Abstract
Through the actuation of vibronic modes in cell-membrane-associated aminocyanines, using near-infrared light, a distinct type of molecular mechanical action can be exploited to rapidly kill cells by necrosis. Vibronic-driven action (VDA) is distinct from both photodynamic therapy and photothermal therapy as its mechanical effect on the cell membrane is not abrogated by inhibitors of reactive oxygen species and it does not induce thermal killing. Subpicosecond concerted whole-molecule vibrations of VDA-induced mechanical disruption can be achieved using very low concentrations (500 nM) of aminocyanines or low doses of light (12 J cm−2, 80 mW cm−2 for 2.5 min), resulting in complete eradication of human melanoma cells in vitro. Also, 50% tumour-free efficacy in mouse models for melanoma was achieved. The molecules that destroy cell membranes through VDA have been termed molecular jackhammers because they undergo concerted whole-molecule vibrations. Given that a cell is unlikely to develop resistance to such molecular mechanical forces, molecular jackhammers present an alternative modality for inducing cancer cell death.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The source data used during this study are uploaded to the Zenodo database, accessible at https://doi.org/10.5281/zenodo.8271482. The datasets generated and/or analysed during the current study are available from the corresponding author on request. Source data are provided with this paper.
References
García-López, V. et al. Molecular machines open cell membranes. Nature 548, 567–572 (2017).
Ayala Orozco, C. et al. Visible-light-activated molecular nanomachines kill pancreatic cancer cells. ACS Appl. Mater. Interfaces 12, 410–417 (2020).
Guentner, M. et al. Sunlight-powered kHz rotation of a hemithioindigo-based molecular motor. Nat. Commun. 6, 8406 (2015).
Santos, A. L. et al. Hemithioindigo-based visible light-activated molecular machines kill bacteria by oxidative damage. Adv. Sci. 9, 2203242 (2022).
Weissleder, R. A clearer vision for in vivo imaging. Nat. Biotechnol. 19, 316–317 (2001).
Liu, D. et al. Near-infrared light activates molecular nanomachines to drill into and kill cells. ACS Nano 13, 6813–6823 (2019).
Weber, H. P. Two-photon-absorption laws for coherent and incoherent radiation. IEEE J. Quantum Electron. 7, 189–195 (1971).
Gerwien, A., Mayer, P. & Dube, H. Green light powered molecular state motor enabling eight-shaped unidirectional rotation. Nat. Commun. 10, 4449 (2019).
Mustroph, H. & Towns, A. Fine structure in electronic spectra of cyanine dyes: are sub-bands largely determined by a dominant vibration or a collection of singly excited vibrations? ChemPhysChem 19, 1016–1023 (2018).
Cui, Y. et al. Molecular plasmon−phonon coupling. Nano Lett. 16, 6390–6395 (2016).
Kong, F. F. et al. Probing intramolecular vibronic coupling through vibronic-state imaging. Nat. Commun. 12, 1280 (2021).
Chapkin, K. D. et al. Lifetime dynamics of plasmons in the few-atom limit. Proc. Natl Acad. Sci. USA 115, 9134–9139 (2018).
Santos, A. L. et al. Light-activated molecular machines are fast-acting broad-spectrum antibacterials that target the membrane. Sci. Adv. 8, eabm2055 (2022).
Orlandi, G. & Siebrand, W. Theory of vibronic intensity borrowing. Comparison of Herzberg–Teller and Born–Oppenheimer coupling. J. Chem. Phys. 58, 4513–4523 (1973).
Melikyan, A. & Minassian, H. On surface plasmon damping in metallic nanoparticles. Appl. Phys. B 78, 453–455 (2004).
Caruso, F., Novko, D. & Draxl, C. Phonon-assisted damping of plasmons in three- and two-dimensional metals. Phys. Rev. B 97, 205118 (2018).
