speci"c cell membranereceptors, such as integrins [12,13]. Radiolabeledparticles are most applicable for studying short-time mucocilliary clearance in human and animallungs. All these studies require spherical monodis-perse test particles.

0304-8853/01/$ - see front matter � 2001 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 8 8 5 3 ( 0 0 ) 0 1 2 2 1 - X

Iron-oxide particles in the micrometer size rangecan be produced by either a crystallization process[14] or by nebulization of a colloidal solution [15].Particles obtained by crystallization are smallerthan 2�m. During crystallization the particles tendto form aggregates, which cannot be redispersed. Ininhalation studies, the aggregates show di!erentbehavior from single particles and deposit in di!er-ent sites in the lungs. For this reason the techniqueof nebulization of an aqueous solution was prefer-red, because it allows a direct voluntary inhalation.Two systems are available, the vibrating ori"ce andthe spinning top, which can both form monodis-perse droplets in the micrometer size range. Undernormal conditions the nebulization techniqueyields spherical particles. The primary particles areporous, but they can be sintered in a furnace, yield-ing particles with high chemical stability. A furtheradvantage of the nebulization technique is thatcolloids of di!erent compositions can be mixed.This allows labeling the iron-oxide particles withcolloids of radiotracers ��Tc, ��In or ��Au. Thespinning top nebulization technique is easy to con-trol and can be run over several hours unlike thevibrating ori"ce, since the ori"ce tends to clogdue to impurities. The composition of iron oxidecan be changed from �-Fe

�O

�(hematite, red) to

ferrimagnetic Fe�O

�(magnetite, black) and back

to ferrimagnetic �-Fe�O

�(maghemite, brown) by

using speci"c carrier gas compositions and temper-atures [1,16,17].

2. Materials and methods

2.1. Chemical preparation

The preparation of iron-oxide particles requiresseveral experimental steps: "rst, a non-magnetic�-Fe

�O

�colloid was produced by hydrolysis of

FeCl�[18]. Four millilitres of a 32wt% FeCl

�solution were prepared in deionized water. Thissolution was added slowly (over 20min) to 250mlof boiling deionized water while stirring, and aninsoluble Fe(OH)

�colloid was formed spontan-

eously. In order to remove the chloride ions, thesolution was repeatedly washed by dialysis.A chloride-free colloidal iron hydroxide solution

was obtained, which was stable over several monthwhen stored at 43C. Iron hydroxide can be assumedto be a representation of iron oxide: 2Fe(OH)

�"

Fe�O

�#3H

�O.

2.2. Improved spinning top assembly

An air-driven spinning top STAG MK I (Re-search Engineers Ltd., London, UK) [19] was usedin this study. The spinning top has 2.3 cm diameterand was driven by compressed N

�at a constant

pressure of 1.7�10�Pa (0.7 bar). The rotationalspeed of the top was about 1000 rotations/s. Thecolloidal solution was fed at 30ml/h through a hy-podermic needle to the center of the rotating topand propelled along the surface of the top to theedge by centrifugal force. At the edge, droplets areformed when the centrifugal force acting on theliquid overcomes the surface tension of the liquid.The droplets leave the top tangentially at highspeed, and can be picked up by the main air-streamafter reaching the stopping distance R

, which can

be approximated as [19]

R(cm)&0.25�D

�(�m), (1)

where D�is the diameter of the droplet. A salt

solution of known concentration was used to deter-mine the mean diameter of the primary droplets,which was 27.5�m. The stop distance of the maindroplets was 7 cm. About 10% of the nebulizedsolution forms smaller droplets (satellites) that areone-fourth of the main droplet diameter. UsingEq. (1), the satellite's stopping distance is approx-imately 1.75 cm. Because of the air-driven rotor,a negative pressure rises near the rotor edge, aspir-ating air from the main air-stream. This compensa-tion #ow (60 l/min) catches the satellite's dropletsand removes them into the satellite exhaust.Fig. 1 shows the arrangement of the air-drivenspinning top in the generator assembly. The com-pensation #ow enters from top between the stopdistance of the satellite and the primary droplets.This ensures a complete removal of the satelliteswhile allowing all primary droplets to be kept in themain air stream (dilution #ow). The N

�gas con-

sumption was reduced by a feed-back of the satel-lite exhaust into the compensation #ow channel.

