domain: sdv spi

text:
Gaëtan Mary+

Aurore Van De Walle+

Dr Jose Efrain Perez

Dr Tomofumi Ukai

Toru Maekawa

Prof Nathalie C Luciani

Claire Wilhelm
email: claire.wilhelm@univ-paris-diderot.fr

Dr G Mary

Dr A Van De Walle

Wilhelm

Prof. T Maekawa

High-Throughput Differentiation of Embryonic Stem Cells into Cardiomyocytes with a Microfabricated Magnetic Pattern and Cyclic Stimulation

Keywords: magnetic nanoparticles, stem cell differentiation, cardiomyogenesis, magnetic cell stimulation, microfabrication

Pluripotent stem cells are central tools to many regenerative medicine strategies due to their ability to differentiate towards the three embryonic germ layers. One challenge remains in providing control over their differentiation into specific lineage, such as cardiac commitment.

Here, the possibility of directing cardiomyogenesis of embryonic stem cells (ESCs) using a microfabricated magnetic pattern is demonstrated. The stem cells are labeled with magnetic nanoparticles, aggregated into embryoïd bodies (EBs) onto the pattern, and stimulated with a local magnetic force applied via the pattern. The EBs formed on this magnetic device experience the same differentiation profile than the ones created by the common hanging drop approach, while it allows high-throughput production of hundreds of EBs. Further on/off cyclic magnetic force stimulation mediated by the same device is sufficient to enhance cardiomyogenesis in a way that almost all EBs develop spontaneous beating, confirmed by the overexpression of -actin and troponin proteins, and by the upregulation (2 to 5-fold) of genes involved in mesoderm differentiation (Nkx2.5, Gata4, and Gata6), and more specifically cardiac lineage (Tnnt2, Myh6 and Myl-2). Beyond holding high application-level potential, this work confirms that physical forces, and specifically on/off dynamic ones can be sufficient to govern cell function.

Introduction

The production of in vitro three-dimensional multicellular assemblies, or spheroids, has been of recent interest for tissue engineering and regenerative cell therapies. It stands as a pivotal biological process to initiate the differentiation of stem cells into specific pathways, such as chondrogenesis or cardiomyogenesis. The most common spheroid formation techniques rely on well established procedures that include the aggregation of cells via their culture in suspension [1] or on non-adherent surfaces [2] , their pelleting by centrifugation, [3] or their selfassembling by bioprinting. [4] In parallel, material and nanotechnological means have advanced not only to control cell patterning but also to direct cell function such as stem cells differentiation, using for examples nanoparticles or nanoelectrodes, [5] or specific substrates, [6] combined or not with nanomaterials (e.g. nanoelectrodes, nanofibers, nanowires, nanotopography, nanopillars) [7] . Among them, iron oxide nanoparticles have recently emerged as magnetic cellular label that has a double advantage. On one hand, it can be exploited to promote cell aggregation via localized magnetic forces and organize cells into engineered tissues. [8] As such, it has been employed for the formation of scaffold-free cell-spheroids [8b, 9] , combined with standard techniques such as the hanging drop method [10] or agarose-well patterning, [11] and used as well to seed cells within 3D scaffolds. [12] On the other hand, it offers the possibility to control cell functions, including their differentiation. [13] Spheroids based on embryonic stem cells (ESCs), termed embryoïd bodies (EBs), are particularly relevant as these cells can differentiate into lineages representative of the three embryonic germ layers and their EBs not only mimic early stages of embryonic development but also can be directed into specific organs due to their pluripotency. Despite most recent advances, it is still difficult to control the differentiation of pluripotent stem cells and maintain simultaneously a high production of differentiated or precursors cells, both being of particular relevance for application in regenerative medicine, such as in cardiac regeneration. [14] One emerging approach for cardiac repair, alternative to cardiac transplantation and implantable mechanical-assisted devices in case of severe heart failure, is to transplant cells. [15] When stem cells (typically mesenchymal ones [16] or pluripotent ones [17] ) were administered after myocardial infarction, cardiac performance was improved, but with low tissue integration and contraction generation. [18] Approaches have been tested to increase contraction, by aligning cardiac cells using electrospun nanofibers [7b, 7c] or micropatterned surfaces, [19] or by orienting cells using soft lithography; [20] however, these approaches are two-dimensional, based on adult cardiac cells, with low output.

Iron oxide nanoparticles have recently provided a solution for the differentiation of ESCs as cardiomyocytes. [13d] Upon internalization of these magnetic nanoparticles into ESCs, the cells were manipulated via magnetic forces to form three-dimensional EBs on a one-by-one basis, which were then stretched periodically using a magnetic attractor. This purely mechanical stimulation prompted the differentiation of the cells, reaching differentiation levels challenging those achieved by the more traditional chemical stimulation. This initial study thus shed light on the potential of a magnetically-induced stimulation for cardiac regeneration.

Yet, it could perform only on a single EB at a time, and could not provide translatable conditions to produce clinically relevant numbers of functional cells.

Herein, a magnetic stimulation of EBs is accomplished as well, but at a high-throughput level thanks to the design of an original, high precision, all-integrated microfabricated pattern consisting of 900 magnetic microtips. This micro-pattern allows the controlled formation of 900 EBs in only one pipetting step and offers the possibility of a totally remote stimulation of said EBs, with no direct contact between the cells and the magnetic attractors. Remarkably, using this design, we demonstrate the directed cardiomyogenesis of ESCs upon remote (electromagnetic) cyclic stimulation of ESCs labelled with magnetic nanoparticles, surpassing the cardiomyogenesis achieved in the previous study, [13d] and confirming the importance of mechanical factors in the differentiation of stem cells. [21]

Results and Discussion

The photolithography-patterned nickel microtips device was fabricated to achieve a spatially controlled and highly localized magnetic attraction (Figure 1A and Figure S1). The end device accommodates 900 microtips, each 1 µm in height and 1 mm apart (Figure 1B). The magnetic characterization of the microtips is shown in Figure S2, providing a magnetization of 4x10 5 A/m. The device is positioned upon a permanent magnet or an electromagnet, both developing a 0.2 T magnetic field perpendicular to the device substrate. The magnetic field concentration by each microtip was computed and is presented in Figure S3A. It results in a magnetic field gradient in the 100 T/m range 100 µm apart from the microtips surface (Figure S3B). Magnetic nanoparticles were internalized in the ESCs upon a 30 minutes incubation at

[Fe]=2 mM, these same conditions having been previously reported as having no impact on ESC viability or differentiation profiles. [13d] After cellular uptake, the nanoparticles were localized in the cell endosomes (Figure 1C). Magnetically-labelled ESCs were then organized into EBs either by the typical hanging drop method (Figure 1D), with EBs being formed over three days of drop confinement, or under a set of three conditions using the magnetic microtips pattern (Figure 1E-G). For all three conditions, a magnet was placed below the device to magnetize the tips and achieve localized cell attraction and EB formation.

