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Chamber for testing metered-dose propellant-driven aerosols of immunologically relevant proteins
JOURNALOF IMMUNOLOGICAL METHODS ELSEVIER
Journal of ImmunologicalMethods 176 (1994) 203-212
Chamber for testing metered-dose propellant-driven aerosols of immunologically relevant proteins Alan R. Brown
a,,, J o h n A . P i c k r e l l b
a Department of Pathology and Microbiology, Collegeof Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA b Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA
Received 20 May 1994; revised 5 July 1994; accepted 6 July 1994
Abstract
A small aerosol chamber was developed for testing and delivery of aerosols of immunologically important proteins to the respiratory tracts of rodents. The chamber was designed to accommodate the small aerosol volumes produced by metered-dose propellant-driven aerosol canisters. Metered bursts of protein aerosols released into the chamber could be sampled for their particle sizes or used to expose the noses of up to six mice to the aerosols. The chamber consisted of a polyethylene tank with two removable plexiglass end plates. One end plate accommodated the propellant-driven, metered-dose, aerosol vial. The other end of the tank was fitted with a plate accepting aerosol sampling devices or a plate containing mouse restrainers. Uniform concentrations of aerosolized proteins were obtained at different positions in the chamber when sampled for particles of respirable size. Respirable-sized protein particles produced by propellant-driven aerosols ranged from 5 to 50% of total aerosolized protein. Propellant-driven aerosols of proteins released in the chamber produced aerosol particles equivalent to 15-26 Izg of total protein exposure to the respiratory tract of each mouse. The chamber permitted aerosol releases without risk of operator exposure. This aerosol chamber will permit the testing of protein aerosols for their immunologic consequences to the respiratory tract. Potential proteins for testing in this device include immunizing vaccine antigens, immunomodulating cytokine proteins, and passive antibody aerosol therapies against respiratory infections. Keywords: Aerosol; Propellant; Protein; Immunomodulation; (Lung); (Respiratory)
I. Introduction
I m m u n e responses in the respiratory tract are of great importance for both protection against infections of this system (Welliver and Ogra, 1988; Lipscomb, 1989) and for their involvement in respiratory allergies and asthma (Platts Mills et
* Corresponding author.
al., 1991; Corrigan, 1992). Effective targeting of immunomodulating reagents to the respiratory tract could be of benefit in increasing local immunity to respiratory pathogens or decreasing immune-mediated respiratory pathology. Metered-dose inhalers (MDI) are aerosol-generating devices that use the pressure and flashing of volatile propellants to deliver measured quantities of aerosolized drugs into the respiratory tract (Wolff and Niven, 1994). They particularly
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00192- 8
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have been used to deliver anti-asthmatic treatments (Matthys, 1990). We are interested in metered-dose propellant-driven aerosols as a means of delivering vaccine antigens, cytokines, or other therapeutic proteins to the respiratory tract. To produce and test such aerosols, we needed a chamber for testing the small aerosol volumes produced by metered-dose, propellant-driven, aerosol canisters while accommodating both aerosol sampling devices and aerosol exposures to rodents. We also needed a chamber in which aerosol flows could be small because we wanted to produce aerosols of potentially expensive proteins (i.e., cytokines). Unfortunately, most described aerosol chambers have been fabricated for testing large-volume aerosol particle outputs of 6 or more liters/minute (1/min) associated with jet or ultrasonic aqueous nebulizers (Davis et al., 1986; Newman et al., 1987; Johnson et al., 1989; O'Doherty et al., 1989; Gilmour et al., 1989) or 100-200 1/min from solid particle or vapor generators (Decker et al., 1982; Griffis et al., 1983). Here we describe and test a low-flow aerosol chamber suitable for studying propellantdriven aerosols of proteins.
plastic-coated, aerosol vials (#WG-1045-005, Wheaton Coated Products, Mays Landing, NJ) and 'cold-filled' with dimethylether (DME) (Aldrich, Milwaukee, WI) as propellant. The D M E was liquified by passage through a small condenser made of polyethylene tubing chilled in an e t h a n o l / d r y ice bath, measured, and placed into chilled plastic-coated aerosol vials. The 20 mm diameter, metered-dose, aerosol valves ( # MP20CP) delivered 40/zl/actuation, and were kindly provided by Emson Research (Bridgeport, CT). Aerosol valves were crimped to the chilled propellant-containing vials with a makeshift crimper. Valves were placed on the vials; a large bolt was placed over the valve stem to protect it, and the valve was seated on the vial by pressing it against a solid upper surface using a hydraulic auto jack. The valve was crimped manually to the vial with needle-nosed pliers. Emson A-7 model aerosol actuators with a 0.150 in depth and 0.013 in inserts were used on the metering valves. Liquid pick-up 'dip' tubes for this valve/vial combination were 34 mm long. Aerosols were released into the aerosol chamber 6-30 times at 15 or 30 s intervals with agitation between each actuation. Actuators were usually changed between each series of aerosol releases.
2. Experimental procedures 2.3. Aerosol sampling 2.1. A n i m a l s
C 5 7 B L / 6 J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were approximately 4 months old when used. Their average weight was 23.7 g. Rectal temperatures of mice were taken by a Becton-Dickinson digital thermometer. 2.2. Aerosols
Propellant-driven aerosols were preparations of the protein, bovine gamma globulin (BGG) (Calbiochem-Behring, La Jolla, CA), which served as a prototype aerosolized protein. It is a model for either an immunizing antigen protein or a passive therapeutic antibody delivered to the respiratory tract. B G G and one of several surfacrants were put into 20 mm diameter, 10 ml,
Unfiltered room air was the source of air flow in the aerosol chamber. The flow of aerosols in the chamber was maintained by a vacuum port, which was attached to either a battery-operated vacuum pump (BGI, Waltham, MA) or an electric pump (Model 13152, Gelman Instruments, Ann Arbor, MI) at a flow rate of 2.2 1/min determined using a rotameter (BGI, Waltham, MA). A unique feature was that all of the chamber's air flow was sampled. Respirable-sized aerosolized particles in the chamber were determined by their capture in a British cyclone (metal construction) or Casella cyclone (plastic construction) aerosol sampler (BGI, Waltham, MA) operated with an air flow rate of 2.2 l/rain and capturing particles of _< 4 ~ m median mass aerodynamic diameters (MMAD) (Bartley et al., 1994). Particles were trapped in the cyclone on 25 mm
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diameter vinyl-acrylic, filtration discs (GM-800, Gelman, Ann Arbor, MI) with a pore size of 0.8 /xm. Total aerosol particles also were sampled using the above filters mounted in a Millipore Swinnex filter unit (Millipore, Bedford, MA). This filter unit was attached to a Drummond pipet-aid vacuum pump (Drummond, Broomall, PA) with the vacuum restricted by an in-line valve to a flow rate of 26 m l / m i n . This flow rate was measured by the volume of water the pump moved along a horizontal tube. The flow rate was consistent for three sequential 1 min tests and again when the flow rate was tested after cumulatively running for more than 2 h. This flow rate through the filter approximated the respiratory air intake per rain, 24 ml, of a mouse (Bernstein, 1966). This filter estimated the total respiratory aerosol exposure to a mouse. Aerosols in the chamber were sampled during their release and for 5 min after the final aerosol actuation. In most experiments the chamber was operated within a chemical fume hood having a flow rate of 4200 l / r a i n with the aerosol chamber oriented with aerosol delivery box at the front of the hood. After air sampling with the cyclone, filter discs were removed and recovered protein quantitated. In some experiments aerosols in the chamber were sampled for respirable protein with the cyclone's inlet port positioned 6 cm in front of the sampling plate and at six different positions (four corners and upper and lower middle locations). Sampled locations circumscribed a 10 cm wide by 8 cm high rectangle within the chamber.