W. Mo( ller et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 8}16 9

Fig. 1. Diagram of the spinning top device with gas and liquid supply and feedback system for capturing of the satellite droplets. Thedrying column was prepared from copper to reduce particle losses due to electrostatic charge buildup on the particles.

This feedback line consists of an oil-free pump,a silica-gel drying column and a "lter. With thisfeedback system a closed gas circuit was built,where only the output #ow of the STAG needs tobe provided. A 50-l bottle with compressed N

�(200�10�Pa, 200 bar) was su$cient for 4 h of runtime.The drying funnel had a volume of 10 l. In thisvolume, the mean residence time of the droplets isapproximately 10 s, which is su$cient for completeevaporation of the solvent [20]. During nebuliz-ation of the aqueous liquid, a considerable amount

of charge is produced on the droplets, which caninduce high wall losses. To prevent this, the dryingfunnel was prepared from copper and the entiresystem was grounded. At the output of the funnela radioactive Americium source (� emitter) wasmounted, which maintained an equilibrium chargedistribution of the aerosol.

2.3. Concentration and reduction stage

A one-stage virtual impactor was used to concen-trate the aerosol and to reduce the #ow rate from

10 W. Mo( ller et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 8}16

Fig. 2. (A) Arrangement of the centripeter for increasing the particle number concentration and reducing the #ow of the aerosol. Theconcentrated aerosol was mixed with the reducing gas and fed through the furnace at 8003C, where the particles are changed into theferrimagnetic form of iron oxide. (B) Detailed view of the aerosol concentrator design as a virtual impactor.

40 l/min to 1 l/min (Fig. 2a). This was necessary forincreasing the residence-time in the reduction stage(furnace) and to facilitate "lter collection. The vir-tual impactor is shown in Fig. 2b and consists ofa nozzle and an ori"ce, both having a mean dia-meter of 2.3mm. The nozzle and ori"ce are ar-ranged coaxially at a distance of 0.5mm. Theaerosol #ow of 40 l/min through the nozzle stronglyaccelerates the particles. Due to their inertia, theparticles penetrate into the ori"ce, while the major-ity of the gas (39 l/min) is separated radially into theslit between nozzle and ori"ce by a vacuum pump.The diameter of the ori"ce and the nozzle wasoptimized for particles with an aerodynamic dia-meter between 2�m and 5�m. Behind the ori"cethe particles are concentrated 20-fold to a #owrate of 1 l/min. A second concentrator device with

a 4mm diameter of ori"ce and nozzle was construc-ted for concentrating larger particles.The �-Fe

�O

�was changed to ferrimagnetic

Fe�O

�by adding H

�gas as a reducing agent and

by heating the material to 8003C [17]

3�Fe�O

�#H

�P2Fe

�O

�#H

�O. (2)

While Fe�O

�,FeOFe

�O

�, one-third of the iron

is reduced in this process. It was necessary to pro-duce the particles in an oxygen-free atmosphere, sothe generator was driven with compressed N

�. The

reducing agent was added as a gas mixture, con-taining 98% N

�and 2% H

�. All gases used had

a high purity of at least 99.999%. The reductiontook place in the furnace at 8003C. Behind thefurnace, the particles were collected on "lters orused for direct inhalation studies.


Table 1Solubility of magnetite particles (in %/day) and leakage of ��Tc from iron oxide particles (after 3 h to 24 h) in di!erent solvent

Solubility LeakageSolvent Fe

�O

��Tc-�-Fe

�O

��Tc-Fe

�O

�

Physiological NaCl 0.11%/day 12% 0.8%Deionized water 0.18%/day 14% 1.4%1MHNO

�11.5%/day 14% 0.6%

Human lung 0.8%/day� 2% (0.1%

�This solubility was estimated from long-term clearance measurements with mean half time of 120 day for healthy non-smokers [4].

