Comparison of nanotube-protein corona composition in cell culture media

Abstract

In biological environments, nanomaterials associate with proteins forming a protein corona (PC). The PC may alter the nanomaterial's pharmacokinetics and pharmacodynamics, thereby influencing toxicity. Using a label-free mass spectrometry-based proteomics approach, the composition of the PC is examined for a set of nanotubes (NTs) including unmodified and carboxylated single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), polyvinylpyrrolidone (PVP)-coated MWCNT (MWCNT-PVP), and nanoclay. NTs are incubated for 1 h in simulated cell culture conditions, then washed, resuspended in PBS, and assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for their associated PC. To determine those attributes that influence PC formation, the NTs are extensively characterized. NTs had negative zeta potentials in water (SWCNT-COOH < MWCNT-COOH < unmodified NTs) while carboxylation increases their hydrodynamic sizes. All NTs are also found to associate a common subset of proteins including albumin, titin, and apolipoproteins. SWCNT-COOH and MWCNT-COOH are found to bind the greatest number of proteins (181 and 133 respectively) compared to unmodified NTs (<100), suggesting covalent binding to protein amines. Modified NTs bind a number of unique proteins compared to unmodified NTs, implying hydrogen bonding and electrostatic interactions are involved in PC formation. PVP-coating of MWCNT did not influence PC composition, further reinforcing the possibility of hydrogen bonding and electrostatic interactions. No relationships are found between PC composition and corresponding isoelectric point, hydropathy, or aliphatic index, implying minimal roles of hydrophobic interaction and pi-stacking.

Figure 1

Scanning electron microscopy images of nanoclay, unmodified single walled carbon nanotubes (SWCNT-Raw), carboxylated SWCNT (SWCNT-COOH), unmodified multi-walled carbon nanotubes (MWCNT-Raw), pure MWCNT (MWCNT-Pure), carboxylated MWCNT (MWCNT-COOH), and polyvinylpyrrolidone-coated MWCNT (MWCNT-PVP) samples confirming the dimensions of all carbon NTs used in this study.

Figure 2

Characterization of nanoclay, unmodified single walled carbon nanotubes (SWCNT-Raw), carboxylated SWCNT (SWCNT-COOH), unmodified multi walled carbon nanotubes (MWCNT-Raw), pure MWCNT (MWCNT-Pure, carboxylated MWCNT (MWCNT-COOH), and polyvinylpyrrolidone-coated MWCNT (MWCNT-PVP) samples. 2A) Hydrodynamic size for each NT was assessed in both water and DMEM cell culture media via dynamic light scattering. 2B) Zeta potentials for each NT were determined in water via electrophroretic light scattering.

Figure 3

Total number and number of unique proteins found to associate with NTs after incubation in DMEM cell culture media containing 10% fetal bovine serum. Samples were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and proteins and peptides were identified using the UniProtKB Bos Taurus (Bovine) database and validated by PeptideProphet. Only proteins with a probability ≥ 0.9, or peptides with a probability ≥ 0.8, and a peptide weight ≥ 0.5 were used in the quantitation algorithm. 3A) The total number of constituent proteins detected in each NT protein corona. 3B) The number of unique proteins detected in each NT protein corona.

Figure 4

Grand average of hydophathicity (GRAVY) scores for all identified proteins found to associate with NTs after incubation in DMEM cell culture media containing 10% fetal bovine serum compared to their relative abundance. The ten most abundant peptides/proteins are denoted in red within the graph. GRAVY scores were calculated for a peptide or protein based on the sum of hydropathy values for all amino acids and divided by the number of residues in the sequence.