Liau, Y. H., Unterreiner, A. N., Chang, Q. & Scherer, N. F. Ultrafast dephasing of single nanoparticles studied by two-pulse second-order interferometry. J. Phys. Chem. B 105, 2135–2142 (2001).
Yang, T.-S. et al. Femtosecond pump-probe study of molecular vibronic structures and dynamics of a cyanine dye in solution. J. Chem. Phys. 110, 12070–12081 (1999).
Mishra, A., Behera, R. K., Behera, P. K., Mishra, B. K. & Behera, G. B. Cyanines during the 1990s: a review. Chem. Rev. 100, 1973–2012 (2000).
Li, Y., Zhou, Y., Yue, X. & Dai, Z. Cyanine conjugates in cancer theranostics. Bioact. Mater. 6, 794–809 (2021).
Shi, C., Wu, J. B. & Pan, D. Review on near-infrared heptamethine cyanine dyes as theranostic agents for tumor imaging, targeting, and photodynamic therapy. J. Biomed. Opt. 21, 050901 (2016).
Lange, N., Szlasa, W., Saczko, J. & Chwiłkowska, A. Potential of cyanine derived dyes in photodynamic therapy. Pharmaceutics 13, 818 (2021).
Bilici, K., Cetin, S., Aydındogan, E., Yagci Acar, H. & Kolemen, S. Recent advances in cyanine-based phototherapy agents. Front. Chem. 9, 707876 (2021).
Huang, Z. et al. Direct observation of delayed fluorescence from a remarkable back-isomerization in Cy5. J. Am. Chem. Soc. 127, 8064–8066 (2005).
Huang, Z. et al. Spectral identification of specific photophysics of Cy5 by means of ensemble and single molecule measurements. J. Phys. Chem. A 110, 45–50 (2006).
Mahoney, D. P. et al. Tailoring cyanine dark states for improved optically modulated fluorescence recovery. J. Phys. Chem. B 119, 4637–4643 (2015).
Misawa, K. & Kobayashi, T. Wave-packet dynamics in a cyanine dye molecule excited with femtosecond chirped pulses. J. Chem. Phys. 113, 7546–7553 (2000).
Mustroph, H. et al. Relationship between the molecular structure of cyanine dyes and the vibrational fine structure of their electronic absorption spectra. ChemPhysChem 10, 835–840 (2009).
Yang, J.‐P. & Callender, R. H. The resonance Raman spectra of some cyanine dyes. J. Raman Spectrosc. 16, 319–321 (1985).
Yu, J. H. et al. Noninvasive and highly multiplexed five-color tumor imaging of multicore near-infrared resonant surface-enhanced Raman nanoparticles in vivo. ACS Nano 15, 19956–19969 (2021).
Štacková, L. et al. Deciphering the structure–property relations in substituted heptamethine cyanines. J. Org. Chem. 85, 9776–9790 (2020).
Zhao, J., Wu, W., Sun, J. & Guo, S. Triplet photosensitizers: from molecular design to applications. Chem. Soc. Rev. 42, 5323–5351 (2013).
Itri, R., Junqueira, H. C., Mertins, O. & Baptista, M. S. Membrane changes under oxidative stress: the impact of oxidized lipids. Biophys. Rev. 6, 47–61 (2014).
Riske, K. A. et al. Giant vesicles under oxidative stress induced by a membrane-anchored photosensitizer. Biophys. J. 97, 1362–1370 (2009).
Sakaya, A. et al. Singlet oxygen flux, associated lipid photooxidation, and membrane expansion dynamics visualized on giant unilamellar vesicles. Langmuir 39, 442–452 (2023).
Zonios, G., Bykowski, J. & Kollias, N. Skin melanin, hemoglobin, and light scattering properties can be quantitatively assessed in vivo using diffuse reflectance spectroscopy. J. Invest. Dermatol. 117, 1452–1457 (2001).
Haussmann, P. B. et al. Melanin photosensitization by green light reduces melanoma tumor size. J. Photochem. Photobiol. 9, 100092 (2022).