In the first case, the magnet was removed after five minutes (MAG-short, Figure 1E), whereas in the second one it was removed after three days (MAG-long, Figure 1F). In the last condition, the magnet was maintained over the three first days, but was replaced with an electromagnet for 6 hours per day, imposing a cyclic magnetic field at a frequency of 1 Hz (MAG-cycl, Figure 1G). It should be stressed that the magnetic stimulation was applied during the first three days of EB formation and growth, when the differentiation pathways are setting up. Besides, in a more practical term, the cellular magnetic forces are significant only over the first cell divisions within the EBs.

EB formation (1000 ESCs per EB) is immediate when using the magnetic microtips, with cell aggregation taking place within seconds of seeding (Figure 2A). At day 3, the EBs were collected from the drops or from the magnetic pattern. All four tested conditions, the three magnetic ones and the hanging drop, were then transferred in non-adherent dishes and left to mature as spheroids for 2 more days. At day 5, the EBs were transferred to adherent tissue culture wells, and left there to finalize their differentiation pathway. Figure S4 summarizes all steps occurring upon the 10 days of ESCs differentiation, and presents the different analyses performed at various time points. EBs size was measured at days 0, 1, 3, and 5 (only 3 and 5 for the hanging drop condition). After 1 day of magnetic seeding (Figure 2B, 2G), no size difference was observed over the three magnetic conditions. At day 3 and day 5, EBs diameter were similar across all four conditions (including the hanging drop), at approximately 350 and 500 µm, respectively (Figure 2C-F, 2G). At day 7 and day 10, each EB was observed to record the occurrence of spontaneous beatings. The beating events can be seen on supplementary Movie S1, for all conditions. Figure 2H-K shows representation illustrations of these beating events for each conditions, with spatio-temporal analysis (kymographs). If there is little or no cell displacement (i.e. beating), the position of the cells in the area selected on the images can be seen as unaltered over time, defined as straight lines along the time axis of the kymographs (vertical in Figures 2H-K); however, if beating takes place, it translates into regular perpendicular lines (at given time interval). The beating frequency was then systematically extracted from all kymographs, appears distributed in the 1-2 Hz range (Figure 2L), and is very similar across all the conditions, showing that there is no effect of EBs magnetic formation and stimulation on the beating phenomenon itself, once triggered. Finally, the percentage of beating EBs was quantified across all conditions, at days 7 and 10 (Figure 2M). It is remarkably increased for the MAG-cycl condition, at both times, reaching 92% of beating EBs at day 10, percentage rarely achieved even with the use of the most efficient chemical factors for cardiomyogenic differentiation induction of ESCs. [22] The response of the EBs to the magnetic forces originates from the magnetic nanoparticles that are internalized and confined intracellularly, within endosomes. To explore the impact of the magnetic force at the magnetic endosome level, electron microscopy of EBs submitted to the magnetic field for 6 hours was performed and measurements were made alongside the direction of the magnetic field (Figure S5). It shows that the localisation of the magnetic endosomes is not impacted by the magnetic force. This is consistent with the value of the magnetic force generated on each endosome by the magnetic field gradient created by the nickel microtip, below 0.1 pN, negligible in comparison to active trafficking and cytoplasm viscous friction. Some endosomal cause for cardiomyogenesis cyclic activation is thus unlikely, and nanoparticles confined within endosomes should be seen as embedded in a viscoelastic matrix, simply providing the cell with a magnetic moment. The mechanical response from the cyclic stimulation is then generated at the EB scale. It is confirmed by The expression of six genes characteristic of the endo/mesoderm (the transcription factor Gata6), and/or more specifically of the cardiac mesoderm (the transcription factors Gata4 and Nkx2.5, and regulating proteins directly specific to cardiac contraction troponin T (Tnnt2), α myosin heavy chain (Myh6), and myosin regulatory light chain 2 (Myl2)) was assessed at days 5, 7 and 10, and normalized to the same gene expression at day 0 (Figure 3C and Figure S14). It first evidences that differentiation of the ESCs toward the cardiac mesoderm takes place for the hanging drop control, with a time-dependent increase of all selected gene expression. This was expected as this technique has specifically been developed for allowing cardiac differentiation of ESCs through aggregation of the cells and their growth in a confined environment. [23] Results show that the EBs formed on the magnetic pattern with no further magnetic compaction (MAG-short) display a similar gene expression than the hanging drop method, sometimes very slightly downregulated, but never more that 1.5-fold. When magnetic compaction was applied all along the first days of differentiation initiation (MAG-long), mesoderm and cardiac mesoderm gene expression was sometimes slightly upregulated, but never more than 2-fold. By contrast, what was highly significant is the upregulation of all genes involved in cardiac differentiation when cells were cyclically stimulated (MAG-cycl), always in the range of a 2 to 5-fold increase.

An additional experiment was performed to confirm that the effect observed is not due to the application of a cyclic magnetic field only. To do so, EBs made of cells not labeled with the magnetic nanoparticles were formed via the hanging drop approach and subjected to the cyclic magnetic field at days 2 and 3. Gene expression analyzed at days 5, 7, and 10 is shown in Figure S15. It evidences no difference between EBs subjected or not to a cyclic magnetic field, and further indicates that the cyclic magnetic field alone is not sufficient to have an effect on the differentiation of ESCs. We also verified that the cyclic magnetic field was not triggering an increase in magnetic nanoparticles intracellular degradation. In this case EBs containing magnetically labeled cells and exposed or not to the cyclic stimulation were measured by magnetometry. Results are displayed in Figure S16 and indicate a similar slowly decreasing rate of the EBs magnetic moments, demonstrating that the cyclic stimulation does not impact the fate of the nanoparticles inside the cells.

Finally, the cardiomyogenesis triggered here by cyclic electromagnetic stimulation should be compared to the proof of concept study exploring the role of a magnetically-induced mechanical stimulation on EB. [13d] When observing the gene expression of proteins involved in cardiac contractility, such as troponin T (Tnnt2), myosin regulatory light chain 2 (Myl2), and α-myosin heavy chain (Myh6), an upregulation of 8, 4 and 2-fold is reached, respectively, when comparing the EBs stimulated by the magnetic pattern device to those stimulated in the previous stretching device. Therefore, not only the magnetic micro-pattern set-up allows for the production of thousands of EBs, but also it appears to streamline the cardiac differentiation of ESCs.

