The major goal of this study was to evaluate the suitability of utilizing Survanta®
as a dispersing agent for SWCNT to aid study of their bioactivity. The rationale behind its use include its simplicity and rapidity of nanoparticle dispersion (i.e., it is a single step process), biocompatibility (i.e., it has been approved for human clinical use), and commercial availability particularly as a sterile preparation which greatly facilitates in vivo
and in vitro
studies that require sterile conditions. In addition, one would argue that inhaled SWCNT would initially interact with alveolar lining fluid, which is modeled by Survanta®
suspension. However, the effectiveness of this preparation in dispersing nanoparticles and its possible interfering effect on the bioactivities of SWCNT are not known, and, therefore, are investigated in the present study. The data presented demonstrate that Survanta®
when used at the indicated concentrations is effective in dispersing SWCNT, yielding nanoparticles with dimensions similar to those observed after aerosolization of dry SWCNT or acetone/sonication dispersion of SWCNT [5
]. Non-dispersed SWCNT form large agglomerates in phosphate-buffered saline (PBS) with an average width of 12.35 μm and an average length of 27.72 μm. In contrast, Survanta®
-dispersed SWCNT form much smaller structures with an average width of 0.38 μm and an average length of 1.42 μm. The majority of the dispersed SWCNT is in the form of small bundles with no or minimum detectable individual nanotubes. The reported average diameters of aerosolized dry SWCNT and acetone/sonication-dispersed SWCNT are 0.24 μm and 0.6 μm, respectively [5
]. These results are in good agreement with previous reports showing count median aerodynamic diameter of dry CNT generated aerosols for inhalation studies [14
], structure size distribution found in the workplace [17
], and CNT structures produced by surfactant dispersion [18
]. The data indicate that aerosolized dry SWCNT and surfactant-dispersed SWCNT form much smaller aggregates than SWCNT suspended in PBS. Previous animal studies also showed that well-dispersed SWCNT after pulmonary administration exhibited similar deposited structures and dimensions (e.g., submicron-sized aggregates) as those observed in this study [5
Ideally, in vivo
pulmonary exposure studies should be performed using an inhalation method, since it best mimics the human exposure condition. However, the need for specialized facilities and equipment, trained personnel, and large quantities of nanoparticles has limited the use of this technology. Pulmonary aspiration represents an alternative method that has proven useful in many pulmonary toxicity studies. This method is simple, economical, uses small amounts of nanoparticles, and provides deep lung deposition as well as high correlation to the administered dose [19
]. Recent studies by our group have shown that CNT administered by this method produced pulmonary fibrosis in lab animals similar to that observed after inhalation of CNT [5
]. These studies suggest that the aspiration method is a reasonable alternative method to inhalation for the study of pulmonary fibrosis induced by nanoparticles. Present SWCNT-size analysis further supports the aspiration method using SD-SWCNT in which the average diameter is comparable with aerosolized dry SWCNT using for inhalation study.
The use of Survanta®
for nanoparticle dispersion provides an additional advantage over other methods of dispersion for pulmonary studies as it better mimics the natural lung condition. This is particularly important for in vitro
studies which normally lack lung surfactants that could have an effect on cell interaction and bioactivity of nanoparticles. Previous studies have shown that lung surfactants aid in the displacement of particles from air to the aqueous phase and towards the lung epithelium [19
]. In addition, when particles are present in peripheral airways and alveoli they exist in a completely immersed, wetted state below the surfactant film [20
]. These studies suggest that experiments using lung surfactants may be more physiologically relevant than non-surfactant systems.
A key concern about the use of Survanta®
is its possible adverse effects on cells and tissues or masking effect on the exposed particles [21
]. Our results show that Survanta®
, when used at the indicated concentrations, had no significant cytotoxic effect on lung cells in vitro
and did not induce collagen production or mask the fibrogenic effect of SWCNT either in vitro
or in vivo
. The results of this study also indicate that dispersion status of SWCNT is a key determinant of its biological activities to induce cell proliferation and enhance collagen production.
Another key finding of this study is the correlation between in vitro and in vivo fibrogenic responses to SWCNT and control particles under different dispersion conditions. This finding suggests the potential utility of in vitro lung fibroblasts as a predictive model for in vivo fibrogenicity testing of CNT and other nanomaterials. Fibrogenicity testing of nanomaterials is usually performed using animals. However, this method of testing is time-consuming, laborious, and costly. This combined with the rapid growth in nanotechnology, which produces an uncountable number and variety of nanomaterials, makes it impractical to test all of these materials using animals. The in vitro model described here represents an alternative method that could serve as a rapid screening tool for fibrogenicity testing of a large number of nanomaterials. This model can also be used to conduct detailed mechanistic studies of the fibrogenic effect of nanoparticles, which may not be achievable in vivo.