Liu, D., Huang, H., Zhao, B. & Guo, W. Natural melanin-based nanoparticles with combined chemo/photothermal/photodynamic effect induce immunogenic cell death (ICD) on tumor. Front. Bioeng. Biotechnol. 9, 635858 (2021).
Weinberger, A. et al. Gel-assisted formation of giant unilamellar vesicles. Biophys. J. 105, 154–164 (2013).
Malcıoğlu, O. B., Gebauer, R., Rocca, D. & Baroni, S. TurboTDDFT—a code for the simulation of molecular spectra using the Liouville–Lanczos approach to time-dependent density-functional perturbation theory. Comput. Phys. Commun. 182, 1744–1754 (2011).
Giannozzi, P. et al. QUANTUM ESPRESSO: a modular and open-source software project for quantum simulations of materials. J. Phys. Condens. Matter 21, 395502 (2009).
Momma, K. & Izumi, F. VESTA 3 for three-dimensional visualization of crystal, volumetric and morphology data. J. Appl. Crystallogr. 44, 1272–1276 (2011).
Acknowledgements
J.M.T. acknowledges financial support from Nanorobotics, the Discovery Institute and the Welch Foundation (C-2017-20220330). We thank A. Santos for helpful discussions. We thank H. Xiao at Rice University for kindly hosting C.A.-O. and sharing his laboratory to culture the A375 cancer cells. We thank C. Kittrell for helpful discussions. We thank D. James for revising the manuscript. J.M.S. acknowledges the Lannater and Herb Fox Professorship. We also thank A. Budi Utama for confocal microscopy training and H. Deshmukh for flow cytometry training at the Rice Share Equipment Authority. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Author information
Authors and Affiliations
Contributions
The idea to use VDA to permeabilize cell membranes was suggested by C.A.-O. and discussed with J.M.T. C.A.-O. conducted all the experiments to demonstrate the permeabilization of cells by molecular vibrations, flow cytometry studies, confocal microscopy, in vivo studies, temperature measurements, ROS measurements, crystal violet assay and studies in GUVs under the supervision of J.M.T. C.A.-O. designed and conducted the in vivo experiments. A.C. injected the cancer cells to generate the tumours and assisted in monitoring the mice. R.R. and J.N.M. oversaw the in vivo experiments, provided the mice and established the B16-F10 tumour model. D.G.-A. and J.M.S. conducted the TD-DFT calculations of molecular plasmons in Cy7.5-amine. C.A.-O. and J.M.T. wrote the manuscript. All authors read and approved the manuscript.
Corresponding authors
Ethics declarations
Competing interests
Rice University owns the intellectual property on the use of MJH coupled with VDA for the permeabilization of cell membranes. C.A.-O. is a former subcontractor to Nanorobotics, the possible licensee of this technology from Rice University. J.M.T. is a stockholder in Nanorobotics, but not an officer, director or employee. Conflicts are mitigated through regular disclosure to and compliance with the Rice University Office of Sponsored Programs and Research Compliance. The remaining authors declare no competing interests.
Peer review
Peer review information
Nature Chemistry thanks Weian Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Calculated TD-DFT absorption spectrum and induced charge density plots of the molecular plasmons in Cy7.5-amine.
(a) Total and partial, by the orientation of the electric field component (Ei), absorption spectra calculated by time-dependent density-functional theory (TD-DFT) using the Lanczos approach. The electric field is used to simulate the optical excitation of the Cy7.5-amine. The partial components of the spectrum are oriented along the transversal molecular plasmon resonance (red), longitudinal (blue) and perpendicular (teal) axis of Cy7.5-amine. (b) Absorption spectra comparison between the experimental (top) and the TD-DFT calculation (bottom). The dashed lines represent the position of the wavelengths at which the induced charge density maps were calculated for molecular plasmon resonances. The experimental shoulder at 730 nm for the vibronic mode in Cy7.5-amine is observed at 750 nm in the theoretical transversal component of the spectrum, but it is less obvious in the total spectrum. (c) Total induced charge densities [Δρ(r)] at 430, 530, 750 and 809 nm wavelengths for molecular plasmon resonance. (d) Induced charge densities [Δρ(r)] by electric field (Ei) components at 430, 530, 750 and 809 nm wavelengths oriented along the transversal, longitudinal and perpendicular axis of Cy7.5-amine. The vectors on the rightmost represent the orientation of the electric filed components. The long alkyl-amine arm in Cy7.5-amine structure was not included in the electronic structure calculation because it has negligible contributions to the conjugation of the core structure. Instead, a methyl group was substituted for the long alkyl-amine in Cy7.5-amine.