Conclusion

The advent of microfabrication offered promising materials tools to engineer stem cells into matured cells such as cardiomyocytes depending on substrate topography or rigidity variations. [24] Herein, a microfabricated magnetic pattern was specifically designed for the high-throughput production of EBs and enhanced cardiomyogenesis. Upon cell labeling with magnetic nanoparticles, 900 EBs could be magnetically formed in a single pipetting step, instead of one by one for the common hanging drop method. Without any further stimulation, these EBs have the same differentiation potential toward the mesodermal pathway than when formed by hanging drop in optimal conditions. The even more unprecedented advantage of the magnetic pattern device resides in the possibility of further stimulating the EBs magnetically. When dynamic (cyclic), this stimulation drove the EBs towards cardiac differentiation with a remarkable efficiency. Indeed, after 10 days, more than 90% of the EBs were beating, a much higher percentage than with the state-of-the-art hanging drop approach. Also, when comparing with previous studies that used growth factors to increase the differentiation, such as TGF and BMP2, the cardiomyogenesis enhancement obtained here without any additive of any sort seems even more effective. [25] This remarkable result not only indicates applicative relevance of the magnetic device but also beautifully underlines the control that purely mechanical stimulation may have on the differentiation of stem cells.

In conclusion, the magnetic microtips pattern provides a high-throughput approach for EBs formation. It allows their remote mechanical cyclic stimulation, induced magnetically, sufficient to direct almost totally ESCs differentiation towards the cardiac lineage. It could be considered as a next potent platform for high scale production of stem cells-derived cardiomyocytes for regenerative medicine therapies.

Experimental Section

Magnetic pattern: Magnetic pattern was fabricated on a silicon substrate via electroplating and UV photolithography. Titanium and copper films were formed on a silicon substrate using a sputtering system (E-200S-TY, CANON ANELVA Corp.) and reversal patterns of magnetic dots were obtained by a negative tone UV photoresist (KMPR, KAYAKU Advanced Materials, Inc.) via UV lithography. A nickel film was then deposited on the substrate via electroplating and the UV photoresist was removed by Remover PG (KAYAKU Advanced Materials, Inc.). The titanium and copper films were wet-etched and finally silicon oxide was deposited using the sputtering system. The pattern was magnetized by placing it over either a permanent neodymium magnet (50 mm x 50 mm x 20 mm) creating a 0.2 T magnetic field in the device plane, or over an electromagnet to generate an on/off magnetic field. The electromagnet was composed of a custom-made water-cooled coil (6Ω, 14 mH) wrapped around a soft iron polar piece (diameter 55mm, height 85mm). 4A-on/off current (1Hz) was delivered to the coil using a TGA 1242 signal generator (Aim&TTi) and amplifier (Kebco BOP), creating an on/off 0.2 T magnetic field on the device.

ESCs and magnetic labeling: CGR8 mouse embryonic stem cell were purchased from Sigma (07032901) and were cultured on 0.1% gelatin-coated surfaces in fresh GMEM medium (Sigma, G5154) supplemented with 10% fetal calf serum, 2 mM glutamine, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol, 1 mM sodium pyruvate, 1% penicillin/streptomycin and 1000 U/ml of leukemia inhibitor factor (LIF, Thermo Fisher Scientific). They were passaged every 2 days at a density of 10 4 cells per cm², and were systematically used at passage 5 to 8 (passage zero being initial purchased batch). An older CGR8 cell line was also tested (Figure S14), corresponding to another cell batch, having experienced over 50 passages, and less competent for cardiomyogenesis. The magnetic nanoparticles used were made by aqueous co-precipitation of iron salts, they have a mean diameter of 8 nm, and a polydispersity index of 0.35, and are the most commonly used in biomedicine applications.

EBs formation and differentiation:

At day 0, ESCs were labeled with the magnetic nanoparticles (30 min, [Fe]=2mM), then detached and resuspended in differentiation medium (same as culture medium, but with 20% fetal calf serum and without LIF factor). EBs were next formed either by the hanging drop method (DROP, considered as control, seeding of 1000 ESCs in a 30 µL drop, plated on Petri dish cover gently turned over on the dish filled with PBS) or onto the magnetic pattern. ESCs (100 000) were then transferred to 12 mm diameter glass-bottom dishes, previously treated with PLL-PEG (SuSos) diluted in 10 mM HEPES (30min, 10 mg/ml) to prevent adhesion, and placed over the magnetic pattern device.

Each dish covers 100 magnetic dots (1000 ESCs available per dot), and multiple dishes can be placed together on the device. The device is then placed over the permanent magnet, and the dishes are left on site for 5 min (MAG-short) or until day 3 (MAG-long). For the MAG-cycl condition, the device is placed on the electromagnet for on/off magnetic stimulation cycling at a 1 Hz frequency for 6 hours per day, the first 3 days. At day 3, all EBs (hanging drops and magnetically formed ones) are collected and transferred to non-adhesive Petri dishes for 2 days for 2-days maturation before being individually seeded at day 5 in 0.1% gelatin-coated 24-well plates. Figure S2 illustrates the 10-days differentiation process.

Immunohistology: After paraformaldehyde (4% in PBS) fixation and Triton permeabilization (15 min), cell samples were incubated sequentially with blocking solution (BSA 5%) for 1 hour at room temperature, primary antibodies overnight at 4°C (mouse monoclonal anti-actinin, Sigma Aldrich, A7811, dilution 1:400; mouse monoclonal (IgG1) anti cardiac Troponin T, Thermofisher, MA5-12960, dilution 1:200), goat anti-mouse IgG secondary antibody for 2 hours at room temperature (Sigma-Aldrich, 43394, IgG-Atto550, dilution 1:500), and DAPI for 30 min at room temperature (Sigma-Aldrich, D9564, 1 µg/mL), and mounted (Fluoromount, Sigma Aldrich, F4680) on slides. Images were acquired with Olympus JX81/BX61 device/Yokogawa CSU device spinning-disk microscope (Andor Technology) using 60X oil objective (Olympus).

