3 Materials and Methods
4.2 In vivo Experiments
4.2.1 Intratracheal Instillation
Intratracheal instillation as a low-cost screening-method allowed to investigate up to 4 exemplary types of nanoparticles (commercially available; reference data existing in the literature) in vivo. Thus, the ag-/deglomeration behaviour of various nanoparticle types could be analysed.
Table 4.2 Intratracheal instillations: Experimental design
Intratracheal instillation, single dose e.g. approx. 0.3 mg per rat in phosphate buffer;
e.g. TiO2, amorphous SiO2, carbon black, ZnO, constantan a; all as nanoparticles Comparison of a maximum of 5
dif-ferent commercial nanoparticle types
Dispersion e.g. in physiological phosphate buffer (Ultra-Turrax; ultrasonification; target diameter: e.g. approx. 100 nm)
Time-point of investigation after acute exposure (days)
Instillation of a nanoparticle suspen-sion with number-based median of approx. 100 nm
28 5 Measurements • Before administration: Determination of the particle
ag-glomerate size using the ZetaSizer®
• At scheduled dates:
o Bronchoalveolar lavage (BAL) with determination of agglomerate sizes c
o Investigation of lung sections/LALN as well as blood, urine and remote organs, i.e. spleen, liver and kid-neys on nanoparticles; whole body perfusion fixation post mortem with uranyl acetate to guarantee the optimal consistence of the surfactant ultrastructure (SEM/TEM method)
a Constantan is a copper-nickel alloy usually consisting of 55 % copper,44 % nickel and 1 % mangane. Its main feature is its resistance which is constant over a wide range of temperatures.
Note: During the BAuA-ITEM meeting on April 2, 2009 silver or copper were discussed as candidates for the spark generator aeroal; however, technical trials revealed that both metals where not optimal.
b The originally appointed days 3 and 7 have been merged to day 5.
c Supplementing method to the scheduled acellular in vitro surfactant models
Originally the 100 nm size range should be compared to the 500 nm size range. However, instillation of a nanoparticle suspension with number-based median of approx. 500 nm was technically difficult because at this particle size range the stability of agglomerate sizes was not achievable.
On the next pages the measurements on particle stock suspensions including control of the stability are presented for TiO2 P25, TiO2 T805 and constantan.
Hydrodynamic diameter (nm)
Record 168: P25 Stamm für HV am 07.07.10 2
ζ potential (mV)
Fig. 4.42 Characterisation of stock suspension TiO2 P25 on day of administration Hydrodynamic diameter (nm)
Record 173: P25 Stamm 17h=1d Instillationstag 1
ζ potential (mV)
Fig. 4.43 Characterisation of stock suspension TiO2 P25 after 17 further hours
Hydrodynamic diameter (nm)
Record 60: T805 Stamm 48h 1
ζ potential (mV)
Fig. 4.44 Characterisation of stock suspension TiO2 T805 on day of administration Hydrodynamic diameter (nm)
Record 101: T805 Stamm 3Tage 3
ζ potential (mV)
Fig. 4.45 Characterisation of stock suspension TiO2 T805 after 24 further hours
Hydrodynamic diameter (nm)
Record 74: Konstantan Stamm Endlösung vom 13.07.10 /KontrollMess Utrgerüttelt 2
ζ potential (mV)
Fig. 4.46 Characterisation of stock suspension Constantan on day of administration
Table 4.3 Stock suspensions used for intratracheal instillation of rats with sacrifices used for TEM analysis after 1 hour, 1, 5 and 28 days
Substance/
4.2.2 TEM Analysis
The tissue was investigated in detail by thoroughly looking at it at a magnification of 10.000x. If particles were found resembling nanoparticles the magnification was in-creased up to 80.000x to verify the observation.
TiO2 P25 and TiO2 T805
One hour after instillation most of the particle found were within the lung lining fluid or attached to the cellular surface of the alveolar lining epithelium of alveolar macro-phages. However, some particles were observed within the cytoplasm of alveolar macrophages and pneumocytes type I. The localization of the particles changed in the following time points investigated (1 day, 5 days and 28 days) from extracellular to more or exclusive intracellular.
Measurement of diameter
To acquire the size of the agglomerates found, the diameter of 50 to 200 of these particles were manually measured using the AxioVision 4.8.1 software (Zeiss).
Fig. 4.47 Lung 1 hour after instillation of TiO2 P25
Fig. 4.48 TiO2 P25 nanoparticle within the surfactant 1 hour after instillation
Fig. 4.49 Lung 1 day after instillation of TiO2 P25
Fig. 4.50 Lung 5 days after instillation of TiO2 P25
Fig. 4.51 Lung 28 days after instillation of TiO2 P25
Fig. 4.52 TiO2 P25 nanoparticles within a macrophage 28 days after instillation
Fig. 4.53 Lung 1 hour after instillation of TiO2 T805
Fig. 4.54 TiO2 T805 nanoparticles within the lung lining fluid 1 hour after instillation
Fig. 4.55 Lung 1 day after instillation of TiO2 T805
Fig. 4.56 Lung 5 days after instillation of TiO2 T805
Fig. 4.57 Lung 28 days after instillation of TiO2 T805
Fig. 4.58 TiO2 T805 nanoparticles within a macrophage 28 days after instillation
Constantan
Following instillation of constantan into the lung only single particles were found.