Extended Data Fig. 2 Molecular jackhammer (MJH) model and summary of structures used in this study.
(a) Important MJH structural elements. LMP = longitudinal molecular plasmon. TMP = transversal molecular plasmon. The strength of the molecular plasmon (VDA) is expected to be proportional to the length of the π-conjugation. The π-conjugation can be increased in two ways: 1) increasing the length of polymethine bridge and 2) increasing the size of the polycyclic aromatic hydrocarbon (PAH) fused to the indole. The purple color is to highlight the polymethine bridge. The cyanines are named by the number of carbons in the polymethine bridge, in the example it is C7. The red color is to highlight the structure of the indole, and the orange color is for the benzoindole. The heptamethine bridge (C7) can be chemically conjugated with indole to form Cy7 or with benzoindole to form Cy7.5. These structural elements, polymethine and indole or benzoindole, hybridize to from a coupled system with the molecular plasmon-dominated longitudinally by the polymethine bridge (LMP in purple) and transversally by the indole or benzoindole (TMP in teal). However, these structures are hybridized and the electronic conjugation of the benzoindole influences the polymethine bridge and vice versa. (b) Summary of structures in this study. The observed effect on the cell killing is summarized for each structure, and each lists the common name of the conjugate backbone. The addend function is listed for each.
Extended Data Fig. 3 Binding of MJH into the external cellular membrane and into internal organelle membranes of A375 human melanoma cell line.
(a) Fluorescence confocal microscopy imaging of MJH (Cy7.5-amine) loaded in A375 cells. Cloading = 2 µM, incubation time = 30 min, λex = 640 nm, λem = 663–738 nm. Three fields of view (FOV) were recorded for each concentration, and 10 cells in average were recorded per FOV. Here representative cells are presented for each concentration. Scale bars = 25 µm. (b) Effect of the concentration of acetic acid in the population analysis of Cy7.5-amine-positive cells using flow cytometry. Cells to the right of the dotted line are considered Cy7.5-amine-positive. (c) Effect of the concentration of acetic acid on the population analysis of DAPI-permeabilized cells using flow cytometry. Cells to the right of the dotted line are considered cells permeabilized by DAPI. (d) Effect of the concentration of acetic acid on the binding of Cy7.5-amine to the A375 cells using flow cytometry analysis for quantification. Average of two experiments is shown (n = 2). (e) Effect of the concentration of acetic acid on the percentage of DAPI permeabilized cells using flow cytometry analysis for quantification. Cells were treated with 2 μM Cy7.5-amine, incubated for 30 min, then were illuminated with 730 nm light at 80 mW cm−2 for 60 s. Since acetic acid protonates the phosphates in the phospholipids, the Cy7.5-amine binds less efficiently to lipid membranes. The lower binding of Cy7.5-amine to the cells is reflected in the lower permeabilization of the cells upon NIR light excitation. Average of two experiments is shown (n = 2). Detailed flow cytometry data processing is described in Supplementary Information Fig. 5.
Extended Data Fig. 4 Absorption spectrum of MJH and confocal fluorescence microscopy of A375 cells in the presence of MJH.