Relative quantification of gene expression by real-time PCR

Samples for PCR were harvested at day 5 (5 EBs per sample), day 7 (3 EBs per sample), and day 10 (2 EBs per sample). At least 4 independent samples were analyzed for each independent data point, and two independent experiments were performed (total of 8-10 samples per condition). Total RNA was isolated using the NucleoSpin RNA kit (Machery-Nagel). Complementary DNA (cDNA) was produced using the SuperScript II Reverse Transcriptase kit (Thermo Fisher Scientific) and random hexamers. Real-time quantitative PCR was conducted using the SYBR Green dye and the StepOnePlus detection system (Thermo Fisher Scientific). The expression of the housekeeping gene of the 60S acidic ribosomal protein P0 (RPLP0) was used as a reference transcript. All primer sequences are provided in Table S1.

Supporting Information

Supporting Information is available from the Wiley Online Library. ESCs are attracted to the magnetic microtips and form cohesive 3D embryoid bodies (EBs). These EBs are then stimulated magnetically following three independent protocols: the spheroids are either compacted magnetically E) for a short time-period (5 min; MAG-short) or F) for a long one (3 days; MAG-long) by keeping the permanent magnet under the device, or again G) the spheroids were cyclically stimulated using an electromagnet (3 days; MAGcycl).

Table of contents entry

Using a pattern of magnetic microtips and magnetically-labeled embryonic stem cells, highthroughput cell aggregation into embryoid bodies can be achieved. The magnetic pattern allows an on/off magneto-mechanical stimulation of the stem cells which impressively enhances their differentiation into cardiomyocytes when compared to a conventional hanging drop method, with over 90% of embryoid bodies developing spontaneous beating.

Figure S5:

Impact of magnetic field application on intracellular localization of endosomes filled with magnetic nanoparticles (magnetic endosomes). Embryoïd bodies (EBs) were either kept upon magnetic field for 6-hours (left), corresponding to the duration of the magnetic stimulation or just formed above magnet for 5-min, and then left free without any force application (right). A: Scheme illustrating the EBs either on magnet or left free, with a focus at the cell level to define the two distances measured for magnetic endosomes localization: the distance of each endosome from the membrane at the magnet side (down on the scheme and images), and from the nucleus center (or the cell center if the nucleus is not visible on the observed section). B: Transmission electron microscopy (TEM) images of 70-nm thin sections cut alongside the direction of the magnetic field. Magnetic endosomes clearly appear as dark organelles, filled up with the nanoparticles appearing as black dots because they are dense to electrons. Magnetic endosomes are found distributed within the cytoplasm, and in some cases, they are aligned one to the other (see bottom images) due to dipole-dipole magnetic force, 100 higher than the magnetic force created by the external magnetic field gradient. C: Transmission electron microscopy images without magnetic field application, featuring again the dark magnetic endosomes within the cytoplasm. D-G: Histograms of the distances of all imaged magnetic endosomes to the cell edge, upon magnet application (D) or without (E), or to the cell center, upon magnet application (F) or without (G). It demonstrates that the magnetic endosomes are still distributed all around the cytoplasm upon magnet application, evidencing that the magnetic force created on each endosome is not sufficient to displace them towards the side of the cells towards the microtips, and therefore no forces are exerted inside the cells at the cell membrane during magnetic stimulation. Indeed, less than 20% EBs were beating at day 10 for the control hanging drop condition, as well as for the magnetically patterned EBs without stimulation (MAG-short), while with the new batch, this percent was in the 40% range for both control and MAG-short conditions. Yet, the on/off mechanical cyclic stimulation (MAG-cycl) was again improving greatly cardiomyogenesis, with a more than 2-fold increase in the number of beating EBs at day 10. The positive impact of the cyclic stimulation to direct differentiation was further confirmed with RT-PCR, as shown in B). The mesoderm genes (NKX2.5, Gata4 and Gata6) and cardiac mesoderm Tnnt2 gene were 6 to 10-fold upregulated for the MAG-cycl condition. Note that the level of Tnnt2 expression is much lower than the one reached with the new CGR8 ESCs (see Figure 3C), confirming that this ESC batch was less competent for differentiation to the cardiac lineage. However, these results remarkably evidence that, for two independent ESCs batches used, the cyclic stimulation direct cardiomyogenesis, demonstrating further the robustness of the method.

Figure S15: Gene expression of EBs not labeled with magnetic nanoparticles and stimulated, or not, with a cyclic magnetic field. EBs were formed via the hanging drop method starting with 1000 control (non labeled) cells, and were cultured for 2 days to allow the formation of a cohesive structure. At days 2 and 3, the "stimulated" EBs were subjected to the cyclic electromagnetic field, for 6 hours per day, while the "control" EBs were kept free of any magnetic field. Expression of genes involved in mesoderm differentiation (Nkx2.5, Gata4, Gata6 and Tnnt2) was analyzed at days 5, 7, 10 and expressed relatively to day 0. Results show that there is no difference in gene expression between EBs subjected or not to a cyclic magnetic field when no magnetic nanoparticles are internalized in the cells, indicating that a cyclic magnetic field alone is not sufficient to have an effect on the differentiation of ESCs towards the cardiac lineage.

  direct visualisation of EB cyclic deformation(3-4 % range), shown in FigureS6, or on supplementary Movie S2.The increase of cardiomyogenesis triggered by the cyclic magnetic stimulation was further confirmed by immunolabeling and gene expression. Two structural proteins located in the sarcomere were labeled with fluorescent antibody: α-actinin, typical of muscular cells, and cardiac troponin T, typically expressed in cardiomyocytes. Figures3A and 3Bshow an impressive increase in both labels for the MAG-cycl condition, when compared to the three other conditions, which look similar. Additional analyses are shown in FiguresS7-S8(actinin; MAG-long and MAG-cycl conditions), and in Figures S9-S12 (troponin-T; MAGshort, MAG-long, MAG-cycl and DROP conditions) also presenting F-actin labeling with SiR-actin. All images corresponding to the MAG-cycl condition reveal a striated organization for both proteins, representative of the development of sarcomeres, necessary units for cell contraction. It was less marked for observations made at day 7 (Figure S13, MAG-cycl).

Figure 1 .

 1 Figure 1. Remote, magnetically actuated, mechanostimulation of ESCs using a microfabricated (A) magnetic device. B) Image (left) of the 100 μm diameter microtips pattern composed of 30x30 magnetic nickel dots (900 in total); close-up image in the center showing the 1-mm spacing; and further magnification showing a single microtip observed by scanning electron microscopy. C) Magnetic nanoparticles (8 nm in diameter) are internalized in ESCs and localized within endosomes as illustrated on transmission electron microscopy image. D) Spheroids of ESCs are formed via the common hanging drop method (DROP). E-G) Spheroids of ESCs are formed by seeding the cells in a dish positioned on top of the microtips-device exposed to a magnetic field (magnet or electromagnet under the device).ESCs are attracted to the magnetic microtips and form cohesive 3D embryoid bodies (EBs). These EBs are then stimulated magnetically following three independent protocols: the spheroids are either compacted magnetically E) for a short time-period (5 min; MAG-short) or F) for a long one (3 days; MAG-long) by keeping the permanent magnet under the device, or again G) the spheroids were cyclically stimulated using an electromagnet (3 days; MAGcycl).

