Real-time assessment of cigarette smoke particle deposition in vitro

Abstract

Background: Recently there has been a rapid increase in approaches to assess the effects of cigarette smoke in vitro. Despite a range of gravimetric and chemical methods, there is a requirement to identify simpler and more reliable methods to quantify in vitro whole smoke dose, to support extrapolation and comparisons to human/in vivo dose. We have previously characterised an in vitro exposure system using a Borgwaldt RM20S smoking machine and a chamber exposing cellular cultures to whole smoke at the air-liquid interface. In this study we demonstrate the utility of a quartz crystal microbalance (QCM), using this exposure system, to assess real-time cigarette smoke particulate deposition during a 30 minute smoke exposure. Smoke was generated at various dilutions (1:5-1:400, smoke:air) using two cigarette products, 3R4F Kentucky reference and 1 mg commercially available cigarettes. The QCM, integrated into the chamber, assessed particulate deposition and data generated were compared to traditional chemical spectrofluorometric analysis.

Results: The QCM chamber was able to detect mass differences between the different products within the nanogram range. 3R4F reference cigarette smoke deposition ranged from 25.75 ±2.30 μg/cm2 (1:5) to 0.22 ±0.03 μg/cm2 (1:400). 1 mg cigarette smoke deposition was less and ranged from 1.42 ±0.26 μg/cm2 (1:5), to 0.13 ±0.02 μg/cm2 (1:100). Spectrofluorometric analysis demonstrated statistically significant correlation of particulate deposition with the QCM (p < 0.05), and regression R2 value were 97.4 %. The fitted equation for the linear model which describes the relationship is: QCM = -0.6796 + 0.9744 chemical spectrofluorescence.

Conclusions: We suggest the QCM is a reliable, effective and simple tool that can be used to quantify smoke particulate deposition in real-time, in vitro and can be used to quantify other aerosols delivered to our chamber for assessment.

Figure 1

British American Tobacco’s standard exposure chamber used forin vitroexposures to whole smoke at the ALI [A], and a schematic cross-section [B]. For extraction of deposited particulate matter for spectrofluorescence analysis, * illustrates the level reached by 2 ml extraction solvent when added to the exposed cell culture insert, rising 0.56 cm up the inner wall (diagram adapted from [11]).

Figure 2

A single QCM unit housed within the exposure chamber (side view) [A]; a schematic cross-section of the QCM exposure chamber [B]; and a top view of the chamber base showing that the QCM fits into and replaces the position of one of the three cell support inserts [C2], allowing the remaining two positions to house cell support inserts if required for parallel exposure [C1, 3]. Internal surface area and volume changes upon installation of the single QCM unit were nominal, compared to the original chamber geometry. Crystal ø = 2.5 cm; cell support insert ø = 2.4 cm; crystal’s gold electrode ø = 1.3 cm.

Figure 3

Real-time traces of QCM deposited mass in the exposure chamber, showing a 3R4F cigarette smoked at a dilution of 1:400 [A], and a 1 mg commercially available cigarette smoked at a dilution of 1:100 [B]. Observe the plateau phase after the smoke run has finished, showing that stabilisation of the crystal is constant after approximately 10 minutes.

Figure 4

Individual value plots showing QCM particulate deposition over a range of whole smoke dilutions tested for 3R4F reference cigarettes [A] and 1 mg commercially available cigarettes [B], for a 30 minute whole smoke exposure (n=5).

Figure 5

Fitted line plot comparison of QCM and chemical spectrofluorescence assessment of whole smoke particulate deposition over a range of smoke dilutions for both 3R4F reference cigarette and a 1 mg commercially available cigarette (R² = 97.4%). Solid black line = Regression, dashed red line = 95 % CI (confidence intervals), dotted green line = 95 % PI (prediction intervals). For both cigarette types the relationship between the two methods was statistically significant (p < 0.05); this was based on a sample size large enough (n = 105) to obtain a precise estimate of the strength of the relationship. The fitted equation for the linear model which describes the relationship is: QCM = − 0.6796 + 0.9744 chemical spectrofluorescence.