2.4. Assay for proteins Aerosolized proteins captured on filter discs were quantitated using the BCA (bicinchoninic acid) protein detection assay (Pierce, Rockford, IL). Protein-bearing discs were placed in 30 mm, plastic petri dishes and exposed to 0.5 ml of the BCA reagent for 30 rain at 37 ° C. The purple reaction fluid was mixed to ensure homogeneity, and two 100 /zl samples were transferred into a polystyrene ELISA microtiter plate. The optical densities (OD) for protein assay reaction mixtures were obtained with a Bio-Tek Model EL-309
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microtiter plate spectrophotometer (Bio-Tek, Winooski, VT) read at a wavelength of 570 nm. Duplicate readings for samples were averaged, and protein quantities were estimated based on a standard curve produced by developing discs upon which known B G G protein quantities had been applied directly. The standard error for the means of duplicate standards averaged 4 + 0.6% (SEM, standard error of the mean) of the mean values based on five different standard curves calculated over their entire ranges. All optical densities (OD) for developed proteins were corrected for a background OD obtained when the BCA reagent was incubated with the vinyl-acrylic discs alone. These were subtracted from all standards and experimental values prior to plotting the standard curve or determining experimental values directly from a graph of the standards. Uncorrected backgrounds were the equivalent of 8 - 1 6 / z g of protein depending on the assay. The standard curve was nearly linear over a range of 12.5-400/zg of B G G protein.
2.5. Aerosol chamber construction The chamber was constructed predominantly of common and inexpensive plastic laboratory materials. The chamber's main tank was fashioned from a 5 gallon (18.9 liters) polyethylene container (round-cornered rectangle, 24 cm x 25 c m x 35 cm deep) formerly containing 7 x cleaning solution (#76-670-95, Flow Laboratories, McLean, VA). The top and bottom of the container were removed with a Dremel motorized tool equipped with a cutting disk. A 15 cm square hole was removed from its top and slightly extended to remove the container's pour spout and handle. A 10 cm x 14 cm rectangular section was removed just above a slot in the container's bottom. The removed tank handle was cut to fit in that slot and was wired and glued into position using General Electric silicon sealer (local hardware). This protruding handle served as a support for an attached box in which the propellant aerosol vial was housed and actuated. The tank surface around the edges of these holes were roughened with a Dremel grinding stone to aid adherence of a layer of silicon rubber sealant
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applied to form gaskets around the two open ends of the chamber's tank. These seals were flattened by pressing a piece of glycerol-coated plexiglass against the soft sealant. The glycerol prevented sealant adhesion to the plexiglass during gasket curing. To promote uniform air flow through the chamber, five air inlets were placed strategically in the chamber's tank. One was centered under the aerosol delivery box, and two were on the sides of the chamber 14 cm from the tank bottom and 3 cm in from the aerosol delivery box. These were placed to aid aerosol distribution in the tank. Two additional air inlets, one on each side of the tank, were placed 9 cm from the bottom of the tank and 6 cm in from the sampling plate to disrupt a possible dead zone of air that tends to develop in such chambers (Carpenter et al., 1987). Air inlet tubes were 45 m m long tapered plastic sections, inside diameters 10 mm, cut from used Centriprep concentrating units (Amicon, Danvers, MA). They were positioned with the flared end inside the tank, with the inlet protruding outside the tank. Sheet metal vanes (35 m m L x 10 m m W with a 15 m m ' T ' at one end) fitted into a slot in the inlet tube. Vanes were twisted one turn across their length to swirl inlet air for better aerosol mixing. A removable plexiglass box (15 cm L x 12 cm W x 6 cm D) housed aerosol vials actuated into the chamber's tank. This box was supported on the reattached handle with the open 12 c m x 15 cm side facing into the chamber's tank with its edges sealed against the tank's gasket. The aerosol vial was retained and centered on the shelf of this box by a circular plastic section cut from a 50 ml, screw-cap, centrifuge tube mounted on a thin plastic sheet. This arrangement permitted easy removal of both the vial and the retainer from the box. A 10 m m hole was bored into the top of the box above the vial's actuator button to accommodate the vial h o l d e r / a c t u a t o r assembly. The s h a k e r / a c t u a t o r assembly consisted of an ' L ' shaped steel strap to which a 4 cm diameter adjustable hose clamp was welded to its long arm. The hose clamp secured the aerosol vial with the actuator button under the short arm of the 'L'. A 5 m m hole bored in this short arm above the
Fig. 1. Propellant-driven aerosol canister, canister retainer, actuator assembly, and aerosol-generator box portion of the aerosol chamber. Rubber tubing straps secure the aerosolgenerator box to the chamber's tank.
actuator accommodated a 4 m m diameter x 19 cm long bolt that served as an actuating rod. The head of this bolt was serrated where it contacted the top of the vial's actuator button. Resulting friction permitted the actuator to be twisted using the actuator rod for optimal nozzle alignment. A washer of compressible foam rubber maintained tension on the actuator rod between the actuator and the ' L ' shaped retainer. A steel tube with an inside diameter of 5 m m and a length of 13 cm was welded above the hole in ' L ' for the actuator bolt. This tube served as a guide for the actuating rod and a handle by which the clamped vial could be both agitated and actuated from outside the chamber. This assembly permitted aerosol vials to be independently shaken and actuated from outside the chamber. Fig. 1 illustrates this box and a vial clamped in this shaking/actuating assembly. The opposite end of the tank was fitted with one of two types of plexiglass end plates (0.25 in x 26 cm H x 21 cm W). One plate had two plastic rails glued and screwed to the inside to allow a cyclone or other aerosol sampler to be hung by a clip at adjustable locations within the chamber. Fig. 2 shows this end plate attached to a British cyclone aerosol sampler. A second plexiglass end plate of identical size was constructed to permit the exposure of up to six mice to aerosols within the chamber. Two rows of three 30 m m holes were bored into the
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Fig. 2. The aerosol chamber's end plate mounted with a Casella cyclone aerosol sampler and connected to a batteryoperated, sampling, vacuum pump.
Fig. 3. The chamber's end plate with six mouse-restraining ports and a Casella cyclone mounted to a produce a constant airflow through the chamber.
plate to accept the barrels of six, 60 ml disposable plastic syringes that acted as mouse restrainers. The tip end of each syringe barrel was cut off, and the barrels were mounted nearly flush in the plexiglass plate using plastic hot glue sticks. The bases of the syringes were wired together for rigidity. Sections 15 mm long were cut from the threaded screw cap ends of 50 ml polyethylene centrifuge tubes. These were wired and glued onto the plate over each of the syringe restrainer barrels with the cap threads facing into the chamber. The centers of the tubes' caps were cut out to accommodate insertion of 30 mm diameter steel washers bored with 8 mm holes that served as removable nose only aerosol exposure ports for mice. Nose holes were smoothed to prevent chaffing. The syringe plungers were used to secure the mice in the restrainer; four holes were bored into the plunger's plastic and rubber end to permit ventilation to the rear of the restrainer. Undrilled syringe plungers were used to seal unused mouse-restraining ports. Syringe barrels were slotted from their bases to the 20 ml mark to accommodate the tails of restrained mice. Fig. 3 shows this mouse-restraining assembly with the attached cyclone sampler. The sampling and restrainer end plates each had a polyethylene tube of 6 mm outside diameter was mounted through the upper center of the plate to permit coupling of a sampler inside the tank with an external vacuum pump. Panels on
both ends of the chamber were secured to the chamber's tank by two straps composed of latex rubber hoses bolted to each plate and looped over two sets of stud bolts mounted on each side of the tank at its middle. These tubing loops provided a firm seal against the tank's silicon rubber gaskets. This arrangement allowed the independent removal of end panels. These clear plastic ends permitted easy viewing within the chamber for visual checks on the functions of aerosol canisters and sampling devices. The chamber assembly was screwed to a 2 in x 12 in board 50 cm long to give it stability. Fig. 4 shows the entire assembled chamber with the mouse
Fig. 4. Assembled aerosol chamber comprised of the main tank with the mouse-restraining end plate and aerosol-generator box secured to it. A sampling vacuum pump is attached.
