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Molecular jackhammers eradicate cancer cells by vibronic-driven action

 9 months ago
source link: https://www.nature.com/articles/s41557-023-01383-y
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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.

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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.

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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.

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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.

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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.

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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).

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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.

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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.

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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).

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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.

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