Figure 2 .

 2 Figure 2. EBs magnetic stimulation. A-B) Images of EBs on the magnetic patterns 5 min (A) or 1 day (B; MAG-cycl) after seeding. C-F) Representative EBs images for all seeding approaches, 5 days after EBs formation (day 5). G) Average size of all EBs for the four seeding protocols. H-K) 10 days after seeding (day 10), representative videos of 6 seconds analyzed using Image J, for the four conditions. In each image two defined beating areas (white, full lines) were selected for kymograph analysis (top and bottom raw) which displays the cells beating. M) Average frequency of the beating cells for all conditions, analyzed at day 10. L) Average percentage of beating EBs, at day 7 and day 10. Statistically significant differences are shown between the conditions with *p<0.1, **p<0.05, ***p<0.01.

Figure 3 .

 3 Figure 3. Magneto-mechanical stimulation induced positive expression of the structural proteins A) mesoderm α-actinin and B) cardiac troponin-T. Immunofluorescence images of the specific antibodies (red) are merged with Dapi (green) for nuclei localization. C) Gene expression of proteins involved in mesoderm and cardiac mesoderm differentiation, and in cardiac progenitors, always renormalized by the expression of the same gene at day 0.

  Statistically significant differences are shown as compared to control (DROP), with *p<0.1, **p<0.05, ***p<0.01.

Figure S3 :

 S3 Figure S3: Simulation of the magnetic field density (B, in Tesla) surrounding the microtip. It was computed with COMSOL Multiphysics, upon the constitutive equations between B and H within the nickel microtip: B = μoH+B0 and B = μoH+M, where B0 = 0.2 T is the magnetic field created by the magnet or electromagnet (perpendicular to the substrate), and M = 4.10 5 A/m is the nickel magnetization. The magnetic field is shown color-coded (A), and the magnetic field gradient (B) was extracted along the microtip axis. It is in the 100 T/m range 100 µm apart from the microtip surface (corresponding to the cell seeding plane, separated from the surface by a 100 µm thin glass slide).

Figure S4 :

 S4 Figure S4: Schematic of the methods employed for the differentiation of embryonic stem cells toward the cardiac lineage (top raws) and of some typical genes expressed over the steps of this differentiation (bottom raws).[1][2][3][4] [1] U. Rutishauser, J. P. Thiery, R. Brackenbury, B. A. Sela, G. M. Edelman, Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 577. [2] E. Willems, P. J. Bushway, M. Mercola, Pediatr Cardiol 2009, 30, 635. [3] M. Nakanishi, A. Kurisaki, Y. Hayashi, M. Warashina, S. Ishiura, M. Kusuda-Furue, M. Asashima, FASEB J. 2009, 23, 114. [4] K. Rajala, M. Pekkanen-Mattila, K. Aalto-Setälä, Stem Cells Int 2011, 383709.

Figure S6 :

 S6 Figure S6: Evidence of the mechanical deformation of the magnetic EBs upon cyclic magnetic stimulation at 1 Hz. The images below represent the kymograph obtained in the centre of each EB (yellow line), and demonstrate a cyclic deformation of the EBs (3.5±0.9 %) following the 1 Hz frequency of the applied magnetic field.

Figure S7 :

 S7 Figure S7: Immunofluorescent analysis of -actinin for the condition MAG-long, at day 10. Additional images to the ones showed in Figure 3A. Cells were labeled with -actinin mouse monoclonal antibody, then stained using goat anti-mouse IgG secondary antibody (red, bottom panel) and counterstained with DAPI (blue, middle panel). Top panel is the merged image.

Figure S8 :

 S8 Figure S8: Immunofluorescent analysis of -actinin for the condition MAG-cycl, at day 10. Additional images to the ones showed in Figure 3A. Cells were labeled with -actinin mouse monoclonal antibody, then stained using goat anti-mouse IgG secondary antibody (red, bottom panel) and counterstained with DAPI (blue, middle panel). Top panel is the merged image.

Figure S9 :

 S9 Figure S9: Immunofluorescent analysis of cardiac troponin-T for the condition MAG-short, at day 10. Cells were labeled with cardiac troponin-T mouse monoclonal antibody, then stained using goat anti-mouse IgG secondary antibody (red, second middle panel) and counterstained with DAPI (blue, first middle panel). SiR-actin probe was used (SC001, Spirochrome, 1:1000 in PBS for 2h at room temperature, before DAPI staining) to stain Factin (yellow, bottom panel). Top panel is the merged image.

Figure S10 :

 S10 Figure S10: Immunofluorescent analysis of cardiac troponin-T for the condition MAG-long, at day 10. Cells were labeled with cardiac troponin-T mouse monoclonal antibody, stained using goat anti-mouse IgG secondary antibody (red, second middle panel), then stained with SiR-actin probe (yellow, bottom panel) and counterstained with DAPI (blue, first middle panel). Top panel is the merged image.

Figure S11 :

 S11 Figure S11: Immunofluorescent analysis of cardiac troponin-T for the condition MAG-cycl, at day 10. Cells were labeled with cardiac troponin-T mouse monoclonal antibody, stained using goat anti-mouse IgG secondary antibody (red, second middle panel), then stained with SiR-actin probe (yellow, bottom panel) and counterstained with DAPI (blue, first middle panel). Top panel is the merged image. In the middle raw image corresponding to the Troponin staining, four sections have been zoomed in and are displayed in the right column, showing the appearance of striated structures.

Figure S12 :

 S12 Figure S12: Immunofluorescent analysis of cardiac troponin-T for the condition hanging DROP, at day 10. Cells were labeled with cardiac troponin-T mouse monoclonal antibody, stained using goat anti-mouse IgG secondary antibody (red, second middle panel), then stained with SiR-actin probe (yellow, bottom panel) and counterstained with DAPI (blue, first middle panel). Top panel is the merged image.

Figure S13 :

 S13 Figure S13: Immunofluorescent analysis of cardiac troponin-T for the condition MAG-cycl, at day 7. Cells were labeled with cardiac troponin-T mouse monoclonal antibody, stained using goat anti-mouse IgG secondary antibody (red, second middle panel), then stained with SiR-actin probe (yellow, bottom panel) and counterstained with DAPI (blue, first middle panel). Top panel is the merged image. As compared to the same condition, at day 10 (previous Figure S11), the troponin staining shows less marked striated muscle fiber localization.