2.4. Radiolabeling of the colloid

The radioisotope ��Tc is used extensively innuclear medicine. The half-life of 6 h, and the emis-sion of �-rays of 140 keV make this isotope veryuseful for human medical diagnostics. The detailedprocedure of radiolabeling of the iron oxide colloidis described by Wales et al. [21]. A stock solutionof SnCl

�in 0.1NHCl and ��Tc in physiological

NaCl (sodium pertechnetate) was used. Under N�-

atmosphere this solution was mixed and repeatedlypressure dialyzed. Fifteen milliliters of the solutionwere "lled into an Ultrasart X pressure "ltrationunit (Sartorius, GoK ttingen, Germany) and dialyzedto 7ml. Then deionized water was added to theinitial volume. This dialysis procedure was repeat-ed 15 times until all chloride-ions were removed.

2.5. Coating of magnetite particles

For the investigation of the cytoplasmic mechan-ics of living cells the magnetite particles must bemechanically coupled to the cytoskeleton. In orderto attach the particles to speci"c ligands on the cellmembrane, the particles have to be coated withantigens for these ligands. The mechanical couplingbetween the cell membrane and the cytoskeleton ispartially mediated by integrin receptors [11,12].For example "bronectin with the sequence RGD(Arg}Gly}Asp) is a speci"c ligand for the �

��-

integrin receptor. The coating of the particles with"bronectin or collagen I was done in a 50mMcarbonate bu!er at pH of 9.4. The pH was adjustedwith acetic acid. After "ltering, the bu!er can bestored at 43C. One milligram of particles was addedto 1ml of carbonate bu!er containing 50�g of the

protein. This mixture was rotated at 43C for 24 h.The coated particles can be stored at 43C for manyweeks. Before use in the cell assays, the particleswere washed twice with PBS-bu!er and then redis-persed in the required medium.

2.6. Characterization of the particle properties

The aerodynamic particle diameter was deter-mined in a sedimentation cell, while the geometricdiameter was measured by electron microscopy.The e$ciency of the aerosol-generator and the con-centration system was measured with a laser-aerosol-spectrometer (LAS), working in the for-ward scattering mode [22]. The scattered lightintensity of the particle, a measure of the particlediameter, was ampli"ed by a photomultiplier andclassi"ed using a multi-channel pulse-height ana-lyzer (PHA). The remanent magnetic properties ofthe produced particles were measured with theMPG-system [4]. The particles were dispersed inepoxy to prevent aggregation. The probe was re-peatedly magnetized in increasing magnetic "elds,B, ranging from 2 to 200mT (Fig. 5) and theremanent magnetic moment of the probe wasmeasured by a SQUID sensor (superconductingquantum interference device).For testing the chemical stability, particles werecollected on a "lter in the form of a "ltrate sand-wich [23]. This sandwich was stored in physiolo-gical NaCl solution, in deionized water and in1MHNO

�(Table 1). The loss of magnetic moment

of the "lter was analyzed with the MPG-systemover a period of 155 days. The stability of theradiolabel in the iron-oxide particles was investi-gated by leaching tests either in vitro in di!erent


Fig. 3. (A) The aerodynamic particles diameter of �-Fe�O

�particles after the STAG and of Fe

�O

�after concentration and

chemical reduction. (B) The geometric diameter and particlenumber concentration measured with the laser aerosol spec-trometer LASF (PHA: pulse height analyzer, l.a.: left axis, r.a.:right axis).

solvents, or in vivo after inhalation by human vol-unteers. For the in vitro test, non-magnetic(�-Fe

�O

�) and magnetic (Fe

�O

�) particles were

collected on a "lter as they exited the 8003C fur-nace. The radiolabeled particles were incubated inphysiological NaCl solution, in deionized waterand in 1MHNO

�. After 3, 8 and 24 h of incubation

the particles were separated from the solution bycentrifugation and the activity was analyzed in thesolution and in the particles using a gammacounter. For in vivo tests, the particles were inhaledand the urine of the subjects was collected andanalyzed with a gamma counter.