Therefore, a proper analysis was not possible.
Examples of particles observed: One particle measuring 735 nm in diameter was found in the cytoplasm 5 days after instillation (see figure 4.59).
Fig. 4.59 Constantan nanoparticle within a macrophage 5 days after instillation However, many particles might be missed since the primary particles of constantan were only approximately 5 nm in diameter and not very electron dense (see figure 4.60).
Fig. 4.60 Constantan nanoparticles within lung lining fluid 28 days after instillation
Table 4.4 Stock suspensions used for intratracheal instillation of rats with sacrifices used for BAL analysis after 15 min, 1, 4 and 24 hours
Substance/
particle type
Concentration (%)
Medium Z-Average (nm)
ζ potential (mV)
TiO2 P25
0.2
0.15 %
Na2HPO4 buffer 192 -47
TiO2 T805 168 n.m.
TiO2 T805 after 17 h
0.2
0.15 %
Na2HPO4 buffer
0.1 % Tween 80 169 n.m.
Constantan 0.15 %
Na2HPO4 buffer Printex 90 fine
0.1
Dispersion medium
(Porter et al., 2008) 225 -1,3 Printex 90 coarse
0.1
Dispersion medium
(Porter et al., 2008) 475 n.m.
Hydrodynamic diameter (nm)
Record 94: P25 0,2% frisch 2
ζ potential (mV)
Record 6: TiO2 P25 30Min UT+30Min US 0,2%ig 2
Fig. 4.61 Characterisation of stock suspension TiO2 P25 on day of administration Table 4.5 BAL: TiO2 P25-treated rats
Record 94: P25 0,2% frisch 2 Record 301: Kontroll-BAL PPuffer-Spülung 15 min Att=6 Pos=1,05 2
Fig. 4.62 Control BAL 15 min following intratracheal instillation of 0.15 % phosphate buffer. Lavage: 1 x 5 ml 0.15 % phosphate buffer
0
Record 94: P25 0,2% frisch 2 Record 102: PPuffer-BAL nach P25 0,2% 15min Att=6 1
Fig. 4.63 BAL 15 min following intratracheal instillation of TiO2 P25
0
Record 94: P25 0,2% frisch 2 Record 106: PPuffer-BAL nach P25 0,2% 1h Att=6 2
Fig. 4.64 BAL 1 h following intratracheal instillation of TiO2 P25
0
Record 94: P25 0,2% frisch 2 Record 122: PPuffer-BAL nach P25 0,2% 4h Atten =6 3
Fig. 4.65 BAL 4 h following intratracheal instillation of TiO2 P25
0
Record 94: P25 0,2% frisch 2 Record 144: PPuff-BAl nach P25 0,2% 1d /Att=6 1
Fig. 4.66 BAL 24 h following intratracheal instillation of TiO2 P25
Hydrodynamic diameter (nm)
Record 127: T805 Stammlösung f risch 2
Hydrodynamic diameter after 17 h (nm)
0
Record 127: T805 Stammlösung frisch 2 Record 129: T805 Stammlösung 1Tag 1
Fig. 4.67 Characterisation of stock suspension TiO2 T805 on day of administration Table 4.6 BAL: TiO2 T 805-treated rats
Record 129: T805 Stammlösung 1Tag 1
Record 304: Kontroll-BAL NACL-Spülung 15 min Att=6 Pos=1,05 2
Fig. 4.68 Control BAL 15 min following intratracheal instillation of 0.15 % saline.
Lavage: 1 x 5 ml saline
0
Record 129: T805 Stammlösung 1Tag 1 Record 134: NaCl-BAL nach T805 0,2% 15 min /Att=6 3
Fig. 4.69 BAL 15 min following intratracheal instillation of TiO2 T805
0
Record 129: T805 Stammlösung 1Tag 1 Record 139: NaCl-BAL nach T805 0,2% 1h /Att=6 2
Fig. 4.70 BAL 1 h following intratracheal instillation of TiO2 T805
0
Record 129: T805 Stammlösung 1Tag 1 Record 148: NaCl-BAl nach T805 0,2% 4h /Att=6 2
Fig. 4.71 BAL 4 h following intratracheal instillation of TiO2 T805
0
Record 129: T805 Stammlösung 1Tag 1 Record 153: NaCl-BAL nach T805 0,2% 1d /Att=6 1
Fig. 4.72 BAL 24 h following intratracheal instillation of TiO2 T805
Fig. 4.73 TiO2 P25: BAL 1 h after intratracheal instillation; agglomerate size
inhomogeneous; agglomerates from 166 to 1000 nm; 40.6k magnification
Fig. 4.74 TiO2 T805: BAL 1 h after intratracheal instillation; agglomerate size homogeneous; small agglomerates; 41.2k magnification
Fig. 4.74A TiO2 T805 nanoparticles floating on the lung lining fluid or attached to cellular surfaces 1h after intracheal instillation to lungs
Fig. 4.74B TiO2 T805 nanoparticles intracellular in a macrophage 24 hrs after intracheal instillation to lungs
Hydrodynamic diameter (nm)
Record 231: Printex 90 /Att=6/0.1% im DMedium/50min Utur St3 +3minUSChSt Wdh+20min Uschall 3
Fig. 4.75 Characterisation of stock suspension Carbon black Printex® 90 fine on day of administration
ζ potential (mV): not measured
Table 4.7 BAL: Printex® 90 fine-treated rats
- A reduction in the count rate with time is evident.
