Scavenger Receptor A1 Distinguishes Bovine Serum Albumin-Coated Pristine and Carboxylated Multi-Walled Carbon Nanotubes

This dissertation concerns the interactions of multi-walled carbon nanotubes (MWNTs) with mammalian macrophages, cells that are first responders to foreign invaders in the body. The production and use of MWNTs are rapidly increasing world-wide despite the possible adverse effects they may have on human health. For example, MWNTs pose a human respiratory hazard because they can cause pulmonary fibrosis, which may lead to mesothelioma. How MWNTs trigger such adverse effects is not well understood, especially whether MWNT binding to surface receptors on macrophages occurs. A complicating factor is what effects a protein corona, derived from serum proteins such as bovine serum albumin (BSA), may have on the interaction of MWNTs with cells. Achieving consensus in this research field is hampered by batch-to-batch inconsistencies with commercially synthesized MWNTs. This dissertation first presents a comprehensive physicochemical characterization of two lots of pristine MWNTs (pMWNTs) and carboxylated MWNTs (cMWNTs), which is important since the biological response of MWNTs is related to their physicochemical properties. There were many similarities between the physicochemical properties of the two commercial lots of cMWNTs and neither significantly diminished the 24-h proliferation of RAW 264.7 macrophages up to the highest concentration tested (200 μg cMWNTs/mL). Conversely, several physicochemical properties of the two lots of pMWNTs were different: notably, the newer lot of pMWNTs displayed less oxidative stability, a higher defect density, and a smaller amount of surface oxygen species relative to the original lot. Furthermore, a 72-h half maximal inhibitory concentration of ~90 µg pMWNTs/mL was determined for RAW 264.7 cells with the new lot of pMWNTs. These results demonstrate that subtle physicochemical differences can lead to significantly dissimilar cellular responses, and that production-lot consistency must be considered when assessing the toxicity or biomedical performance of MWNTs. Next, using the lots of well-characterized pMWNTs and cMWNTs, the interaction of MWNTs with class A-type 1 scavenger receptors (SR-A1s) was studied with a direct binding assay under conditions where the influence of nanotube functionalization and protein coronas could be carefully controlled. Both pMWNTs and cMWNTs coated with BSA bound to and were accumulated by RAW 264.7 macrophages, although the cells bound two times more BSA-coated cMWNT than pMWNTs. RAW 264.7 cells that were deleted for SR-A1 using CRISPR-Cas9 gene-editing technology had markedly reduced binding and accumulation of both BSA-coated cMWNTs and pMWNTs, suggesting that SR-A1 was responsible for the uptake of both MWNT types. Moreover, Chinese hamster ovary (CHO) cells that ectopically expressed SRA1 accumulated both MWNT types, whereas wild-type CHO cells did not. One model to explain these results is that SR-A1 can interact with two structural features of BSA-coated cMWNTs, one inherent to the carboxylated nanotubes and the other provided by the BSA corona, whereas SRA1 only interacts with the BSA corona of BSA-pMWNTs. A better understanding of the mechanisms by which MWNTs interact with macrophages should lead to the rational design of nanotoxicity remediation efforts and biomedical applications of carbon nanomaterials.