(a) Absorption spectrum of MJH showing the position of the excitation lasers that were used in the confocal microscope (λex = 405 nm with λem = 425–475 nm or λex = 640 nm with λem = 663–738 nm). (b) Expansion of the x and y axis of the absorption spectrum to observe the expected TMP (transversal molecular plasmon). The Cy7.5-amine shows a strong TMP (strong hybridization of longer C7 heptamethine bridge and larger benzoindole). Cy7-amine shows a weaker TMP and slightly shifted to ~375 nm because the C7 is hybridized to the smaller indole. Cy5.5-amine shows a strong TMP (larger benzoindole) but shifted to ~360 nm because of the hybridization with a weaker LMP (shorter C5 pentamethine). Cy5-amine shows little TMP because of the poor hybridization of the shorter C5 and smaller indole; this is the weakest combination because of the poor plasmonicity on both components. (c) Cells in the absence of dyes. (d) Cells in the presence of 2 µM Cy7.5-amine, 30 min of incubation. The emission of TMP mode can be observed at λex = 405 nm in blue color. The excitation at 640 nm excites the tail of the LMP and produces a weak emission from Cy7.5-amine. (e) Cells in the presence of 2 µM Cy7-amine, 30 min of incubation. The emission from the LMP in Cy7-amine, since it is blue-shifted relative Cy7.5-amine, can be observed more intense in the red (λem = 663–738 nm). (f) Cells in the presence of 2 µM Cy5.5-amine, 30 min of incubation. The emission from the LMP is clearly visible in red. (g) Cells in the presence of 2 µM Cy5-amine, 30 min of incubation. The emission from LPM is clearly visible in red. The emission from TMP (at λex = 405 nm) is not observed or is very weak in signal in e-g panel since the TMP is not present or shifted to other wavelengths on those molecules (Cy7, Cy5.5, and Cy5). For panels c-g, 75 cells in average were analyzed in each condition in the confocal microscope in 5 different locations. Two independent experiments were conducted. Here representative images are shown. Scale bars = 25 µm.
Extended Data Fig. 5 Excitation of the 680 nm vibronic shoulder in Cy7 improves the MJH effect for opening cell membranes in A375 cells.
(a) Absorption spectra of Cy7-amine and overlaid with the spectral output of two LED lights: 730 nm and 680 nm. (b) Structure of Cy7-amine. (c) Flow cytometry analysis to measure the permeabilization of A375 cells in the presence of Cy7-amine without light. (d) Flow cytometry analysis to measure the permeabilization of A375 cells in the presence of Cy7-amine with 730 nm LED activation. (e) Flow cytometry analysis to measure the permeabilization of A375 cells in the presence of Cy7-amine with 680 nm LED activation. In all the flow cytometry analyses the red line represents the gating to discriminate between DAPI negative and positive cells (permeable). In all the cases the incubation with the cyanine was for 30 min and irradiation was with an equal light dose of 80 mW cm−2 for 10 min. The light dose was calibrated with an Optical Power Meter from Thorlabs, sensor model S302C and console model PM100D. (f) Percentage of permeabilized cells, the numbers are obtained from the flow cytometry. 10,000 cells are analyzed per each concentration. Detailed flow cytometry data processing is described in Supplementary Information Fig. 5.
Extended Data Fig. 6 Temperature of the cell suspension while under light treatment.