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restrainer and vacuum pump attached. The overall dimensions of the chamber (27 cm W × 30 cm H x 60 cm L) permitted it to be operated lengthwise in a chemical fume safety hood with a 60 cm depth. The distance from the aerosol vial's actuator to the aerosol sampling plates in the assembled chamber was 37 cm. The cost of materials to fabricate the chamber were estimated to be $50.
Table 1 Respirable particles within the aerosol chamber when operated, inside or outside of a chemical fume hood Aerosol a
3.1. Uniformity of aerosols within the chamber The construction of the chamber allowed the actuation of propellant-driven aerosol vials in an enclosed environment with only intake air vents open for delivering the vacuum-mediated inward flow of unfiltered room air through the chamber. We anticipated producing aerosols of unknown toxicity and possibly using radioactive aerosols. For safety purposes, the aerosol chamber was designed for use in a chemical fume hood. However, we were concerned that the high flow rates of air within a hood might adversely affect the quality or flow of aerosols produced within the chamber. Consequently, we compared the level of respirable B G G particles generated in the chamber while operated in ambient room air with that generated in the chamber while in a chemical fume hood. Table 1 shows that in seven of ten tests performed, more respirable protein was present when the chamber was operated outside the fume hood. The average reduction in respirable protein recovered by the cyclone when the chamber was operated under the hood in those seven tests was 31 + 6.1% ( x + SEM). This indicated that the high velocity of air passing by and possibly through, the intake ports of the tank reduced the respirable protein particles recoverable from the aerosol chamber. However, the average micrograms of respirable proteins for the ten tests inside the hood, 105 + 23/xg (SEM), and outside the hood, 103 + 11 /zg (SEM), were not significantly different. An average of 22 + 2.5% (SEM) of the total expected protein released in the
Respirable protein (~g) b Chamber operated
Ac
Bc
3. Results
Test
Bd
1 2 3 1 2 3 1 2 3 4
Inside hood
Outside hood
240 96 98 220 60 71 66 45 72 84
144 94 170 84 100 114 92 86 94 56
a Aerosol vials containing l0 mg of BGG with two different surfactants (aerosols A and B) were actuated six times at 30 second intervals with a 5 min terminal air sampling period by a British cyclone. For each test, the chamber was used first inside the hood and then outside the hood. b Protein quantities were assayed as described in the materials and methods section. Expected protein/actuation was 80 /zg x 6 actuations = 480 /zg of expected total protein released in the chamber. c Tests #3 of both aerosols A and B were from different vials of the same aerosol formulation. d Two identically prepared aerosols of B were compared against each other instead of using one aersol vial for both inside and outside the hood. Data were corrected proportionally for initial differences in delivered respirable proteins between the two vials when both were operated inside the hood prior to tests outside the hood.
chamber was recovered from the British cyclone as respirable-sized particles. Depending on the type and quantity of surfactant used, 9 - 5 0 % of the released proteins were of respirable size. Despite the indication of diminished respirable protein when the chamber was operated within a hood, the unknown toxicity of these protein-containing aerosols required us to operate the chamber inside a hood. Room air was not sampled for possible escape of protein from the chamber when operated outside the hood. We were equally interested in determining if respirable particles in the chamber could be sampled uniformly with the cyclone mounted in different positions on the sampling plate. Table 2 shows that no appreciable difference occurred in the quantity of respirable protein recovered from
A.R. Brown, J.A. Pickrell /Journal of lmmunological Methods 176 (1994) 203-212 Table 2 Uniformity of respirable aerosols within the aerosol chamber Chamber location
Respirable protein (/xg) a Experiment 1
Experiment 2
Mean (SEM)
Upper left Upper center Upper right Lower left Lower center Lower right
53 46 72 64 52 46
52 52 52 53 60 48
53 (0.7) 49 (4.2) 62 (14.1) 59 (7.8) 56 (5.2) 47 (1.4)
a Six actuations (40 ~1 each) of aerosolized BGG were delivered within the aerosol chamber at 30 s intervals with a final 5 minute terminal sampling by a British Cyclone. (SEM)= standard error of the mean.
t h e British cyclone, r e g a r d l e s s o f w h e r e on th e c h a m b e r ' s s a m p l i n g p l a t e t h e c y c l o n e was mounted. The average amount of respirable-sized p r o t e i n r e c o v e r e d by th e British cyclone for all t he positions o v e r two e x p e r i m e n t s was 54 + 2.3 ( S E M ) /zg, o r 11% o f t h e e x p e c t e d , total, a e r o s o l i z e d p r o t e i n r e l e a s e d into t h e c h a m b e r . This e s t a b l i s h e d t h a t u n i f o r m i t y o f r e s p i r a b l e p r o t e i n p a r t i c l e s existed w i t h i n t h e c h a m b e r w h e n all the c h a m b e r ' s air flow was c h a n n e l e d t h r o u g h t he cyclone.
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3.2. Function o f the chamber's m o u s e restrainer. W e a n t i c i p a t e d e x p o s i n g m i c e to p r o p e l l a n t driven aerosols o f i m m u n o l o g i c a l l y i m p o r t a n t p r o t e i n s using this a e r o s o l c h a m b e r . T h e r e f o r e , it was i m p o r t a n t to d e t e r m i n e w h e t h e r t h e six-position m o u s e r e s t r a i n e r co u l d h u m a n e l y restrain m i c e for 30 m i n or m o r e w h i l e exposing only t h e i r noses to aerosols within t h e c h a m b e r . O f particular c o n c e r n was w h e t h e r m i c e m i g h t b e c o m e o v e r h e a t e d in t h e c h a m b e r ' s r e t a i n i n g tubes. T e m p e r a t u r e s o f m i c e w e r e c o m p a r e d b e f o r e an d af t er r e s t r a i n for up to 30 m i n with t h e c h a m b e r inside a f u m e hood. R e s u l t s s h o w e d that m i c e e x p e r i e n c e d an a v e r a g e 2.0°C. t e m p e r a t u r e d r o p e v e n af t er v e n t i l a t i o n holes in t h e sides of t h e r e s t r a i n e r t u b es w e r e sealed. R e d u c e d t e m p e r a t u r es w e r e d u e to the high airflow in t h e hood, however, mice removed from the chamber were i m m e d i a t e l y active, with n o e v i d e n c e h y p o t h e r mia. Fig. 5 shows m i c e h e l d in t h e c h a m b e r ' s r est r ai n er .