Figure S14 :

 S14 Figure S14: Use of another batch of CGR8 ESCs, having experienced a large number of passages (over 50) prior to use. As shown in A), where the number of beating EBs observed at day 7 and day 10 are plotted for each condition, they were less competent to cardiomyogenesis than the batch newly purchased (Sigma, 07032901) and used in the work. Indeed, less than 20% EBs were beating at day 10 for the control hanging drop condition, as well as for the magnetically patterned EBs without stimulation (MAG-short), while with the new batch, this percent was in the 40% range for both control and MAG-short conditions. Yet, the on/off mechanical cyclic stimulation (MAG-cycl) was again improving greatly cardiomyogenesis, with a more than 2-fold increase in the number of beating EBs at day 10. The positive impact of the cyclic stimulation to direct differentiation was further confirmed with RT-PCR, as shown in B). The mesoderm genes (NKX2.5, Gata4 and Gata6) and cardiac mesoderm Tnnt2 gene were 6 to 10-fold upregulated for the MAG-cycl condition. Note that the level of Tnnt2 expression is much lower than the one reached with the new CGR8 ESCs (see Figure3C), confirming that this ESC batch was less competent for differentiation to the cardiac lineage. However, these results remarkably evidence that, for two independent ESCs batches used, the cyclic stimulation direct cardiomyogenesis, demonstrating further the robustness of the method.

Figure S16 :

 S16 Figure S16: Magnetometry performed at the EB level after their formation, over time (day 1 to day 5). Herein, the EBs were prepared by magnetic attraction of the cells for 5 min (30 000 cells were used per EB to be in a range measurable by magnetometry). They were then left free after formation (condition MAG-short) or kept upon magnet and stimulated for 6-hours per day during the first two days (condition MAG-cycl). Parts a and b show typical magnetization curves for control (A) or stimulated (B) EBs obtained by placing 3 EBs within cuvettes that were measured via vibrating sample magnetometry, at 300 K. Part C shows the average of the saturation magnetic moment per EB, including over 4 independent measurements (with different EBs). It shows that the nanoparticles slowly degrade within the ESCs, but that the cyclic magnetic stimulation doesn't change the degradation rate.

Acknowledgements

This work was supported by the European Union (ERC-2014-CoG project MaTissE #648779). We thank Aude Michel (Sorbonne Université, CNRS, PHENIX, UMR8234) for nanoparticles synthesis, Christine Péchoux from Mima2 platform-Inra (Jouy en Josas) for TEM analysis, and Myriam Reffay for discussion.

Received: ((will be filled in by the editorial staff))

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Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2018.

Supporting Information

High-Throughput Differentiation of Embryonic Stem Cells into Cardiomyocytes with a

Microfabricated Magnetic Pattern and Cyclic Stimulation

Gaëtan Mary+, Aurore Van de Walle+, Jose Efrain Perez, Tomofumi Ukai, Toru

Supplementary Table

Genes

Primers sequences

RPLP0

Fwd: GCC-AGC-TCA-GAA-CAC-TGG-TCT-A Rev: ATG-CCC-AAA-GCC-TGG-AAG-A

Nkx2.5

Fwd: CTC-CGC-CAA-CAG-CAA-CTT-C

Rev: GGA-CTC-TGC-ACG-GTG-TTC-AA

Gata4

Fwd: CCT-CCC-GCA-CGA-TTT-CTG

Supplementary Figures

Figure S1: Fabrication procedure of the nickel micropatterns covered with a silica layer using a photolithography method.

Supplementary Experimental Section

Transmission Electron Microscopy (TEM):

The embryoïd bodies were fixed in 5% glutaraldehyde diluted in 0.1 M cacodylate buffer for 1 hour at room temperature, and then washed two times before storing them in 0.1 M cacodylate buffer. They were then contrasted with Oolong Tea Extract (OTE) 0.5% in 0.1 M Na cacodylate buffer, post-fixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate, gradually dehydrated in ethanol (30% to 100%) and gradually embedded in epoxy resins. Note that epoxy inclusion was achieved by maintaining the direction of the magnet in the vertical direction. Ultrathin sectioning (70 nm) was then performed, in the direction of the magnetic field for the ones placed upon magnet, sections were collected onto 200 mesh copper grids and observed with a Hitachi HT 7700 TEM operated at 80 kV. Sample preparation and observations were performed by Christine Péchoux at the facility of MIMA2 MET -GABI, INRA, Agroparistech, 78352 Jouy-en-Josas, France.

Magnetometry of EBs:

EBs were fixed at different maturation times and measured by magnetometry using a PPMS device equipped with a vibrating sample magnetometer (VSM) option (Quantum Design). The analysis was performed at 300 K and provided the magnetic moment of the EBs (expressed in emu) as a function of the applied field (between 0 and 20 000 Oe). The diamagnetic contribution of the cells and substrate, which appears as a negative slope over the saturation region, was then removed, and the magnetization at saturation was obtained.


domain: sdv chim

text:
Anouchka Plan Sangnier

Aurore Van De Walle

Romain Aufaure

Magali Fradet

Laurence Motte

Erwann Guénin

Yoann Lalatonne
email: yoann.lalatonne@aphp.fr

Claire Wilhelm
email: claire.wilhelm@univ-paris-diderot.fr

Erwann Guenin

M Fradet Imagoseine

Endosomal Confinement of Gold Nanospheres, Nanorods, and Nanoraspberries Governs Their Photothermal Identity and Is Beneficial for Cancer Cell Therapy

