Polymer Nanoparticle-Based Controlled Pulmonary Drug Delivery
3.4 Isolated, Perfused, and
Ventilated Lung Model (IPL)
3.4.1 Surgical Procedure for IPL Preparation
5. Expose the trachea by blunt dissection, and insert and fix a cannula (diameter: 3 mm) in the trachea. Subsequently, venti-late the animal with room air (tidal volume: 10 ml/kg; fre-quency: 30 strokes/min).
6. After mid-sternal thoracotomy, spread the ribs, incise the right ventricle, and immediately place a fluid-filled perfusion cathe-ter into the pulmonary arcathe-tery, which needs to be secured with a ligature.
7. After insertion of the catheter, start the perfusion with the cooled perfusion medium (10–20 ml/min), and then cut the heart open at the apex.
8. Excise the trachea, lungs, and heart en bloc from the thoracic cage.
9. Introduce a second catheter with a bent cannula (diameter:
4 mm) via the left ventricle into the left atrium and fix it in this position (see Note 13).
10. Place the isolated organs in a temperature-equilibrated hous-ing chamber, freely suspended from a force transducer for con-tinuous monitoring of the organ weight.
11. After rinsing the lungs with at least 1,000 ml of perfusion medium for washout of blood, close the perfusion circuit for recirculation. Meanwhile, increase the flow rate slowly to 100 ml/min. At the same time, elevate the temperature of the perfusion medium and housing chamber to 40 °C.
12. In parallel, change to the ventilation gas mixture (positive end-expiratory pressure of 1 cmH2O) (see Note 14) to maintain the pH of the perfusion fluid in the range between 7.35 and 7.45.
13. After a 30 min steady-state period, exchange the perfusion fluid once by fresh perfusate (volume: 300 ml). Adjust the pH of the perfusion medium (see Note 15).
14. Adjust the left atrial pressure to 1.5 mmHg by changing the hydrostatic pressure caused by the height of the venous part of the system.
15. Monitor the pH of the perfusion medium, pressures in the pulmonary artery, left atrium, and trachea, as well as organ weight throughout the experiment.
For the analysis of the pulmonary pharmacokinetics of the drug- loaded nanoparticles, formulations need to be delivered to the lung model via the intratracheal route by nebulization (Fig. 2) [10, 18]:
1. Connect the nebulizer to the inspiratory tubing between the ventilator and the isolated organ.
2. Nebulize 3 ml of filtrated formulation containing a CF con-centration of 50 μg/ml into the IPl.
3.4.2 Pulmonary Absorption and Distribution Characteristics
of Nebulized Formulations in the IPL
3. Take 800 μl samples from the venous part of the system (e.g., 10, 20, 30, 45, 60, 90, 120, 180, and 240 min after nebulization).
4. Perform a bronchoalveolar lavage at the end of the absorption experiment. Instill and reaspirate three times 50 ml of isotonic NaCl solution through the trachea.
5. Centrifuge all samples at 300 × g for 10 min to remove cells.
6. determine the sample concentrations by fluorescence spec-troscopy (see Subheading 3.5) (see Note 16).
To determine the percentage of model drug absorbed into the perfusate and distributed within the IPl, the deposited amount of aerosol needs to be determined in separate experiments [10].
7. Connect the nebulizer to the IPl model as described above.
8. dissolve 99mTc in 10 ml of isotonic NaCl solution.
9. Nebulize 3 ml of this solution into the IPl.
10. Clamp the lung and stop perfusion during aerosol delivery.
11. determine the radioactivity of the expiratory filter (exhaled fraction) and lung (lung deposition) by gamma counting (see Note 17).
Fig. 2 Schematic depiction of the rabbit lung model useful for pharmacokinetic studies of drug-loaded polymeric nanoparticle formulations. Reproduced with permission from [10]. Copyright 2009 Elsevier
1. dilute the CF stock solution (50 μg/ml) with PBS pH 7.4 to reach a range of CF concentrations between 5 and 50 ng/ml.
2. dilute samples with PBS pH 7.4 if necessary.