3.3. Aerosol exposure at the m o u s e restrainer ports. E i t h e r a British or Casel l a cyclone air s a m p l e r was m o u n t e d inside t h e c h a m b e r b e t w e e n t h e
Table 3 Aerosolized BGG protein delivery to individual mouse restraining ports of the aerosol chamber Experiment
Aerosol a
Actuations
1
A
80 e
1
B
120 e
3 4
B A
120 f 120 f
Mouse port location b
MR ML ML UR UL BR BL
Aerosolized protein (/xg) Respirable in chamber c
Total at mouse port d
1 064 1 170 836 485 622 483 804
22 26 26 26 16 15 15
a Aerosols A and B used two different surfactants with dimethyether as propellant. b Mouse port locations: T = top, M = middle, B = bottom, R = right, L = left while facing retaining. c Protein recovered from British Cyclone filters (sum of 2 or 3) changed at 40 actuations each while operating at 2.2 l/min as the predominant source of air flow through the chamber. d Total aerosolized protein recovered from a filter unit drawing 26 ml/min of air to approximate the air intake of a singe mouse. Only one mouse port was sampled at a time. e Acutations delivered at 30 s intervals after vial agitation. f Acutations delivered at 15 s intervals.
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port aerosol sampling (483-1064 /zg). Respirable proteins represented 5 - 1 6 % of the total, expected, aerosolized protein released into the chamber in this experiment.
4. Discussion
Fig. 5. Casella cyclone sampler and mice restrained in the chamber's m o u s e restraining end plate as seen through the tank end where the aerosol-generator box is attached.
two rows of mouse restrainers and maintained air flow through the chamber at 2.2 1/min. Several positions for this sampler were tested, and the most even aerosol distribution to the four corner ports was obtained when the cyclone's intake port was 10.5 cm from the bottom of the tank. Using the cyclone to provide overall air flow in the chamber allowed for sampling of all the respirable particles in the chamber while mice were exposed to aerosols. The percent of the chamber's air flow being used by six mice under these conditions was calculated to be 6.5% of the total airflow in the chamber (6 mice × 24 m l / m i n minute volume = 144 ml, 144/2200 m l / m i n = 6.5%). We wished to determine if mice in the individual restrainer ports could be expected to receive equal aerosol exposure in the aerosol chamber. A second filter with an air flow equivalent to the air intake of a mouse was mounted successively in each of the six positions of the mouse restrainer to approximate the total aerosol inhaled by a mouse at each port. Table 3 shows that nearly equal quantities of total aerosolized protein were captured at each of the six sampling ports (15-26 /~g). This represented 13-29% of the theoretical maximum amount of protein that the mouse-port filter could have captured while sampling = 1% of total air flow in the chamber. This was despite a broad range of respirable particles recovered from the British cyclone operated during mouse-
A small, inexpensive aerosol chamber is described that is suitable for sampling the small volumes of aerosols that are generated by metered-dose, propellant-driven, aerosol canisters. Removable end panels for the chamber allowed easy access to sampling devices, restrained animals, and aerosol generators. The chamber was constructed of common laboratory materials. The low cost of materials and 2-3 h of construction time makes it affordable to have several chambers devoted to specialized aerosols; for example, isotopically labeled or infectious aerosols. The chamber's predominantly plastic construction permitted easy cleaning/decontamination, but not autoclave sterilization. Distributions of aerosols in the chamber were found to be quite uniform, when propellant-driven aerosols of proteins were sampled with a British cyclone at several positions in the chamber. Similar levels of total aerosolized proteins also were demonstrated at each of the mouse-restrainer ports, when sampled air flows simulated the air intake of an individual mouse. The small size of the chamber permitted a cyclone air sampler functioning at 2.2 1/min to serve as the sole source of airflow through the chamber. This allowed sampling of all the air passing through the chamber for respirable ( < 4 ~ m MMAD) aerosol particles that had been released into it. The chamber's small size permitted its operation within a chemical fume hood, making it attractive for testing potentially hazardous aerosols. However, operating the chamber in a high-flow (4200 1/min) hood reduced the concentrations of respirable protein aerosols in the chamber by = 30%. The cause of this reduction in small particles in the chamber is unclear. It probably was due to either increased impaction of aerosols on the chamber's walls or aerosols being drawn from the chamber by the high air
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flows around it. Sampling propellant-driven aerosols of proteins in the chamber using a British cyclone resulted in respirable-sized protein particles consisting of 5-50% of the total expected protein aerosolized. The small dimensions of this chamber and the low aerosol flow rates that it requires also make it ideal for testing propellantdriven aerosols of expensive materials. We are aware of no commercial aerosol chamber of this size or versatility. A six-position restrainer when attached to the chamber's main tank, permitted aerosol exposures to just the noses of up to 6 mice. The metal washers serving as nose exposure ports deterred gnawing, and the screw-off detachment of these ports eased mouse removal and cleaning of the restrainer. The restrainer was well ventilated and did not cause elevated temperatures. The results showed that mice could be restrained humanely and safely in the aerosol chamber for 30 min or more without overheating or undue physical stress. We are using this chamber in developing propellant-driven aerosols of proteins that could influence immune responses in the lung. Metereddose propellant-driven aerosols have long been use to deliver propellant-soluble or suspendable drugs to the respiratory tract (Hallworth, 1987). Here and elsewhere we have shown that respirable-sized aerosols of antigenically intact proteins can be produced with metered-dose propellant-driven aerosol canisters (Brown and Pickrell, 1994). We have also established that respirablesized aerosols of a functional enzyme and monoclonal antibody can be produced by propellantdriven aerosols (Brown and Slusser, 1994). The aerosol chamber described here will permit testing of metered-dose propellant-driven aerosols of proteins like immunizing antigens, immuno-modulating cytokines, and passive anti-pathogen antibodies for their immunologic effects on the respiratory tract.
Acknowledgments The authors would like to thank Joyce Slusser for excellent technical services. This work was
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supported by Kansas Agricultural Experimental Station grants KAN00055 and KAN81872 (A.R.B.). Contribution no. 94-198-J from KAES.
References Bartley, D.L., Chen, C.C., Song, R. and Fischbach, T.J. (1994) Respirable aerosol sampler performance testing. AIHA J., in press. Bernstein, S.E. (1966) Physiological Characteristics. In: E.L. Green (Ed.), Biology of the Laboratory Mouse, Vol. 2. McGraw-Hill, New York, p. 337. Brown, A.R. and Pickrell, J.A. (1994) Propellant-driven aerosols of proteins as vaccines or therapeutic agents in the respiratory tract. Aerosol. Med., submitted. Brown, A.R. and Slusser, J.G. (1994) Propellant-driven aerosols of functional proteins as potential therapeutic agents in the respiratory tract. Immunopharmacology, in press. Carpenter, R.L., Kimmel, E.C., Flemming, C.D. and Doarn, C.R. (1987) Toxicant distribution in the Thomas domes. Toxicology 47, 95. Corrigan, C.J. (1992) Allergy of the respiratory tract. Curr. Opin. Immunol. 4, 798. Davis, J.K., Thorp, R.B., Parker, R.F., White, H., Dziedzic, D., D'Arcy, J. and Cassell, G.H. (1986) Development of an aerosol model of murine respiratory mycoplasmosis in mice. Infect. Immun. 54, 194. Decker, J.R., Moss, O.R. and Kay, B.L. (1982) Controlled-delivery vapor generator for animal exposures. Am. Ind. Hyg. Assoc. J. 43, 400. Gilmour, M.I., Taylor, F.G., Baskerville, A. and Wathes, C.M. (1989) The effect of titanium dioxide inhalation on the pulmonary clearance of Pasteurella haemolytica in the mouse. Environ. Res. 50, 157. Griffis, L.C., Pickrell, J.A., Carpenter, R.L, Wolff, R.K., McAllen, S.J. and Yerkes, K.L. (1983) Deposition of Crocidolite asbestos and glass microfibers inhaled by the Beagle dog. Am. Ind. Hyg. Assoc. J. 44, 216. Hallworth, G.W. (1987) The formulation and evaluation of pressurised metered-dose inhalers. In: D. Ganderton and T. Jones (Eds.), Drug Delivery to the Respiratory Tract. Ellis Horwood, Chichester, UK, p. 87. Johnson, M.A., Newman, S.P., Bloom, R., Talaee, N. and Clarke, S.W. (1989) Delivery of albuterol and ipratropium bromide from two nebulizer systems in chronic stable asthma. Efficacy and pulmonary deposition. Chest 96, 6. Lipscomb, M.F. (1989) Lung defenses against opportunistic infections. Chest 96, 1393. Matthys, H. (1990) Inhalation delivery of asthma drugs. Lung 168 (suppl.), 645. Newman, S.P., Pellow, P.G. and Clarke, S.W. (1987) In vitro comparison of DeVilbiss jet and ultrasonic nebulizers. Chest 92, 991. O'Doherty, M.J., Thomas, S., Page, C., Clark, A.R., Mitchell,
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D., Heduan, E., Nunan, T.O. and Bateman, N.T. (1989) Does 99Tcm human serum albumin alter the characteristics of nebulized pentamidine isethionate? Nucl. Med. Commun. 10, 523. Platts Mills, T.A., Chapman, M.D., Pollart, S., Luczynska, C.M. and Ward, G.W.J. (1991) Specific allergens evoking
immune reactions in the lung: relationship to asthma. Eur. Resp. J. 13 (suppl.), 68s. Welliver, R.C. and Ogra, P.L. (1988) Immunology of respiratory viral infections. Annu. Rev. Med. 39, 147. Wolff, R.K. and Niven, R.W. (1994) Generation of aerosolized drugs. J. Aerosol Med. 7, 89.