- Curves at different timepoints are similar (no change in size) - BAL fluid did barely contain Printex® 90; in the cell sediment a very slight black discoloration was visible
0
Record 231: Printex 90 /Att=6/0.1% im DMedium/50min Utur St3 +3minUSChSt Wdh+20min Uschall 3 Record 286: Kontrolle DM 15 min Att=6 Pos=Auto 2
Fig. 4.76 Control BAL 15 min following intratracheal instillation of 0.15 % saline.
Lavage: 1 x 5 ml dispersion medium (DM)
0
Record 231: Printex 90 /Att=6/0.1% im DMedium/50min Utur St3 +3minUSChSt Wdh+20min Uschall 3 Record 243: Printex 90 0.1% in DM BAL 15 min 3
Fig. 4.77 BAL 15 min following intratracheal instillation of Printex® 90 fine
0
Record 231: Printex 90 /Att=6/0.1% im DMedium/50min Utur St3 +3minUSChSt Wdh+20min Uschall 3 Record 264: Printex 90 0.1% in DM BAL 1h , Att=6 Pos=1,05 2
Fig. 4.78 BAL 1 h following intratracheal instillation of Printex® 90 fine
0
Record 231: Printex 90 /Att=6/0.1% im DMedium/50min Utur St3 +3minUSChSt Wdh+20min Uschall 3 Record 268: Printex 90 0.1% in DM BAL 4h , Att=6 Pos=1,05 1
Fig. 4.79 BAL 4 h following intratracheal instillation of Printex® 90 fine
0
Record 231: Printex 90 /Att=6/0.1% im DMedium/50min Utur St3 +3minUSChSt Wdh+20min Uschall 3 Record 308: fein Printex 90 0.1% in DM BAL 1d, Att=6 Pos=1.05 2
Fig. 4.80 BAL 24 h following intratracheal instillation of Printex® 90 fine
Fig. 4.81 Printex® 90 fine: stock solution
Hydrodynamic diameter (nm)
Record 279: grob Printex 90 0.1% in DM 13min UschB+8 min Utr/St3 1
Fig. 4.82 Characterisation of stock suspension Carbon black Printex® 90 coarse on day of administration
ζ potential (mV): not measured
Table 4.8 BAL: Printex® 90 coarse-treated rats
- A reduction in the count rate with time is evident.
- Curves at different timepoints are similar (no change in size) - BAL fluid did barely contain Printex® 90; in the cell sediment a very slight black discoloration was visible
0
Record 279: grob Printex 90 0.1% in DM 13min UschB+8 min Utr/St3 1 Record 286: Kontrolle DM 15 min Att=6 Pos=Auto 2
Fig. 4.83 Control BAL 15 min following intratracheal instillation of 0.15 % saline.
Lavage: 1 x 5 ml dispersion medium (DM)
0 5 10 15 20
0.1 1 10 100 1000 10000
Intensity (%)
Size (d.nm) Size Distribution by Intensity
Record 279: grob Printex 90 0.1% in DM 13min UschB+8 min Utr/St3 1 Record 291: grob Printex 90 0,1% / 15 min Att=6 Pos=1.05 1
Fig. 4.84 BAL 15 min following intratracheal instillation of Printex® 90 coarse
0 5 10 15
0.1 1 10 100 1000 10000
Intensity (%)
Size (d.nm) Size Distribution by Intensity
Record 279: grob Printex 90 0.1% in DM 13min UschB+8 min Utr/St3 1 Record 296: grob Printex 90 0,1% / 1h Att=6 Pos=Auto 2
Fig. 4.85 BAL 1 h following intratracheal instillation of Printex® 90 coarse
0 5 10 15
0.1 1 10 100 1000 10000
Intensity (%)
Size (d.nm) Size Distribution by Intensity
Record 279: grob Printex 90 0.1% in DM 13min UschB+8 min Utr/St3 1 Record 300: grob Printex 90 0.1% /4h Att=6 Pos=1,05 1
Fig. 4.86 BAL 4 h following intratracheal instillation of Printex® 90 coarse BAL 24 h following intratracheal instillation of Printex® 90 coarse not measured