Keywords: gold nanoparticles, cell uptake, endosomes, photothermal therapy, cancer cells

come

Introduction

Due to their unique plasmonic properties and their low toxicity, [1][2][3] gold nanoparticles have been used for multiple biomedical applications such as biological sensing, [4][5][6][7] drug delivery, [8][9][10] cellular imaging [11,12] or photothermal cancer therapy. [1,[13][14][15][16][17][18][19][20][21] For long, lasermediated heating was not considered as a potential modality for tumor treatment, firstly because light was not specifically absorbed by cancerous tissues, leading to unavoidable side effects on surrounding healthy tissues, and secondly because biological tissues present a strong extinction coefficient in the visible range, limiting the treatment to superficial or optical fibre reachable tumors. [22,23] The use of gold nanoparticles as thermal enhancers has addressed both issues. First, they are able to accumulate within tumors, either by active or passive targeting. [24][25][26] Then, they can be activated by a laser, allowing sufficient heat generation at low laser powers to induce irreversible cell damages by denaturing proteins and disrupting cell membranes directly within the tumor, minimizing side effects. [23] The on-going challenge has then been to design nanoparticles that absorb at wavelengths within the biological windows, defined as ranges in which the absorbance is minimal for biological tissues [27] . That way, the heating is specific to the nanoparticles leading to localized therapeutic efficacy with minimal side effects. Moreover, it improves laser penetration within the body allowing to reach deeper tumors even though the maximal optical penetration depth is estimated to few centimeters. [28] Biological windows are located in the near infrared (NIR) region and range from 650 to 980 nm for the first one (NIR-I), and from 1000 to 1400 nm for the second one (NIR-II). [22] In practice, NIR-I is less optimal due to limited tissue penetration depth lower than 2 cm. [29] NIR-II is recognized to offer more efficient tissue penetration when considering absorption and scattering effects in tissue. [30][31][32][33] To modulate the plasmonic resonance to the NIR, anisotropic gold nanoparticles such as nanorods, nanostars and nanoshells have been proposed and experimented for cancer thermotherapy. [34][35][36] However photothermal agents within NIR-II are still rare; [START_REF] Cao | Highly Efficient NIR-II Photothermal Conversion Based on an Organic Conjugated Polymer[END_REF][START_REF] Anselmo | Delivering Nanoparticles to Lungs while Avoiding Liver and Spleen through Adsorption on Red Blood Cells[END_REF][START_REF] Chang | [END_REF] among them, multicore gold nanoparticles were recently described as very effective NIR-II nanoheaters. [40,41] As the final goal of photothermal therapy (PTT) is to destroy cancer cells, nanoparticles need to be delivered at the cellular level, within malignant cells, their ultimate target. While the cellular internalization of nanoparticles depends directly on their specific features including shape and surface charge, [42,43] the endosomal confinement resulting from this internalization could have an immediate effect on their specific functions. Herein, the goal is to understand the influence of intracellular processing and endosomal confinement of gold nanoparticles on their heating efficiencies. Indeed, when gold nanoparticles are in close proximity (less than 0.5 nm apart), a plasmonic coupling occurs, leading to a red-shift of the surface plasmon [44,45] as previously evidenced both theoretically and experimentally. [45] As a consequence, the cellular uptake of gold nanoparticles and further aggregation within the endosomes [46] could result in shifting the absorbance towards infrared wavelengths. [44,45,47] We selected a panel of four gold nanoparticles in order to assess their nano-therapeutic potency in situ, in the cellular target environment, by means of heat generation. First, small gold nanospheres were assessed that are advantageous for their simplicity and small size (10nm range), but their plasmon is located in the visible range, therefore they are generally not preferred for photothermal therapy. Gold nanorods with narrow plasmons at 680 and 808 nm were then chosen as state-of-the-art photothermal gold nanoparticles. [48] Finally, gold nanoraspberries were produced, of size small enough (25-nm range), with the advantage of covering both the first and second NIR windows. [41] To assess the impact of cellular internalization on the nanoparticle thermal efficiency, both absorption spectra and heat generation were measured after internalization in cancer cells and compared with measurements in aqueous dispersion, under the same settings. Next step has then been to quantify therapeutic efficiency in terms of induced cancer cell death. Overall, all results converge towards a dramatic change of the gold nanoparticle photothermal fingerprints in cells, which can be highly beneficial for the ones initially absorbing only in the visible region, but harmful for other with initial narrow plasmon band within the NIR-I window.

Results

Structure and absorbance of a panel of four gold nanoparticles

Transmission electron microscopy (TEM) images of all nanoparticles, nanospheres, nanorods and nanoraspberries, are shown in Figure 1A. Nanospheres are single-core nanoparticles composed of a spherical core measuring 9.3±0.6 nm in diameter. Gold nanoraspberries were produced by growing gold over one of these single-core nanoparticles, leading to a multi-core structure with diameter 25±3 nm range. [41] The dimensions (length x diameter) of the two gold nanorods measure 48±5 nm x 16±1 nm (called nanorods-1) and 53±6 nm x 11±1 nm (called nanorods-2).

The nanospheres, nanorods-1 and nanorods-2 all exhibit a narrow plasmon resonance peak as observed on the absorbance spectra of Figure 1B, with a maximum at 520, 680 and 800 nm, respectively. By contrast, the nanoraspberries exhibit a much larger absorbance, covering both NIR-I and NIR-II regions, with a maximum at 550 nm. These spectra correspond to aqueous dispersions of the nanoparticles, further called "in water" conditions.

Internalization in cancer cells

The nanoparticles were then internalized in cancer cells, by supplementing the culture medium with nanoparticles at varying incubation concentrations. The uptake and the resulting dose of nanoparticles internalized in the cells is strongly correlated to this extracellular concentration as shown in Figure 2A. For concentrations of gold between 1 µM and 50 µM, the resulting intracellular dose of gold lies between 0.1 and 6 pg per cell. The absorbance spectra of the cells loaded with nanoparticles (for the high cellular dose of nanoparticles of 2-6 pgAu per cell) were then measured as shown in Figure 2B (condition further called "in cells"). For all nanoparticles, a red-shift of their plasmon absorbance is observed in cells, predicting an impact on the thermal efficiency. Figure 2C shows TEM images of cancer cells following internalization of the gold nanoparticles, for low (0.1-0.4 pgAu/cell) and high (2-6 pgAu/cell) doses of nanoparticles. All nanoparticles are confined within cellular endosomes and, in all situations, they are found in close proximity to one another.