3. Results and discussion

3.1. Performance of the aerosol generatorand particle shape properties

A driving pressure of 0.7 bars and a liquid supplyof 30ml/h are the optimal running conditions forobtaining the lowest particle size variance and thehighest output yield [15]. The particles were foundto be compact spheres with a rough surface, indic-ating a porous structure. Fig. 3a shows the aerody-namic diameter of the �-Fe

�O

�particles, collected

from the STAG, and of Fe�O

�particles, collected

after reduction in the 8003C furnace. The concen-tration and reduction steps do not change the aero-dynamic properties. Passing the aerosol throughthe centripeter causes a loss of smaller and biggerparticles with a reduction of the geometric standarddeviation from �

�"1.14 to �

�(1.1. The measure-

ments with the LAS indicate a reduction of thegeometric diameter of the �-Fe

�O

�particles after

passing the concentrator and the 8003C furnace(Fig. 3b). The LAS measurements of the magnetiteparticles suggest an additional diameter reductioncompared to �-Fe

�O

�, which could not be veri"ed

in the sedimentation cell. The apparent diameterreduction may be caused by a change of the opticalproperties of the particles from �-Fe

�O

�(red) to

Fe�O

�(black). A comparison of the aerodynamic

and the geometric diameter was used to estimatethe particle density. �-Fe

�O

�particles originating

from the STAG had a density of 3.2 g/cm� [15].When passing through the 8003C furnace, the geo-

metric diameter of the particles decreased, whichre#ects a further removal of crystal water togetherwith an increase of the particle density to 4.9 g/cm�,which is only slightly lower than the density of bulkiron oxide (5.2 g/cm�).


Fig. 4. Relative remanent magnetic moment, m(B)/m��, of Fe

�O

�particles embedded in epoxy in increasing external magnetizing "elds

B together with the model "t described in Eq. (3).

A high performance of the STAG requires a con-tinuous #ow of the #uid over the rotating top.A sensitive system to adjust the height of the needleover the top was constructed. The housing over therotating top was prepared from Plexiglas in orderto allow a visual control of the spreading of the#uid over the top. With a liquid feed of 30ml/h andan output aerosol #ow rate of 40 l/min, the max-imum e$ciency of 100% corresponds to a particlenumber concentration of 1200/cm�. After runningfor 30min, a constant, high performance of thegenerator was achieved with an output concentra-tion near 1000 particles/cm�, corresponding to ane$ciency of more than 80%. The particle numberconcentration of the "nal magnetite aerosol isabove 10,000/cm�.

3.2. Magnetic properties

The saturation magnetic moment m��, related to

the mass of material, was S�

+6Am�/kg. Thecourse of saturation was approximated by:

m(B)

m��

"�� tanh

ln (B/B��)

ln b�

#1�, (3)

where B��is the external "eld, producing 50%

saturation and b�describes the gradient at B

��. The

"t of the model (Eq. (3)) is shown in Fig. 4 withparameters B

��"60.3$1.2mT and b

�"1.82$

0.07. Commercially available polydisperse magnet-ite dust (mass median diameter 0.4�m, geometricstandard deviation �

�+1.8, Ventron GmbH,

Karlsruhe, Germany) has model parameters ofS�+9Am�/kg, B

��"37.1$1.1mT and b

�"

2.11$0.03. Ferromagnetic particles used for re-cording materials have an ellipsoidal or "ber-likeshape (acicular). This shape causes a high shapeanisotropy and thereby is responsible for a highremanent magnetic moment. The use of the par-ticles as rheological markers in cell studies and theapplication of models require spherical particles,which have no shape anisotropy. In this case, crys-tal anisotropy can stabilize the remanent magnet-ism. Because the particles being produced by thenebulizing technique consists of many smallersubunits, we expected only low remanent magneticproperties. Fig. 5 shows the remanent magnetiz-ation curves for the 1.4�m diameter sphericalmonodisperse magnetite particles (left graph) incomparison with the polydisperse magnetite par-ticles (right graph), which had been produced bycrystallization and have a cubic shape. Prior tomagnetization both probes were embedded in ep-oxy in order to prevent particle rotation andaggregation. The speci"c saturation magnetic