Temperature on the cell killing experiment using Cy7.5-amine. (a-b, d-e) No detection of heat production by Cy7.5-amine under NIR light treatment in the cell suspension (A375 cells) above the control. Temperature of the cell suspension (A375 cells) with 2 μM Cy7.5-amine and under illumination with 730 nm NIR light (80 mW cm−2 for 10 min). The temperature of the media was recorded when the experiment was done at room temperature (a-c) and when the cell suspension was placed in an ice bath (d-f). A picture of the experimental set up when done at room temperature is shown in c and in ice bath is shown in f. In d ‘water + ice’ is the increase of the temperature because of the melting of ice without irradiation. In e the change of temperature is corrected by subtracting the temperature increase due to the melting of ice without illumination. The temperature of the cell suspension treated with NIR light and without Cy7.5-amine (DMSO + NIR light) correlates well with temperature profile in the suspension treated with NIR light containing 2 μM Cy7.5-amine (Cy7.5 + NIR light). There is no photothermal effect of Cy7.5-amine beyond the minimal heating caused by the light alone of ~0.5 °C. (g) Percentage of cells permeabilized to DAPI when the treatment was done at room temperature versus when was done placing the cell suspension on ice bath. Detailed flow cytometry data processing is described in Supplementary Information Fig. 5. t-test, two-tail, * p < 0.05, ** p < 0.01, *** p < 0.001 Statistical significance p < 0.05, ns = not significant. The exact p values obtained were 0.0036 (**), 0.006 (**), 0.16 (ns), 0.31 (ns) when comparing the permeabilization at room temperature versus in ice bath for the DMSO, DMSO + L, Cy7.5-amine, and Cy7.5-amine+L groups, respectively. In a and b 3 independent samples were processed and measured (n = 3). In d and e 4 independent samples were processed and measured (n = 4). In g 3 independent samples were processed and measured by flow cytometry (n = 3). In all, the data are presented as mean values ± SD, respectively. For ‘water + ice’ control in panel d data as mean values are presented (n = 4).
Extended Data Fig. 7 ROS effects on the cell killing using Cy7.5-amine.
ROS scavengers do not retard the permeabilization of A375 cells to DAPI when treated with 2 μM Cy7.5-amine under illumination with 730 nm NIR light (80 mW cm−2 for 10 min). (a) Effect of 10 mM NAC (N-acetylcysteine). The exact p values obtained were 0.0004, 0.0005, 0.0969 and 0.6005 for the DMSO, DMSO + L, Cy7.5-amine, and Cy7.5-amine+L groups, respectively. (b) Effect of 100 mM TU (thiourea). The exact p values obtained were 0.9071, 0.8021, 0.4631, and 0.5918 for the DMSO, DMSO + L, Cy7.5-amine, and Cy7.5-amine+L groups, respectively. (c) Effect of 2.5 mM SA (sodium azide). The exact p values obtained were 0.3712, 0.2751, 0.4267, and 1.0 for the DMSO, DMSO + L, Cy7.5-amine, and Cy7.5-amine+L groups, respectively. (d) Effect of ROS scavengers at variable irradiation time of 730 nm NIR light at 80 mW cm−2. Five different scavengers were used: TU 100 mM, azide 2.5 mM, NAC 1 mM, Vit C (vitamin C) 5 mM and Met (methionine) 5 mM. DMSO control contains 0.1% DMSO in the media because DMSO is used to pre-solubilize the Cy7.5-amine stock solution at 2 mM and diluted to 1:1000 to obtain 2 μM Cy7.5-amine in media containing 0.1% DMSO. Experimental groups are: DMSO = 0.1% DMSO, DMSO + L = 0.1 % DMSO + NIR light treatment, Cy7.5 = 2 μM Cy7.5-amine and Cy7.5 + L = 2 μM Cy7.5-amine + NIR light treatment. In all the plots 3 independent samples were processed and analyzed by flow cytometry (n = 3). In all plots data are presented as mean values ± SD, respectively. t-test, two-tail, * p < 0.05, ** p < 0.01, *** p < 0.001 Statistical significance p < 0.05, ns = not significant. Detailed flow cytometry data processing is described in Supplementary Information Fig. 5.
Extended Data Fig. 8 Quantification of ROS and singlet oxygen (SO) levels and their effect on the cell killing using Cy7.5-amine versus the cell-membrane-targeting DiR dye (a strong photosensitizer control).
