Photothermal modulation of human stem cells using light-responsive 2D nanomaterials

Abstract

Two-dimensional (2D) molybdenum disulfide (MoS₂) nanomaterials are an emerging class of biomaterials that are photoresponsive at near-infrared wavelengths (NIR). Here, we demonstrate the ability of 2D MoS₂ to modulate cellular functions of human stem cells through photothermal mechanisms. The interaction of MoS₂ and NIR stimulation of MoS₂ with human stem cells is investigated using whole-transcriptome sequencing (RNA-seq). Global gene expression profile of stem cells reveals significant influence of MoS₂ and NIR stimulation of MoS₂ on integrins, cellular migration, and wound healing. The combination of MoS₂ and NIR light may provide new approaches to regulate and direct these cellular functions for the purposes of regenerative medicine as well as cancer therapy.

Keywords: cell adhesion; cell–nanoparticles interactions; human mesenchymal stem cells; two-dimensional (2D) nanomaterials; whole-transcriptome sequencing.

Fig. 1.

Physicochemical characterization of exfoliate 2D MoS₂ nanosheets. (A) XRD of bulk and exfoliated MoS₂. (B) AFM measures micrometer-sized nanosheets with nanometer thickness, confirming 2D shape. (C) TEM images of ultrathin MoS₂ sheets displayed overlapping layers; each individual layer is numbered. Further, electron diffraction corroborated the formation of the 1T phase following lithium intercalation. (D) Atomic composition of bulk and exfoliated MoS₂ determined via elemental analysis. (E) Crystallographic transformations from bulk to exfoliated MoS₂ were monitored with XPS analysis with shifts from 2H to 1T phases observed. (F) Raman spectroscopy and (G) photoluminescent measurements likewise indicated structural modifications had occurred, seen through changes in vibrational energy bands and luminescent intensity, respectively. (H) Response to NIR light was determined using an IR camera and change in temperature over time. (I) Zeta potential and hydrodynamic size of exfoliated MoS₂ in water, PBS, and media. (J) ANS assay shows protein structures are intact in the presence of exfoliated MoS₂.

Fig. 2.

Cellular interactions of 2D MoS₂ with hMSCs. (A) A protein corona forms when 2D MoS₂ is suspended in media and adheres to the cell surface and is subsequently internalized by cells. (B) IC₅₀ of MoS₂ determined using metabolic assays. (C) Cellular internalization of MoS₂ determined using optical, electron, and fluorescence microscopy. (D) MoS₂ adhere to cell membrane and internalize via micropinocytosis and clathrin-mediated endocytosis. (E) Effect of MoS₂ on metabolic activity over 7 d. (F) Effect of Mo and MoS₂ nanosheet at equivalent molarities on cell viability (day 3 and day 7 on the left and right, respectively). (G) Effect of MoS₂ on cell cycle. (H) Cell viability when treated with NIR, MoS₂, and MoS₂_NIR. *P < 0.05; not significant (n.s.) > 0.05.

Fig. 3.

Understanding the effect of NIR, MoS₂, and MoS₂_NIR on hMSCs using RNA-seq. (A) Pearson correlation coefficient of expressed genes between replicates of different conditions (r = 0.98 for hMSC controls, r = 0.99 for hMSCs_MoS_2, r = 0.99 for hMSCs_NIR, and r = 0.98 for hMSCs_MoS₂_NIR). (B) MA plot highlighting differentially regulated genes (FDR-adjusted P < 0.05) of the treatment groups against the untreated hMSCs and extent of expression change (gray: all of the expressed genes, red: up-regulated genes, blue: down-regulated genes). Venn diagram indicating differential genes overlap between pairwise comparison of treatment groups. (C) Heat map of row normalized FPKM z-scores of differentially expressed genes across all pairwise comparisons against the untreated hMSCs (red: up-regulated, blue: down-regulated).

Fig. 4.

Functional effect of NIR, MoS₂, and MoS₂_NIR on cells. (A) GO terms obtained by gene enrichment analysis of DEGs (FDR-adjusted P < 0.05) due to MoS₂_NIR treatment. (B) GeneMANIA analysis produced a network of coexpression interactions for differentially expressed genes within the hMSCs_MoS₂_NIR population (red: up-regulated, blue: down-regulated; size increases with significance). (C) A volcano plot for the wound healing (GO:0042060), gray: all of the expressed genes, blue: genes associated with the GO term with no significant change in expression, red: genes associated with the GO term that show significantly differential expression due to treatment. (D) Scratch assay used to determine the effect of NIR, MoS₂, and MoS₂_NIR on migration, cell proliferation, and wound healing of hMSCs, hSMCs, and MCF-7. The dotted lines represent scratch generated using pipette tip. The cell migration across the wound area was determined by imaging the sample after 12, 24, and 48 h. (E) Three-dimensional invasion assay demonstrating ability of HUVECs treated with MoS₂ and MoS₂_NIR to invade collagen gel.

Fig. 5.

Role of integrin expression on cell adhesion due to NIR, MoS₂, and MoS₂_NIR treatment. (A) Schematic showing the potential interaction between MoS₂ nanosheets and integrin molecules at the cell surface. (B) Quantification of unbound integrin beta-1 (CD29) in cells treated with and without MoS₂ nanosheets. (C) A volcano plot for the cell adhesion (GO:0007155), gray: all of the expressed genes, blue: genes associated with the GO term with no significant change in expression, red: genes associated with the GO term that show significant difference in expression due to treatment. (D) Gene track showing normalized mRNA expression of integrin alpha-7 (ITGA7) and smooth muscle alpha-2 actin (ACTA2) for hMSCs, hMSCs_NIR, hMSCs_ MoS₂, and hMSCs MoS₂_NIR. (E) Protein expression level of ITGA7, FAK, vinculin, paxillin, and smooth muscle actin (ACTA2) determined using Western blot for hMSCs, hMSCs_NIR, hMSCs_ MoS₂, and hMSCs MoS₂_NIR. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is internal control. *P < 0.05, ***P < 0.001.