Fig. 5. Magnetization curve and coercive "eld of 1.4 �m dia-meter spherical magnetite particles (A) compared to polydis-perse commercial magnetite particles (B).

moment of 6.3Am�/kg for the spherical particleprobe and of 9Am�/kg for the polydisperse particleprobe is smaller compared to the moment of syn-thetic magnetite particles (42Am�/kg) [1]. Thelower values originate from the multi-domain be-havior and the spherical shape. From the graphs,we can estimate the coercive "eld of H

�"

40 000A/m for the spherical particles and ofH

�"24 000A/m for the polydisperse particles. The

coercive "eld and the remanent saturation magnet-izationM

�can be used to estimate the anisotropy

constant K�for spheres from [1]

�K��"

H�M

a, (4)

where K�"7.5�10� J/m� for the spherical par-

ticles and as K�"2.1�10� J/m� for the polydis-

perse particles (a"0.64 for random arrays anda"2 for aligned particles) in comparison toK�"1.1�10� J/m� for synthetic magnetite par-

ticles [1]. In our measurements, the particles andtheir crystalline axes are randomly oriented.Additionally, the spherical particles have to beconsidered as multi-domain particles, becausethey were originally produced as a paramagneticiron oxide, without any particle alignment duringthe aggregation process. As a result, these particleshave no macroscopic crystalline structure and lacka preferred direction of magnetization.

3.3. Chemical stability of the particles and of99mTc radiolabeling

The use of the particles for alveolar clearancemeasurements required a high chemical stability inbody #uid. The data in Table 1 show that theparticles are very stable in physiological NaCl-solution and in deionized water. Even in1MHNO

�, the particles can resist degradation for

several days. The estimated rate of dissolution inthe human lungs from long-term clearancemeasurements results in half times of 120 days[4]. In this estimation, the fraction of mechan-ical clearance is neglected and it is assumed that allparticles are removed by dissolution in macro-phages.The results of the leaching tests of ��Tc from theiron oxide particles are summarized in Table 1. Themaximum leakage was achieved after 3 h and didnot change thereafter. Because of the di!erences inthe ��Tc leakage, inclusion of the radiolabel isdi!erent between Fe

�O

�and Fe

�O

�particles. The

chemical reduction to Fe�O

�appears to melt the

particles, which decreases in porosity. This im-proves the inclusion of the radiolabeled colloidalunits and further increases particle stability. Themaximum leakage of less than 1% makes the��Tc radiolabeled magnetite particles very useful as


Fig. 6. SEM image of 1.4 �m magnetite particles on top ofa J774A.1 mouse macrophage (bar"10�m). The inset showsa TEM image of a phagocytized particle within a phagosome.

tracers for lung deposition and clearance studies[24,25].

3.4. Application in cell-magnetometry

Fig. 6 shows the application of the magnetiteparticles in cell-magnetometry, where the ferrimag-netic particles are used as a probe for the investiga-tion of phagocytosis, phagosome motions andcytoskeletal integrity. The SEM view shows themagnetite particles on top a J774A.1 mouse macro-phage. The particles have a smooth spherical shapewith few irregularities. The inset of Fig. 6 showsa scanning electron microscopic image of a spheri-cal particle within a phagosome of a J774A.1 mac-rophage. A characteristic feature of these particlesis that the membrane of the phagosome is in closecontact with the particle surface [5]. Twisting ofthe particles within the cells by magnetic forcesrotates the whole phagosome and allows an invest-igation of the mechanical coupling of the phago-somes to the cytoskeleton. Because of the sphericalshape of the particles, mathematical models can beapplied and qualitative results of mechanical cellproperties and cell rheology can be given [26]. Inaddition, the site of deposition of the particlescan be controlled in inhalation studies because of

the monodispersity. Also, the quantitative resultsabout the clearance mechanisms form the di!erentsites of the lungs can be obtained because of theparticles' chemical stability. Therefore, the particlesare very useful in inhalation diagnostics and biolo-gical cell research.

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preparation of spherical monodisperse ferrimagnetic iron-oxide microparticles between 1 and 5 μm...

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