3. Measure 200 μl of each sample for its fluorescence intensity (see Note 18).
4. Calculate the CF content using a calibration curve (R2 > 0.995).
4 Notes
1. Biodegradable charge-modified branched polyesters were synthesized and characterized as described in detail elsewhere [26]. These polymers comprise short poly(d,l-lactide-co- glycolide) (PlGA) chains grafted onto an amine-substituted poly(vinyl alcohol) (PVA) backbone. As abbreviation A-PVA(x)-g-PlGA (1:y) is used. A is the abbreviation of the type of amine substitution (dEAPA for 3-(diethylamino)pro-pylamine), x represents the total average number of amine functions on the PVA backbone, and y is the PlGA side-chain length [10]. The employed polyesters are hygroscopic and undergo rapid degradation upon contact with water. The pack-aged polymer samples need to reach room temperature before opening in order to minimize water uptake. Optimally, poly-mers are handled under a glove box in a water-free, inert gas atmosphere.
2. CMC acts as a colloidal stabilizer during nanoparticle prepara-tion as well as nebulizaprepara-tion [10, 13, 29].
3. dilution of nanosuspensions to a final nanoparticle concentra-tion of ≤1 mg/ml is important to avoid multiscattering events.
4. A constant ionic strength improves ζ-potential measurements.
5. AFM analysis in intermittent contact mode minimizes damage of the sample surface [30].
6. The CF entrapment efficiency is calculated indirectly by deter-mining the non-encapsulated CF amount.
7. Nanosuspension without CF and pure model drug should be incubated under the same conditions.
8. Positioning of the nebulizer to the instrument avoids vignett-ing (loss of light scattered at large angles) and mouthpiece interference with the expanded laser beam [31]. Moreover, air extraction behind the laser beam ensures that the aerosol does not reenter the laser sensing zone.
9. The optical properties of the samples are as follows for data anal-ysis: real part of the refractive index, 1.33; complex part of the refractive index (i.e., absorption), 0. The value obtained for 3.5 CF Quantification
by Fluorescence Spectroscopy
the VMd of produced aerosol droplets might be converted to the MMAd using the sample density (MMAd = VMd × (ρp/ρw)1/2).
The GSd is calculated from the laser diffraction values (GSd = d84%/d16%1/2).
10. Consider the drying effect of the aerosol during collection for determination of the model drug entrapment efficiency.
11. Only lungs that have a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis, a constant mean pulmonary artery and peak ventilation pressure in the normal range (i.e., 4–10 and 5–8 mmHg, respectively), and are iso-gravimetric during an initial steady-state period are considered for experiments [18, 32].
12. Take care that no air bubbles are introduced into the system.
13. The system should not reveal any leakage or obstructions.
14. “Positive” pressure ventilation of the IPl allows for a homoge-neous and efficient aerosol deposition within the lung.
15. Artificial perfusion medium (e.g., Krebs-Henseleit buffer) is only adequate for hydrophilic drugs. Presence of albumin relieves the analysis of hydrophobic drug substances [18].
16. In order to consider loss of perfusate during the IPl experi-ments (~10–15 ml/h), the measured CF perfusate concentra-tion needs to be corrected using the following formula [10, 11]:
c t c t V t V V t c t
V
c t V t
corr
( )
=( )
×( )
+éë( )
-( )
ùû ×( )
( )
=( )
×( )
p p p
p
0 p
2
0 2 VVp 0 1
( )
+é ëê ê
ù ûú ú where ccorr(t) is the corrected CF concentration in perfusate after time t, c(t) is the measured CF concentration in perfusate after time t, Vp(t) is the perfusate volume after time t, and Vp(0) is the perfusate volume at the beginning of the experiment.
17. Calculate the deposition fraction (dF) as follows: dF = ld/
IF = ld/(ld + EF), where ld is the lung deposition, EF is the exhaled fraction, and IF is the inhaled fraction = ld + EF [10].
18. Fluorescence intensity of samples is measured at λex = 490 nm (slit: 5.0 nm) and λem = 520 nm (slit: 5.0 nm) for 1 s.
Acknowledgements
This work was supported by grants from the “deutsche Forschungsgemeinschaft” (BE 5308/1-1), “Universitätsklinikum Giessen und Marburg” (1/2012 GI), and “Wirtschafts- und Infrastrukturbank Hessen” (Nanosurfact).
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Kewal K. Jain (ed.), Drug Delivery System, Methods in Molecular Biology, vol. 1141, DOI 10.1007/978-1-4939-0363-4_9, © Springer Science+Business Media New York 2014