(a) Measurement of ROS levels in A375 cell suspensions using 2’,7’-dichlorodihydrofluorescein diacetate as the ROS probe in the presence of 2 µM Cy7.5-amine versus 2 µM DiR and 20 µM DiR with and without light (L). The number of samples processed and measured were n = 4. (b) Percentage of DAPI positive cells as a function of the concentration quantified from the flow cytometry analysis. DiR that produced 10–20-fold more ROS than Cy7.5-amine did not permeabilize the A375 cells. This continues to support that the DiR structure is a weaker MJH with poorer VDA (See Extended Data Fig. 2.) And more importantly, that ROS is not responsible for the permeabilization of DAPI into the cells. 5,000 cells were analyzed by flow cytometry for each concentration (n = 1). (c) Quantification of SO levels by the decomposition rate of DPBF (1,3-diphenylisobenzofuran) under light illumination in the presence of four cyanine dyes: 2.6 μM Cy7.5-amine, 2.6 μM Cy7-amine, 2.6 μM DiR and 2.6 μM ICG. DiR and ICG produces more SO than Cy7.5-amine or Cy7-amine yet DiR was unable to permeabilize the cells (number of samples n = 1). (d) ROS levels in cells in the presence of Cy7.5-amine versus Cy7-amine. Cy7.5-amine and Cy7-amine produced approximately the same levels of SO and ROS yet Cy7.5-amine is a much stronger MJH in cell permeabilization (Fig. 2). The number of samples processed and analyzed were n = 12. (e) Shutting down the levels of SO generation by adding thiourea (TU = 100 mM) or sodium azide (SA = 2.5 mM) or a combination of TU 100 mM and SA 2.5 mM into the 2 µM Cy7.5-amine solution under LED illumination (number of samples n = 1). (f) Effect of ROS scavenger combo (100 mM TU and 2.5 mM SA) on the percentage of DAPI positive cells as quantified from the flow cytometry analysis in the presence of 2 µM Cy7.5-amine with and without illumination: no difference observed in the cell permeabilization to DAPI (number of samples n = 3). Unless otherwise specified, the light irradiations doses were 80 mW cm−2 for 10 min using a 730 nm LED. Except, in b the LED light (L) was a 740 nm light from Keber Applied Research Inc. at the same dose of 80 mW cm−2 for 10 min. This was done with a different LED because the data was collected in the early stage of the research, and we later moved to use the 730 nm LED for all the experiments. In all, the data are presented as mean values, and the error bars are the standard deviations, respectively. For panel c and f, detailed flow cytometry data processing is described in s.
Extended Data Fig. 9 Quantification of cell death by crystal violet assay and clonogenic assay.
A375 cells treated with Cy7.5-amine and 80 mW cm−2 of 730 nm NIR light for 10 min. (a) Representative microscopy picture of each condition in the crystal violet assay (n = 4). Experimental groups consist of: Cy7.5-amine + L = 2 μM Cy7.5-amine + 80 mW cm−2 of 730 nm NIR light for 10 min; Cy7.5-amine = 2 μM Cy7.5-amine; DMSO + L = 0.1% DMSO + 80 mW cm−2 of 730 nm NIR light for 10 min; and DMSO = 0.1% DMSO. Scale bar = 0.5 mm. (b) Crystal violet assay. Plot showing the quantification of the cell viability from the absorbance of crystal violet. Sample repetitions n = 4 for each condition in a 24 well plate (independent samples). Data are presented as mean values ± SD. t-test, two-tail, * p < 0.05, ** p < 0.01, *** p < 0.001 Statistical significance p < 0.05. The exact p values obtained were p = 0.0012 (**) and p = 0.26 (ns), respectively. (c) Clonogenic assay. Representative pictures showing the growth of cell colonies in the controls (0.1% DMSO with or without light) and complete eradication of A375 cells when treated with 1 μM Cy7.5-amine + light (80 mW cm−2 of 730 nm NIR light for 10 min). (d) Clonogenic assay. Quantification of the number cells forming colonies. The survival is the percentage of cells that formed colonies. The results are normalized relative to the DMSO control. Sample repetitions n = 3 (independent samples). Data are presented as mean values ± SD. t-test, two-tail, * p < 0.05, ** p < 0.01, *** p < 0.001 Statistical significance p < 0.05. The exact p values obtained were p = 0.002 (at 0.5 µM), p = 0.005 (at 1 µM) and p = 0.002 (at 2 µM).
