Selective assembly of DNA-conjugated single-walled carbon nanotubes from the vascular secretome

Abstract

Colloidal dispersion of single-walled carbon nanotubes (SWCNTs) is often the first processing step to many of their unique applications. However, dispersed SWCNTs often exist in kinetically trapped states where aggregation can be of concern. Recent work revealed prominent DNA-SWCNT aggregation following intravascular injection. In this study, we performed detailed analysis of DNA-SWCNT aggregate formation, structure, and composition in the context of endothelial cell condition media. Interestingly, we found that aggregates formed within condition media from cells that have undergone a stress response differ in size and organization from that of the control. We also found that temperature increases also promote DNA-SWCNT associations. A mathematical model was developed to describe the kinetics of SWCNT extraction from solution. Through orthogonal optical analysis and imaging modalities, we verified that proteins form the bulk of the aggregate structure and dictate aggregate assembly at multiple levels of organization. Finally, physiochemical analysis indicated preferential extraction of low-abundance hydrophobic and charged proteins. The formed aggregates also remain relatively stable in solution, making them potential macroscopic indicators of solution content.

Keywords: DNA; aggregation; endothelial; secretome; self-assembly; single-walled carbon nanotubes.

Figure 1

Aggregate formation conditions. (A) Different solutions exhibit different degrees of SWCNT aggregation. Media components having no effect were ruled out. (B) Visual comparison of aggregation formation of starving media and conditioned media. Aggregations formed are stable after 2 weeks. (C) Strong nIR signal from aggregates showed that SWCNT DNA wrapping is likely undisturbed.

Figure 2

Photoluminescence. (A) Example emission spectra from laser excitation. (B) Integrated PL emissions from five lasers normalized to preincubation PBS control (the PBS plotted is after experimental incubation). (C) Example peak-shift between control and conditioned media. (D) Peak-shifts from control. Dotted line indicating approximate instrument sensitivity.

Figure 3

Raman spectroscopy. Radial breathing mode (RBM) region of the SWCNT Raman spectra shows changes in band gap energy consistent with differences in aggregation structure.

Figure 4

Bright-field image analysis. (A) Inverted bright-field images of SWCNT aggregates formed from different media conditions, showing differences in size and structure. (B) Work flow of image analysis code. (C) Resulting D₃ fractal coefficients from calculation show conditioned media aggregates with higher structural complexity.

Figure 5

SEM imaging. SEM of SWCNTs with or without gold coating showed aggregates consisted of both protein and an underlying network of SWCNTs. Starving media aggregates show consistently less density and interconnectedness.

Figure 6

SPT temperature response. (A) SPT of (AT)₁₅–SWCNT shows aggregation formation following a change from 24 °C to 37 °C, a phenomenon not present with SDS–SWCNTs. (B) SPT analysis of particle size distribution for temperature changes and solution disturbance. Imaging of a SWCNT dimer shows two connected but distinct elements. Linear fitting of size peaks shows each SWCNT contributes 183 nm in particle size to the aggregate.

Figure 7

Aggregation kinetics. (A) SPT shows aggregation extractions of total SWCNTs from solution. (B) Approximating total SWCNT volume through 8 h of aggregation at 37 °C shows significant SWCNT extraction. (C) Comparison of protein concentration through BCA assay for two media types. (D) Amount of free protein postaggregation as a function of SWCNT concentration for conditioned media.

Figure 8

Gel quantification. (A) Silver stain of proteins within SWCNT aggregates from full media, conditioned media, and in the presence of cells are compared along with control. A ∼500 kDa protein aggregate band is also observed, which can be eliminated via sonication. (B) Quantification of band intensities normalized to albumin shows differential SWCNT preference for certain protein species.

Figure 9

Mass spectroscopy analysis. (A) Top 12 high-concentration proteins near the molecular weight of the gel bands are compared against BSA in a panel of material properties. Results show preferred proteins to be more hydrophobic and contain more charged amino acids. (B) Hydrophobicity versus length for detected protein species. (C) Detected protein cellular distribution, showing a majority to be either cytoplasmic or nuclear. Statistical tests for protein properties between three groups.