Journal of ImmunologicalMethods 176 (1994) 203-212
Chamber for testing metered-dose propellant-driven aerosols of immunologically relevant proteins Alan R. Brown
a,,, J o h n A . P i c k r e l l b
a Department of Pathology and Microbiology, Collegeof Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA b Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA
Received 20 May 1994; revised 5 July 1994; accepted 6 July 1994
Abstract
A small aerosol chamber was developed for testing and delivery of aerosols of immunologically important proteins to the respiratory tracts of rodents. The chamber was designed to accommodate the small aerosol volumes produced by metered-dose propellant-driven aerosol canisters. Metered bursts of protein aerosols released into the chamber could be sampled for their particle sizes or used to expose the noses of up to six mice to the aerosols. The chamber consisted of a polyethylene tank with two removable plexiglass end plates. One end plate accommodated the propellant-driven, metered-dose, aerosol vial. The other end of the tank was fitted with a plate accepting aerosol sampling devices or a plate containing mouse restrainers. Uniform concentrations of aerosolized proteins were obtained at different positions in the chamber when sampled for particles of respirable size. Respirable-sized protein particles produced by propellant-driven aerosols ranged from 5 to 50% of total aerosolized protein. Propellant-driven aerosols of proteins released in the chamber produced aerosol particles equivalent to 15-26 Izg of total protein exposure to the respiratory tract of each mouse. The chamber permitted aerosol releases without risk of operator exposure. This aerosol chamber will permit the testing of protein aerosols for their immunologic consequences to the respiratory tract. Potential proteins for testing in this device include immunizing vaccine antigens, immunomodulating cytokine proteins, and passive antibody aerosol therapies against respiratory infections. Keywords: Aerosol; Propellant; Protein; Immunomodulation; (Lung); (Respiratory)
I. Introduction
I m m u n e responses in the respiratory tract are of great importance for both protection against infections of this system (Welliver and Ogra, 1988; Lipscomb, 1989) and for their involvement in respiratory allergies and asthma (Platts Mills et
* Corresponding author.
al., 1991; Corrigan, 1992). Effective targeting of immunomodulating reagents to the respiratory tract could be of benefit in increasing local immunity to respiratory pathogens or decreasing immune-mediated respiratory pathology. Metered-dose inhalers (MDI) are aerosol-generating devices that use the pressure and flashing of volatile propellants to deliver measured quantities of aerosolized drugs into the respiratory tract (Wolff and Niven, 1994). They particularly
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00192- 8
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have been used to deliver anti-asthmatic treatments (Matthys, 1990). We are interested in metered-dose propellant-driven aerosols as a means of delivering vaccine antigens, cytokines, or other therapeutic proteins to the respiratory tract. To produce and test such aerosols, we needed a chamber for testing the small aerosol volumes produced by metered-dose, propellant-driven, aerosol canisters while accommodating both aerosol sampling devices and aerosol exposures to rodents. We also needed a chamber in which aerosol flows could be small because we wanted to produce aerosols of potentially expensive proteins (i.e., cytokines). Unfortunately, most described aerosol chambers have been fabricated for testing large-volume aerosol particle outputs of 6 or more liters/minute (1/min) associated with jet or ultrasonic aqueous nebulizers (Davis et al., 1986; Newman et al., 1987; Johnson et al., 1989; O'Doherty et al., 1989; Gilmour et al., 1989) or 100-200 1/min from solid particle or vapor generators (Decker et al., 1982; Griffis et al., 1983). Here we describe and test a low-flow aerosol chamber suitable for studying propellantdriven aerosols of proteins.
plastic-coated, aerosol vials (#WG-1045-005, Wheaton Coated Products, Mays Landing, NJ) and 'cold-filled' with dimethylether (DME) (Aldrich, Milwaukee, WI) as propellant. The D M E was liquified by passage through a small condenser made of polyethylene tubing chilled in an e t h a n o l / d r y ice bath, measured, and placed into chilled plastic-coated aerosol vials. The 20 mm diameter, metered-dose, aerosol valves ( # MP20CP) delivered 40/zl/actuation, and were kindly provided by Emson Research (Bridgeport, CT). Aerosol valves were crimped to the chilled propellant-containing vials with a makeshift crimper. Valves were placed on the vials; a large bolt was placed over the valve stem to protect it, and the valve was seated on the vial by pressing it against a solid upper surface using a hydraulic auto jack. The valve was crimped manually to the vial with needle-nosed pliers. Emson A-7 model aerosol actuators with a 0.150 in depth and 0.013 in inserts were used on the metering valves. Liquid pick-up 'dip' tubes for this valve/vial combination were 34 mm long. Aerosols were released into the aerosol chamber 6-30 times at 15 or 30 s intervals with agitation between each actuation. Actuators were usually changed between each series of aerosol releases.