Extended Data Fig. 10 Time-course treatment of DPhPC GUVs with MJHs and under VDA photoactivation.
A strong MJH (Cy5.5-amine) is compared against a weak MJH (Cy5-amine). The photoactivation consisted of continuous exposure to 640 nm confocal microscope laser at 5% power (50 µW power, Plan Apo IR 60x/1.27 water immersion objective). Fluorescence images are recorded by imaging at λex = 640 nm, λem = 663–738 nm, and 5% (50 µW) laser power every 10 s. a) DPhPC GUV treated with 2 µM Cy5.5-amine without VDA photoactivation. b) DPhPC GUV treated with 2 µM Cy5.5-amine and under VDA photoactivation. c) DPhPC GUV treated with 2 µM Cy5-amine without VDA photoactivation. d) DPhPC GUV treated with 2 µM Cy5-amine and under VDA photoactivation. e) DPhPC GUV treated with 0.1% DMSO as control and under photoactivation. The pictures were recorded every 10 s for all the panels. Scale bar 5 µm.
Supplementary information
Supplementary Information
Supplementary Figs. 1–6 and Discussion.
Supplementary Video 1
Movie of the vibrational mode at the 750 nm vibronic shoulder, obtained from DT-DFT calculations.
Supplementary Video 2
Movie of the vibrational mode at the 809 nm LMP, obtained from DT-DFT calculations.
Supplementary Video 3
Movie of the vibrational mode at the 530 nm LMP, obtained from DT-DFT calculations.
Supplementary Video 4
Movie of the vibrational mode at the 430 nm TMP, obtained from DT-DFT calculations.
Supplementary Data 1
Initial atomic coordinates for the DFT calculations.
Supplementary Data 2
Final atomic coordinates for the DFT calculations.
Source data
Source Data Fig. 1
Absorption spectra of Cy7.5-amine and Cy7-amine and spectrum of the 730 nm LED.
Source Data Fig. 2
Flow cytometry source data from FlowJo analysis.
Source Data Fig. 3
Flow cytometry data, percentage of permeabilized cells and spectra of all compounds and LED lights.
Source Data Fig. 4
Confocal microscopy source images in TIFF format and organized into folders, and Excel sheet containing the quantification of DAPI intensity (statistical source data).
Source Data Fig. 5
Statistical source data of in vivo studies.
Source Data Extended Data Fig. 1
DFT calculations source data.
Source Data Extended Data Fig. 2
ChemDraw drawings.
Source Data Extended Data Fig. 3
Flow cytometry results (statistical source data).
Source Data Extended Data Fig. 4
Folder with all confocal microscope images in TIFF format and the absorption spectra of compounds in the Excel sheet.
Source Data Extended Data Fig. 5
Absorption spectra of compounds and spectrum of the 630 nm LED. Flow cytometry results for percentage of DAPI positive cells.
Source Data Extended Data Fig. 6
Temperature measurements and flow cytometry results for the percentage of DAPI positive cells (statistical source data). Effect of temperature.
Source Data Extended Data Fig. 7
Flow cytometry results for the percentage of DAPI positive cells (statistical source data). Effect of ROS inhibitors.
Source Data Extended Data Fig. 8
Statistical source data for the ROS detection experiments and effect of ROS on permeabilization.
Source Data Extended Data Fig. 9
Statistical source data for the cell death assays: crystal violet and clonogenics.
Source Data Extended Data Fig. 10
Confocal microscopy source images in TIFF format.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Ayala-Orozco, C., Galvez-Aranda, D., Corona, A. et al. Molecular jackhammers eradicate cancer cells by vibronic-driven action. Nat. Chem. 16, 456–465 (2024). https://doi.org/10.1038/s41557-023-01383-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41557-023-01383-y
