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Cell Death Versus Cell Survival Instructed by Supramolecular Cohesion of Nanostructures


Cell Death Versus Cell Survival Instructed by Supramolecular Cohesion of Nanostructures

Christina J Newcomb et al. Nat Commun.


Many naturally occurring peptides containing cationic and hydrophobic domains have evolved to interact with mammalian cell membranes and have been incorporated into materials for non-viral gene delivery, cancer therapy or treatment of microbial infections. Their electrostatic attraction to the negatively charged cell surface and hydrophobic interactions with the membrane lipids enable intracellular delivery or cell lysis. Although the effects of hydrophobicity and cationic charge of soluble molecules on the cell membrane are well known, the interactions between materials with these molecular features and cells remain poorly understood. Here we report that varying the cohesive forces within nanofibres of supramolecular materials with nearly identical cationic and hydrophobic structure instruct cell death or cell survival. Weak intermolecular bonds promote cell death through disruption of lipid membranes, while materials reinforced by hydrogen bonds support cell viability. These findings provide new strategies to design biomaterials that interact with the cell membrane.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.


Figure 1
Figure 1. Design of membrane-interacting self-assembling molecules
(a) Chemical structures of PA 1 and PA 2 that conserve the alkyl tail length (hydrophobic tail) and number of lysine residues (cationic charge) but vary the propensity for intermolecular hydrogen bonding adjacent to the tail. (b) Representative fluorescence images of MC3T3-E1 cells that are viable (green, calcein) or dead (red, EthD-1) on coatings of each PA after 4 h of culture (scalebars, 100 μm). (c) Quantification of cell viability from MC3T3-E1 cells on each PA coating including PAs 36 that vary the alkyl tail length and number of lysine residues (Supplementary Fig. 2). One-way ANOVA with Tukey post-test. ***p < 0.001, compared to PA 2. (d) lactate dehydrogenase (LDH) release over time of cells seeded on coatings of either PA 1 (blue) or PA 2 (red). Two-way ANOVA with Bonferroni post-test, ***p ≤ 0.001.
Figure 2
Figure 2. Nanostructure and supramolecular cohesion within PA 1 and PA 2 assemblies
(a) Cryogenic TEM showing representative nanostructures in cell media. Scalebars: 100 nm. (b) X-ray diffraction of solutions of PA 1 (blue) and PA 2 (red). 4.7 Å corresponds to β-sheet hydrogen bonding. (c) Chemical structures of the spin labeled analogues of PA 1 (top) and PA 2 (bottom) with a site specific spin label (TOAC) located at the first amino acid adjacent to the fatty acid tail to probe the β-sheet hydrogen bonding segment of the assemblies. (d) Electron paramagnetic resonance spectra of PA 1 and PA 2 combined with 20% of the corresponding spin labeled analogue. Dashed lines and double headed arrows indicate the spectral broadening that and PA 2 occurs with reduced rotational diffusion (kr). (e) Atomistic modeling of PA 1 demonstrating differences in β-sheet hydrogen bonding (yellow: β-sheet hydrogen bonding, cyan: β-turn, gray: random coil, blue: alkyl tail). (f) Data from modeling experiments to determine the distribution of β-sheet hydrogen bonding within assemblies of PA 1 (blue) and PA 2 (red) as a function of residue number from the alkyl tail.
Figure 3
Figure 3. Interactions between liposomes and peptide amphiphiles
Time-lapse phase contrast microscopy of giant liposomes exposed to either (a) PA 1 or (b) PA 2. Time-points are indicated in the upper right corner. Scalebars: 10 μm. (c) Cryogenic TEM of extruded DPPC liposomes (100 nm diameter) and mixtures with either molecule PA 1 or PA 2 Inset: A less representative image of DPPC with PA 1 where a nanofibre is observed interacting with a liposome (black arrows: bicelles, red arrows: nanofibre,). Scalebars: 100 nm. (d) Differential scanning calorimetry of liposome-PA 1 mixtures (blue), liposome-PA 2 mixtures (red) and liposomes alone (black) where the lipid component is comprised of DPPC (left), DMPC (middle) or DSPC (right). (e) Values of the gel-liquid crystalline phase transition temperatures from each DSC sample.
Figure 4
Figure 4. Cell response to PA with varied β sheet hydrogen bonding
(a) Confocal live cell imaging of cells treated with a membrane dye (DiI, red) and calcein (green) to visualize viability over time on coatings of PA 1. Scale bar: 10 μm. Discontinuities in the cell membrane are indicated by arrows. (b) Confocal images of calcein-treated cells cultured on coatings of fluorescently labeled PA 1 (left) and higher magnification of the cell indicated by the arrow (center). Calcein-treated cells cultured on coatings of fluorescently labeled PA 2 (right) Scalebars: 10 μm. (c) Scanning electron micrographs of cells on coatings of PA 1 and PA 2 after 30 minutes (yellow: coating surface, blue: internal surface of cell, no color: outer cell surface). Scalebars: 500 nm. Arrow indicates the ruptured cell membrane. (d) Transmission electron micrographs of cross sections through both the cell and PA coating to reveal interactions between the cell and coating (C: cytoplasm, N: nucleus, PA: PA coating) after 30 minutes of culture (dashed line: interface between cell and PA coating, arrows: distinct sites of cell attachment to PA coating). Scalebars: 500 nm. (e–g) Time dependent measurement of lactate dehydrogenase (LDH) release: (e) LDH release after inhibition of actin (blue) or myosin (red) were compared to untreated controls (black); (f) LDH release from cells cultured at either 37°C (red) on coatings of PA 1 (circles, solid line) or PA 2 (triangles, dotted line). Cells were also cultured at 4°C (blue) on PA 1 (circles, solid line) or PA 2 (triangles, dotted line); (g) LDH release from cells depleted of ATP (green) and cultured on coatings of PA 1 (solid line) or PA 2 (dotted line) were compared against control (black) cultures on PA 1 (solid line) or PA 2 (dotted line) coatings. Statistical analysis: Two-way ANOVA with a Bonferroni post test: ***p < 0.001, treatments compared against their respective controls; ^^^p < 0.001, PA 1 compared against PA 2.
Figure 5
Figure 5. Creating a cell barrier with PA coatings to compartmentalize cells
(a) A schematic of the experimental setup. A cell-encapsulated hydrogel (palmitoyl-VVAAEE 1%, w/v, hydrogel 1) is surrounded by collagen (hydrogel 2) with PA 1 or PA 2 coating at the interface. The experiment evaluates the cell migration through the hydrogel interface from hydrogel 1 to hydrogel 2 (dashed double headed arrow). (b) A representative confocal slice from the two-compartment hydrogel system (as shown in (a), but without cells) where hydrogel 1 is coated with either fluorescently labeled PA 1 or fluorescently labeled PA 2 (arrows indicate the coating layer which is approximately 10 μm thick). Scalebars: 100 μm. (c) Viability of MC3T3-E1 cells that were encapsulated in the hydrogel 1, coated with either PA 1 or PA 2, and stained after 1 hour with calcein (green, live cells) and propidium iodide (red, dead cells). Scalebars: 100 μm (d) PA 1 or PA 2 coated cell encapsulating hydrogel 1 were further embedded in hydrogel 2 (as in the illustration in (a)) and cultured for 7 days, before staining for actin (phalloidin, green) and nuclei (propidium iodide, red). Cells remained confined within the hydrogel 1 compartment (grey) when coated with PA 1, while cells escaped into the surrounding collagen when coating was done with PA 2. Scalebars: 100 μm.

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