2. Experimental procedures 2.3. Aerosol sampling 2.1. A n i m a l s
C 5 7 B L / 6 J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were approximately 4 months old when used. Their average weight was 23.7 g. Rectal temperatures of mice were taken by a Becton-Dickinson digital thermometer. 2.2. Aerosols
Propellant-driven aerosols were preparations of the protein, bovine gamma globulin (BGG) (Calbiochem-Behring, La Jolla, CA), which served as a prototype aerosolized protein. It is a model for either an immunizing antigen protein or a passive therapeutic antibody delivered to the respiratory tract. B G G and one of several surfacrants were put into 20 mm diameter, 10 ml,
Unfiltered room air was the source of air flow in the aerosol chamber. The flow of aerosols in the chamber was maintained by a vacuum port, which was attached to either a battery-operated vacuum pump (BGI, Waltham, MA) or an electric pump (Model 13152, Gelman Instruments, Ann Arbor, MI) at a flow rate of 2.2 1/min determined using a rotameter (BGI, Waltham, MA). A unique feature was that all of the chamber's air flow was sampled. Respirable-sized aerosolized particles in the chamber were determined by their capture in a British cyclone (metal construction) or Casella cyclone (plastic construction) aerosol sampler (BGI, Waltham, MA) operated with an air flow rate of 2.2 l/rain and capturing particles of _< 4 ~ m median mass aerodynamic diameters (MMAD) (Bartley et al., 1994). Particles were trapped in the cyclone on 25 mm
A.R. Brown, J.A. Pickrell /Journal of lmmunological Methods 176 (1994) 203-212
diameter vinyl-acrylic, filtration discs (GM-800, Gelman, Ann Arbor, MI) with a pore size of 0.8 /xm. Total aerosol particles also were sampled using the above filters mounted in a Millipore Swinnex filter unit (Millipore, Bedford, MA). This filter unit was attached to a Drummond pipet-aid vacuum pump (Drummond, Broomall, PA) with the vacuum restricted by an in-line valve to a flow rate of 26 m l / m i n . This flow rate was measured by the volume of water the pump moved along a horizontal tube. The flow rate was consistent for three sequential 1 min tests and again when the flow rate was tested after cumulatively running for more than 2 h. This flow rate through the filter approximated the respiratory air intake per rain, 24 ml, of a mouse (Bernstein, 1966). This filter estimated the total respiratory aerosol exposure to a mouse. Aerosols in the chamber were sampled during their release and for 5 min after the final aerosol actuation. In most experiments the chamber was operated within a chemical fume hood having a flow rate of 4200 l / r a i n with the aerosol chamber oriented with aerosol delivery box at the front of the hood. After air sampling with the cyclone, filter discs were removed and recovered protein quantitated. In some experiments aerosols in the chamber were sampled for respirable protein with the cyclone's inlet port positioned 6 cm in front of the sampling plate and at six different positions (four corners and upper and lower middle locations). Sampled locations circumscribed a 10 cm wide by 8 cm high rectangle within the chamber.
2.4. Assay for proteins Aerosolized proteins captured on filter discs were quantitated using the BCA (bicinchoninic acid) protein detection assay (Pierce, Rockford, IL). Protein-bearing discs were placed in 30 mm, plastic petri dishes and exposed to 0.5 ml of the BCA reagent for 30 rain at 37 ° C. The purple reaction fluid was mixed to ensure homogeneity, and two 100 /zl samples were transferred into a polystyrene ELISA microtiter plate. The optical densities (OD) for protein assay reaction mixtures were obtained with a Bio-Tek Model EL-309
205
microtiter plate spectrophotometer (Bio-Tek, Winooski, VT) read at a wavelength of 570 nm. Duplicate readings for samples were averaged, and protein quantities were estimated based on a standard curve produced by developing discs upon which known B G G protein quantities had been applied directly. The standard error for the means of duplicate standards averaged 4 + 0.6% (SEM, standard error of the mean) of the mean values based on five different standard curves calculated over their entire ranges. All optical densities (OD) for developed proteins were corrected for a background OD obtained when the BCA reagent was incubated with the vinyl-acrylic discs alone. These were subtracted from all standards and experimental values prior to plotting the standard curve or determining experimental values directly from a graph of the standards. Uncorrected backgrounds were the equivalent of 8 - 1 6 / z g of protein depending on the assay. The standard curve was nearly linear over a range of 12.5-400/zg of B G G protein.
2.5. Aerosol chamber construction The chamber was constructed predominantly of common and inexpensive plastic laboratory materials. The chamber's main tank was fashioned from a 5 gallon (18.9 liters) polyethylene container (round-cornered rectangle, 24 cm x 25 c m x 35 cm deep) formerly containing 7 x cleaning solution (#76-670-95, Flow Laboratories, McLean, VA). The top and bottom of the container were removed with a Dremel motorized tool equipped with a cutting disk. A 15 cm square hole was removed from its top and slightly extended to remove the container's pour spout and handle. A 10 cm x 14 cm rectangular section was removed just above a slot in the container's bottom. The removed tank handle was cut to fit in that slot and was wired and glued into position using General Electric silicon sealer (local hardware). This protruding handle served as a support for an attached box in which the propellant aerosol vial was housed and actuated. The tank surface around the edges of these holes were roughened with a Dremel grinding stone to aid adherence of a layer of silicon rubber sealant
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applied to form gaskets around the two open ends of the chamber's tank. These seals were flattened by pressing a piece of glycerol-coated plexiglass against the soft sealant. The glycerol prevented sealant adhesion to the plexiglass during gasket curing. To promote uniform air flow through the chamber, five air inlets were placed strategically in the chamber's tank. One was centered under the aerosol delivery box, and two were on the sides of the chamber 14 cm from the tank bottom and 3 cm in from the aerosol delivery box. These were placed to aid aerosol distribution in the tank. Two additional air inlets, one on each side of the tank, were placed 9 cm from the bottom of the tank and 6 cm in from the sampling plate to disrupt a possible dead zone of air that tends to develop in such chambers (Carpenter et al., 1987). Air inlet tubes were 45 m m long tapered plastic sections, inside diameters 10 mm, cut from used Centriprep concentrating units (Amicon, Danvers, MA). They were positioned with the flared end inside the tank, with the inlet protruding outside the tank. Sheet metal vanes (35 m m L x 10 m m W with a 15 m m ' T ' at one end) fitted into a slot in the inlet tube. Vanes were twisted one turn across their length to swirl inlet air for better aerosol mixing. A removable plexiglass box (15 cm L x 12 cm W x 6 cm D) housed aerosol vials actuated into the chamber's tank. This box was supported on the reattached handle with the open 12 c m x 15 cm side facing into the chamber's tank with its edges sealed against the tank's gasket. The aerosol vial was retained and centered on the shelf of this box by a circular plastic section cut from a 50 ml, screw-cap, centrifuge tube mounted on a thin plastic sheet. This arrangement permitted easy removal of both the vial and the retainer from the box. A 10 m m hole was bored into the top of the box above the vial's actuator button to accommodate the vial h o l d e r / a c t u a t o r assembly. The s h a k e r / a c t u a t o r assembly consisted of an ' L ' shaped steel strap to which a 4 cm diameter adjustable hose clamp was welded to its long arm. The hose clamp secured the aerosol vial with the actuator button under the short arm of the 'L'. A 5 m m hole bored in this short arm above the
Fig. 1. Propellant-driven aerosol canister, canister retainer, actuator assembly, and aerosol-generator box portion of the aerosol chamber. Rubber tubing straps secure the aerosolgenerator box to the chamber's tank.
actuator accommodated a 4 m m diameter x 19 cm long bolt that served as an actuating rod. The head of this bolt was serrated where it contacted the top of the vial's actuator button. Resulting friction permitted the actuator to be twisted using the actuator rod for optimal nozzle alignment. A washer of compressible foam rubber maintained tension on the actuator rod between the actuator and the ' L ' shaped retainer. A steel tube with an inside diameter of 5 m m and a length of 13 cm was welded above the hole in ' L ' for the actuator bolt. This tube served as a guide for the actuating rod and a handle by which the clamped vial could be both agitated and actuated from outside the chamber. This assembly permitted aerosol vials to be independently shaken and actuated from outside the chamber. Fig. 1 illustrates this box and a vial clamped in this shaking/actuating assembly. The opposite end of the tank was fitted with one of two types of plexiglass end plates (0.25 in x 26 cm H x 21 cm W). One plate had two plastic rails glued and screwed to the inside to allow a cyclone or other aerosol sampler to be hung by a clip at adjustable locations within the chamber. Fig. 2 shows this end plate attached to a British cyclone aerosol sampler. A second plexiglass end plate of identical size was constructed to permit the exposure of up to six mice to aerosols within the chamber. Two rows of three 30 m m holes were bored into the
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207
Fig. 2. The aerosol chamber's end plate mounted with a Casella cyclone aerosol sampler and connected to a batteryoperated, sampling, vacuum pump.
