Multi-Drug/Gene NASH Therapy Delivery and Selective Hyperspectral NIR Imaging Using Chirality-Sorted Single-Walled Carbon Nanotubes

Abstract

Single-walled carbon nanotubes (SWCNTs) can serve as drug delivery/biological imaging agents, as they exhibit intrinsic fluorescence in the near-infrared, allowing for deeper tissue imaging while providing therapeutic transport. In this work, CoMoCAT (Cobalt Molybdenum Catalyst) SWCNTs, chirality-sorted by aqueous two-phase extraction, are utilized for the first time to deliver a drug/gene combination therapy and image each therapeutic component separately via chirality-specific SWCNT fluorescence. Each of (7,5) and (7,6) sorted SWCNTs were non-covalently loaded with their specific payload: the PI3 kinase inhibitor targeting liver fibrosis or CCR5 siRNA targeting inflammatory pathways with the goal of addressing these processes in nonalcoholic steatohepatitis (NASH), ultimately to prevent its progression to hepatocellular carcinoma. PX-866-(7,5) SWCNTs and siRNA-(7,6) SWCNTs were each imaged via characteristic SWCNT emission at 1024/1120 nm in HepG2 and HeLa cells by hyperspectral fluorescence microscopy. Wavelength-resolved imaging verified the intracellular transport of each SWCNT chirality and drug release. The therapeutic efficacy of each formulation was further demonstrated by the dose-dependent cytotoxicity of SWCNT-bound PX-866 and >90% knockdown of CCR5 expression with SWCNT/siRNA transfection. This study verifies the feasibility of utilizing chirality-sorted SWCNTs for the delivery and component-specific imaging of combination therapies, also suggesting a novel nanotherapeutic approach for addressing the progressions of NASH to hepatocellular carcinoma.

Keywords: NASH; chirality separation; drug-gene delivery; near IR hyperspectral imaging; single-walled carbon nanotubes.

Figure 1

Measured and simulated fluorescence spectra of separated (a) (7,5), (b) (7,6) chiral single-walled carbon nanotubes (SWCNTs). The corresponding generated photoluminescence–excitation contour plot of (c) (7,5), (d) (7,6) sorted fractions. Graphene sheet representing the distribution of the emissive species in the respective (e) (7,5) and (f) (7,6) enriched sorted SWCNT samples. The blue-filled portion of hexagons represent the relative abundance of the species.

Figure 2

Near-infrared (NIR) hyperspectral images of (7,5) sorted SWCNTs at (a) 990, (b) 1030, and (c) 1130 nm; and (7,6) sorted SWCNTs at (d) 990, (e) 1030, and (f) 1130 nm. SWCNT fluorescence is only observed at the emission wavelengths of the corresponding sorted SWCNTs.

Figure 3

(a) Fluorescence spectra of raw SWCNTs dispersed with sorting surfactants, raw SWCNTs dispersed with siRNA, and SWCNTs washed/annealed for surfactant removal and re-dispersed with siRNA showing similar peak positions for raw SWCNTs dispersed with siRNA and washed/annealed SWCNTs dispersed with siRNA. (b) Cell viability of HepG2 cells subject to SWCNTs dispersed with sorting surfactants, raw SWCNTs dispersed with siRNA, and SWCNTs washed/annealed for surfactant removal and re-dispersed with siRNA.

Figure 4

Emission spectra of raw CoMoCAT SWCNTs dispersed with (a) PX-866, (b) siRNA. Emission spectra of (c) (7,5) and (7,6) sorted washed/annealed SWCNTs dispersed with PX-866 and CCR5 siRNA, respectively. (d) Visible emission spectrum of PX-866 with 400 nm excitation.

Figure 5

Brightfield/fluorescence overlay images of (a) untreated control HepG2 cells and (b,c) cellular uptake of (7,5) sorted SWCNTs imaged at 1030 nm (red), (7,6) sorted SWCNTs imaged at 1130 nm (blue), and PX-866 imaged at 535 nm (green) after the intracellular release.

Figure 6

MTT assay cell viability of HepG2 cells treated with either SWCNTs, PX-866, or SWCNT/PX866 conjugates.

Figure 7

Downregulation of CCR5 in HepG2 cells by siRNA/nanotubes complex. CCR5 expression was detected by flow cytometry after nanotubes-delivered siRNA-mediated knockdown for 48 h. IgG staining was used for isotype control (left). Control indicated natural expression of CCR5 in HepG2 cells without any treatment (middle) compared to CCR5-targeting siRNA/nanotubes treatment (right).