Toxicodynamics of rigid polystyrene microparticles on pulmonary gas exchange in mice: implications for microemboli-based drug delivery systems

Abstract

The toxicodynamic relationship between the number and size of pulmonary microemboli resulting from uniformly sized, rigid polystyrene microparticles (MPs) administered intravenously and their potential effects on pulmonary gas exchange were investigated. CD-1 male mice (6-8 weeks) were intravenously administered 10, 25 and 45 μm diameter MPs. Oxygen hemoglobin saturation in the blood (SpO(2)) was measured non-invasively using a pulse oximeter while varying inhaled oxygen concentration (F(I)O(2)). The resulting data were fit to a physiologically based non-linear mathematical model that estimates 2 parameters: ventilation-perfusion ratio (V(A)/Q) and shunt (percentage of deoxygenated blood returning to systemic circulation). The number of MPs administered prior to a statistically significant reduction in normalized V(A)/Q was dependent on particle size. MP doses that resulted in a significant reduction in normalized V(A)/Q one day post-treatment were 4000, 40,000 and 550,000 MPs/g for 45, 25 and 10 μm MPs, respectively. The model estimated V(A)/Q and shunt returned to baseline levels 7 days post-treatment. Measuring SpO(2) alone was not sufficient to observe changes in gas exchange; however, when combined with model-derived V(A)/Q and shunt early reversible toxicity from pulmonary microemboli was detected suggesting that the model and physical measurements are both required for assessing toxicity. Moreover, it appears that the MP load required to alter gas exchange in a mouse prior to lethality is significantly higher than the anticipated required MP dose for effective drug delivery. Overall, the current results indicate that the microemboli-based approach for targeted pulmonary drug delivery is potentially safe and should be further explored.

FIGURE 1

Model of a 1 compartment lung. P_xO2 is partial pressure of O₂ and C_xO2 is the concentration of O₂ in compartment x, where x is the arterial (a), venous (v) or mixed pulmonary capillary (c) compartment; Q[dot] is blood flow; F_IO₂ is the fraction of inspired O₂; Shunt is the percentage of deoxygenated blood passing through the lung. Blue indicates deoxygenated blood, red indicates oxygenated blood.

FIGURE 2

Theoretical SpO₂ vs. F_IO₂ curves. Values of shunt increase (0, 5, 10, 20, 30%) resulting in a downward shift. Decreased V_A/Q (1.1, 1.0, 0.9, 0.8, 0.7, 0.6) resulting in a rightward shift. There is little apparent change to the shape of the curves. Dotted line indicates F_IO₂ of room air at sea level.

FIGURE 3

Change of SpO₂ breathing room air (F_IO₂ =21%) between Day −3 (pretreatment) (A) and Day 1 (post-treatment) (B). ANOVA analysis indicates P<0.05 however Dunnett’s post-hoc analysis only indicates the 8000 45 μm group as statistically different.

FIGURE 4

Two typical SpO₂ vs. F_IO₂ curves of animals receiving low (150,000 10 μm MPs/g) or high (550,000 10 μm MPs/g) shown on Day −3 (pretreatment, black) and Day 1 (red). Each symbol represents the same animal on different days. The connecting lines are for clarity in the top (A, B). The best-fit lines are shown in (C, D). There is a rightward shift for the high dose treatment group, indicating a statistically significant decrease in V_A/Q.

FIGURE 5

Time course of V_A/Q and Shunt for MPs of 10, 25 and 45 μm MPs from pre-treatment (Day −3 and Day −1) to post-treatment (Day 1 through Day 7). The black line represents shunt and the red V_A/Q. The connected line is through the mean of the value. Error bars indicate SE. Individual V_A/Q is plotted as open symbols and shunt is closed symbols. Similar shapes indicate the same animal (i.e., a red open box is animal 1’s V_A/Q and a closed black box is animal 1’s shunt).

FIGURE 5

Time course of V_A/Q and Shunt for MPs of 10, 25 and 45 μm MPs from pre-treatment (Day −3 and Day −1) to post-treatment (Day 1 through Day 7). The black line represents shunt and the red V_A/Q. The connected line is through the mean of the value. Error bars indicate SE. Individual V_A/Q is plotted as open symbols and shunt is closed symbols. Similar shapes indicate the same animal (i.e., a red open box is animal 1’s V_A/Q and a closed black box is animal 1’s shunt).

FIGURE 5

Time course of V_A/Q and Shunt for MPs of 10, 25 and 45 μm MPs from pre-treatment (Day −3 and Day −1) to post-treatment (Day 1 through Day 7). The black line represents shunt and the red V_A/Q. The connected line is through the mean of the value. Error bars indicate SE. Individual V_A/Q is plotted as open symbols and shunt is closed symbols. Similar shapes indicate the same animal (i.e., a red open box is animal 1’s V_A/Q and a closed black box is animal 1’s shunt).

FIGURE 6

Data grouped by MP size to look at changes in V_A/Q over time. Left, raw data; Right, normalized data to the individual animal using their average V_A/Q value on Day −3 and Day −1 (pretreatment).

FIGURE 7

Changes in V_A/Q (A, B) and Shunt (C, D) compared to total cross-sectional area/volume defined as Number of MPs times area/volume of an individual MP.

FIGURE 8

Immunohistochemistry staining for PCNA on Day 7. Control animals have little to no staining in the septal walls of the lung (A). Injection of different sized MPs does not change PCNA localization (B, D). Injection of increased number of MPs does not change PCNA relative expression (C, D). Original magnification, 4x.