Fig. 3. The chamber's end plate with six mouse-restraining ports and a Casella cyclone mounted to a produce a constant airflow through the chamber.
plate to accept the barrels of six, 60 ml disposable plastic syringes that acted as mouse restrainers. The tip end of each syringe barrel was cut off, and the barrels were mounted nearly flush in the plexiglass plate using plastic hot glue sticks. The bases of the syringes were wired together for rigidity. Sections 15 mm long were cut from the threaded screw cap ends of 50 ml polyethylene centrifuge tubes. These were wired and glued onto the plate over each of the syringe restrainer barrels with the cap threads facing into the chamber. The centers of the tubes' caps were cut out to accommodate insertion of 30 mm diameter steel washers bored with 8 mm holes that served as removable nose only aerosol exposure ports for mice. Nose holes were smoothed to prevent chaffing. The syringe plungers were used to secure the mice in the restrainer; four holes were bored into the plunger's plastic and rubber end to permit ventilation to the rear of the restrainer. Undrilled syringe plungers were used to seal unused mouse-restraining ports. Syringe barrels were slotted from their bases to the 20 ml mark to accommodate the tails of restrained mice. Fig. 3 shows this mouse-restraining assembly with the attached cyclone sampler. The sampling and restrainer end plates each had a polyethylene tube of 6 mm outside diameter was mounted through the upper center of the plate to permit coupling of a sampler inside the tank with an external vacuum pump. Panels on
both ends of the chamber were secured to the chamber's tank by two straps composed of latex rubber hoses bolted to each plate and looped over two sets of stud bolts mounted on each side of the tank at its middle. These tubing loops provided a firm seal against the tank's silicon rubber gaskets. This arrangement allowed the independent removal of end panels. These clear plastic ends permitted easy viewing within the chamber for visual checks on the functions of aerosol canisters and sampling devices. The chamber assembly was screwed to a 2 in x 12 in board 50 cm long to give it stability. Fig. 4 shows the entire assembled chamber with the mouse
Fig. 4. Assembled aerosol chamber comprised of the main tank with the mouse-restraining end plate and aerosol-generator box secured to it. A sampling vacuum pump is attached.
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restrainer and vacuum pump attached. The overall dimensions of the chamber (27 cm W × 30 cm H x 60 cm L) permitted it to be operated lengthwise in a chemical fume safety hood with a 60 cm depth. The distance from the aerosol vial's actuator to the aerosol sampling plates in the assembled chamber was 37 cm. The cost of materials to fabricate the chamber were estimated to be $50.
Table 1 Respirable particles within the aerosol chamber when operated, inside or outside of a chemical fume hood Aerosol a
3.1. Uniformity of aerosols within the chamber The construction of the chamber allowed the actuation of propellant-driven aerosol vials in an enclosed environment with only intake air vents open for delivering the vacuum-mediated inward flow of unfiltered room air through the chamber. We anticipated producing aerosols of unknown toxicity and possibly using radioactive aerosols. For safety purposes, the aerosol chamber was designed for use in a chemical fume hood. However, we were concerned that the high flow rates of air within a hood might adversely affect the quality or flow of aerosols produced within the chamber. Consequently, we compared the level of respirable B G G particles generated in the chamber while operated in ambient room air with that generated in the chamber while in a chemical fume hood. Table 1 shows that in seven of ten tests performed, more respirable protein was present when the chamber was operated outside the fume hood. The average reduction in respirable protein recovered by the cyclone when the chamber was operated under the hood in those seven tests was 31 + 6.1% ( x + SEM). This indicated that the high velocity of air passing by and possibly through, the intake ports of the tank reduced the respirable protein particles recoverable from the aerosol chamber. However, the average micrograms of respirable proteins for the ten tests inside the hood, 105 + 23/xg (SEM), and outside the hood, 103 + 11 /zg (SEM), were not significantly different. An average of 22 + 2.5% (SEM) of the total expected protein released in the
Respirable protein (~g) b Chamber operated
Ac
Bc
3. Results
Test
Bd
1 2 3 1 2 3 1 2 3 4
Inside hood
Outside hood
240 96 98 220 60 71 66 45 72 84
144 94 170 84 100 114 92 86 94 56
a Aerosol vials containing l0 mg of BGG with two different surfactants (aerosols A and B) were actuated six times at 30 second intervals with a 5 min terminal air sampling period by a British cyclone. For each test, the chamber was used first inside the hood and then outside the hood. b Protein quantities were assayed as described in the materials and methods section. Expected protein/actuation was 80 /zg x 6 actuations = 480 /zg of expected total protein released in the chamber. c Tests #3 of both aerosols A and B were from different vials of the same aerosol formulation. d Two identically prepared aerosols of B were compared against each other instead of using one aersol vial for both inside and outside the hood. Data were corrected proportionally for initial differences in delivered respirable proteins between the two vials when both were operated inside the hood prior to tests outside the hood.
chamber was recovered from the British cyclone as respirable-sized particles. Depending on the type and quantity of surfactant used, 9 - 5 0 % of the released proteins were of respirable size. Despite the indication of diminished respirable protein when the chamber was operated within a hood, the unknown toxicity of these protein-containing aerosols required us to operate the chamber inside a hood. Room air was not sampled for possible escape of protein from the chamber when operated outside the hood. We were equally interested in determining if respirable particles in the chamber could be sampled uniformly with the cyclone mounted in different positions on the sampling plate. Table 2 shows that no appreciable difference occurred in the quantity of respirable protein recovered from
A.R. Brown, J.A. Pickrell /Journal of lmmunological Methods 176 (1994) 203-212 Table 2 Uniformity of respirable aerosols within the aerosol chamber Chamber location
Respirable protein (/xg) a Experiment 1
Experiment 2
Mean (SEM)
Upper left Upper center Upper right Lower left Lower center Lower right
53 46 72 64 52 46
52 52 52 53 60 48
53 (0.7) 49 (4.2) 62 (14.1) 59 (7.8) 56 (5.2) 47 (1.4)
a Six actuations (40 ~1 each) of aerosolized BGG were delivered within the aerosol chamber at 30 s intervals with a final 5 minute terminal sampling by a British Cyclone. (SEM)= standard error of the mean.
t h e British cyclone, r e g a r d l e s s o f w h e r e on th e c h a m b e r ' s s a m p l i n g p l a t e t h e c y c l o n e was mounted. The average amount of respirable-sized p r o t e i n r e c o v e r e d by th e British cyclone for all t he positions o v e r two e x p e r i m e n t s was 54 + 2.3 ( S E M ) /zg, o r 11% o f t h e e x p e c t e d , total, a e r o s o l i z e d p r o t e i n r e l e a s e d into t h e c h a m b e r . This e s t a b l i s h e d t h a t u n i f o r m i t y o f r e s p i r a b l e p r o t e i n p a r t i c l e s existed w i t h i n t h e c h a m b e r w h e n all the c h a m b e r ' s air flow was c h a n n e l e d t h r o u g h t he cyclone.
209
3.2. Function o f the chamber's m o u s e restrainer. W e a n t i c i p a t e d e x p o s i n g m i c e to p r o p e l l a n t driven aerosols o f i m m u n o l o g i c a l l y i m p o r t a n t p r o t e i n s using this a e r o s o l c h a m b e r . T h e r e f o r e , it was i m p o r t a n t to d e t e r m i n e w h e t h e r t h e six-position m o u s e r e s t r a i n e r co u l d h u m a n e l y restrain m i c e for 30 m i n or m o r e w h i l e exposing only t h e i r noses to aerosols within t h e c h a m b e r . O f particular c o n c e r n was w h e t h e r m i c e m i g h t b e c o m e o v e r h e a t e d in t h e c h a m b e r ' s r e t a i n i n g tubes. T e m p e r a t u r e s o f m i c e w e r e c o m p a r e d b e f o r e an d af t er r e s t r a i n for up to 30 m i n with t h e c h a m b e r inside a f u m e hood. R e s u l t s s h o w e d that m i c e e x p e r i e n c e d an a v e r a g e 2.0°C. t e m p e r a t u r e d r o p e v e n af t er v e n t i l a t i o n holes in t h e sides of t h e r e s t r a i n e r t u b es w e r e sealed. R e d u c e d t e m p e r a t u r es w e r e d u e to the high airflow in t h e hood, however, mice removed from the chamber were i m m e d i a t e l y active, with n o e v i d e n c e h y p o t h e r mia. Fig. 5 shows m i c e h e l d in t h e c h a m b e r ' s r est r ai n er .
3.3. Aerosol exposure at the m o u s e restrainer ports. E i t h e r a British or Casel l a cyclone air s a m p l e r was m o u n t e d inside t h e c h a m b e r b e t w e e n t h e
Table 3 Aerosolized BGG protein delivery to individual mouse restraining ports of the aerosol chamber Experiment
Aerosol a
Actuations
1
A
80 e
1
B
120 e
3 4
B A
120 f 120 f
Mouse port location b
MR ML ML UR UL BR BL
Aerosolized protein (/xg) Respirable in chamber c
Total at mouse port d
1 064 1 170 836 485 622 483 804
22 26 26 26 16 15 15
a Aerosols A and B used two different surfactants with dimethyether as propellant. b Mouse port locations: T = top, M = middle, B = bottom, R = right, L = left while facing retaining. c Protein recovered from British Cyclone filters (sum of 2 or 3) changed at 40 actuations each while operating at 2.2 l/min as the predominant source of air flow through the chamber. d Total aerosolized protein recovered from a filter unit drawing 26 ml/min of air to approximate the air intake of a singe mouse. Only one mouse port was sampled at a time. e Acutations delivered at 30 s intervals after vial agitation. f Acutations delivered at 15 s intervals.
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port aerosol sampling (483-1064 /zg). Respirable proteins represented 5 - 1 6 % of the total, expected, aerosolized protein released into the chamber in this experiment.
4. Discussion
Fig. 5. Casella cyclone sampler and mice restrained in the chamber's m o u s e restraining end plate as seen through the tank end where the aerosol-generator box is attached.
two rows of mouse restrainers and maintained air flow through the chamber at 2.2 1/min. Several positions for this sampler were tested, and the most even aerosol distribution to the four corner ports was obtained when the cyclone's intake port was 10.5 cm from the bottom of the tank. Using the cyclone to provide overall air flow in the chamber allowed for sampling of all the respirable particles in the chamber while mice were exposed to aerosols. The percent of the chamber's air flow being used by six mice under these conditions was calculated to be 6.5% of the total airflow in the chamber (6 mice × 24 m l / m i n minute volume = 144 ml, 144/2200 m l / m i n = 6.5%). We wished to determine if mice in the individual restrainer ports could be expected to receive equal aerosol exposure in the aerosol chamber. A second filter with an air flow equivalent to the air intake of a mouse was mounted successively in each of the six positions of the mouse restrainer to approximate the total aerosol inhaled by a mouse at each port. Table 3 shows that nearly equal quantities of total aerosolized protein were captured at each of the six sampling ports (15-26 /~g). This represented 13-29% of the theoretical maximum amount of protein that the mouse-port filter could have captured while sampling = 1% of total air flow in the chamber. This was despite a broad range of respirable particles recovered from the British cyclone operated during mouse-
A small, inexpensive aerosol chamber is described that is suitable for sampling the small volumes of aerosols that are generated by metered-dose, propellant-driven, aerosol canisters. Removable end panels for the chamber allowed easy access to sampling devices, restrained animals, and aerosol generators. The chamber was constructed of common laboratory materials. The low cost of materials and 2-3 h of construction time makes it affordable to have several chambers devoted to specialized aerosols; for example, isotopically labeled or infectious aerosols. The chamber's predominantly plastic construction permitted easy cleaning/decontamination, but not autoclave sterilization. Distributions of aerosols in the chamber were found to be quite uniform, when propellant-driven aerosols of proteins were sampled with a British cyclone at several positions in the chamber. Similar levels of total aerosolized proteins also were demonstrated at each of the mouse-restrainer ports, when sampled air flows simulated the air intake of an individual mouse. The small size of the chamber permitted a cyclone air sampler functioning at 2.2 1/min to serve as the sole source of airflow through the chamber. This allowed sampling of all the air passing through the chamber for respirable ( < 4 ~ m MMAD) aerosol particles that had been released into it. The chamber's small size permitted its operation within a chemical fume hood, making it attractive for testing potentially hazardous aerosols. However, operating the chamber in a high-flow (4200 1/min) hood reduced the concentrations of respirable protein aerosols in the chamber by = 30%. The cause of this reduction in small particles in the chamber is unclear. It probably was due to either increased impaction of aerosols on the chamber's walls or aerosols being drawn from the chamber by the high air
A.R. Brown, J.A. Pickrell/Journal of lmmunological Methods 176 (1994) 203-212
flows around it. Sampling propellant-driven aerosols of proteins in the chamber using a British cyclone resulted in respirable-sized protein particles consisting of 5-50% of the total expected protein aerosolized. The small dimensions of this chamber and the low aerosol flow rates that it requires also make it ideal for testing propellantdriven aerosols of expensive materials. We are aware of no commercial aerosol chamber of this size or versatility. A six-position restrainer when attached to the chamber's main tank, permitted aerosol exposures to just the noses of up to 6 mice. The metal washers serving as nose exposure ports deterred gnawing, and the screw-off detachment of these ports eased mouse removal and cleaning of the restrainer. The restrainer was well ventilated and did not cause elevated temperatures. The results showed that mice could be restrained humanely and safely in the aerosol chamber for 30 min or more without overheating or undue physical stress. We are using this chamber in developing propellant-driven aerosols of proteins that could influence immune responses in the lung. Metereddose propellant-driven aerosols have long been use to deliver propellant-soluble or suspendable drugs to the respiratory tract (Hallworth, 1987). Here and elsewhere we have shown that respirable-sized aerosols of antigenically intact proteins can be produced with metered-dose propellant-driven aerosol canisters (Brown and Pickrell, 1994). We have also established that respirablesized aerosols of a functional enzyme and monoclonal antibody can be produced by propellantdriven aerosols (Brown and Slusser, 1994). The aerosol chamber described here will permit testing of metered-dose propellant-driven aerosols of proteins like immunizing antigens, immuno-modulating cytokines, and passive anti-pathogen antibodies for their immunologic effects on the respiratory tract.
Acknowledgments The authors would like to thank Joyce Slusser for excellent technical services. This work was
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supported by Kansas Agricultural Experimental Station grants KAN00055 and KAN81872 (A.R.B.). Contribution no. 94-198-J from KAES.
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