Photothermal heating in cells differs from heating in water

Figure 3A shows typical IR camera images (Figure S1 describes the set-up) of the samples containing nanoparticles and exposed for 5 min to the three lasers at 0.3 W/cm² covering both the NIR-I and the NIR-II windows (680, 808 and 1064 nm). Nanoparticles are either in aqueous dispersion (in water) or internalized within cells (in cells), for both at a resulting gold concentration of [Au]=0.5 mM. Figure 3B shows most typical temperature curves extracted from the IR camera recording, from the moment the laser is turned on. After a few minutes, a plateau temperature is reached, the value of this plateau being next described as T. For the specific laser exposure selected in this Figure 3B, striking differences of heating efficiency are observed when comparing the nanoparticles in water and in cells, with cell confinement systematically beneficial to the heating. All conditions (three lasers always at 0.3 W/cm², four nanoparticles, and two situations in water and in cells) are further detailed in Figure 4 where data are displayed as T. For aqueous dispersion (in water situation), the nanoparticles were diluted at gold concentrations ranging from [Au] = 0.125 mM to [Au] = 2 mM. This range was selected to reach the window where a therapeutic heating can be achieved. It corresponds also to the concentration range that can be further obtained on a tumor mass having internalized gold nanoparticles within the component cells. It is important first to emphasize that all differences in heating efficiency are mostly detected at low doses, which is the windows of applicability of photothermal therapy. Indeed, at high concentrations ΔT saturates around 20°C whatever the laser wavelength; increasing further the nanoparticle concentration is thus not beneficial for heat generation. We can understand this saturation of temperature observed at high concentrations for all samples by writing the heat to light conversion equation as follows: ΔT = 𝑃 0. .(1-10 -𝐴 ).ɳ 𝑚.𝐶.𝐵 (1) with ΔT = the temperature increase (at plateau), P0 the incident power brought by the incident laser, A the absorbance of the sample, ɳ the light-to-heat conversion coefficient parameter, m the mass of the sample assimilated to the one of water, C the specific heat capacity of the sample approximated to the one of water (4185 J/g/K), and B the constant rate of heat dissipation. At high concentrations, 10 -A becomes negligible, and 𝑃 0. .(1-10 -𝐴 ) 𝑚.𝐶.𝐵 is almost constant. As a consequence, ΔT becomes directly proportional to ɳ. This light-to-heat conversion coefficient ɳ can be calculated from experimental heating curves (see experimental section for calculation details). Figure S2 reports the ɳ values measured at the three wavelengths, for all nanoparticles. It was found in the range of 65 %, with no major differences between the nanoparticles or laser applied. This could be intriguing as clearly they do not have the same capacity for heat generation, but in fact η does not precisely account for the efficiency of an object to convert incident power into heat, but rather denotes the ratio of the power converted into heat to the total power lost in the specular direction of the incident light when interacting with the object. The fact that both nanorods exhibit same η is first due to their similar shape. For the nanoraspberries, it demonstrates that they are equally efficient to the nanorods, and for the nanospheres, similar high values are probably due to lower scattering. As ɳ is similar, it is expected that the saturation corresponds to same range (around 20 °C, for 0.3 W/cm² laser power density) of temperature increase, reached at high concentration. The expected (calculated) plateau temperature values are shown in Figure S3 for all conditions. It evidences the fact that all the studied nanoparticles reach eventually the same maximal temperature (of about 20 °C) and that the only differences are coming from the gold concentration necessary to reach this maximal temperature increase. This concentration is directly imposed by the nanoparticles absorbance: the highest their absorbance, the smallest the concentrations will be required to reach the maximal temperature. Nevertheless, for single-core small nanospheres, the theoretical maximum temperature would be reached at

[Au] = 10 mM for the 680 nm laser, and at about 25 and 80 mM for the 808 and 1064 nm lasers, respectively, concentrations which are far from being clinically relevant. By contrast, the maximum value of ΔT ~ 20°C is reached at a concentration as low as [Au] = 0.5 mM for the three other nanoparticles, with the 680 nm laser for the nanoraspberries and the nanorods-1, and with the 808 nm laser for the nanorods-2.

Important differences in thermal elevations can be observed in water depending on the nanoparticle type and the laser wavelength. In brief, when the nanoparticles are irradiated at 680 nm, nanorods-1 and nanoraspberries exhibit a temperature increase within the same range, with values of 18.6 and 20.3 respectively, almost 2-fold the value of nanorods-2 and 3.5-fold the one of nanospheres. At 808 nm, nanorods-2 provided the highest heating, 1.5 times higher than nanorods-1 and nanoraspberries, and 10 times higher than the nanospheres.

Finally, at 1064 nm, the nanoraspberries show the highest heating, with a 1.5 times increase when compared with the nanorods-2, 7 times with the nanorods-1 and 9 times with the nanospheres. Only nanoraspberries were able to generate a temperature increase over 10 °C whatever the laser when in water, and nanospheres remained the less efficient thermal agents for all the three lasers.

To make similar measurements but in the cell environment, all nanoparticles were incubated with cells (prostatic adenocarcinoma PC3) at various extracellular gold concentrations, so that the amount of internalized nanoparticles (in pg of gold per cell) ranges from 0.2 to 6 pg or gold per cell, according to Figure 2A. Cells were then suspended in phosphate saline buffer (PBS), at a density of about 100 million cells per mL, so that the final cell sample concentrations ranged also from [Au] = 0.125 mM to [Au] = 2 mM. Temperature elevation as a function of gold concentration and for all conditions are displayed Figure 4. First, it has to be noted that the maximum temperature reachable found in aqueous solution (ΔTmax ~ 20°C) remained identical in cells. Then, and as predicted by the red-shift in plasmon absorbance following internalization of the nanoparticles in cells, the cellular confinement impacts the PTT properties of the nanoparticles. In the case of nanoraspberries, heating efficiency in cells was reduced at 680 nm, remained similar at 808 nm and increased at 1064 nm. Additionally, these three lasers led to similar temperature elevation in cells, due to the absorption plateau over the NIR-I and NIR-II regions. In the case of nanospheres, the temperature elevation was higher than in aqueous solutions for all three lasers, especially at 680 nm where they became competitive with nanorods and nanoraspberries. Nanorods-1 exhibited a reduction of their heating efficiency at 680 nm compared to water but an increase for the other two lasers, making them good candidates for PTT even at 1064 nm. Finally, nanorods-2 presented a slight reduction of their heating efficiency at 680 and 808 nm and a small increase at 1064 nm. As a consequence, the intracellular confinement was beneficial to the heating potential of the four studied nanoparticles.

Another way to quantify the heating is with the concentration-normalized specific absorption rate (SAR), which corresponds to the heating capacity of the material expressed in watts per gram of gold. The SAR calculated for all concentrations ranging from 0.125 to 2 mM after exposure to lasers 680, 808 and 1064 for nanospheres, nanoraspberries and nanorods 1 and 2 are reported in Figure 5. Theoretically predicted SAR are shown in Figure S4. The SAR values are logically decreasing with increasing concentration. As expected, nanospheres have the lowest SAR values for all three lasers, but it massively increases after cellular internalization, especially for the 680 nm laser. Nanoraspberries are the most polyvalent, both in water and in cells, with only few variations due to cell internalization, and impressive SAR in the NIR-II. Nanorods are strongly impacted by the cellular confinement, which diminishes the interest of having a strong but very fine plasmon resonance perfectly adjusted to one laser wavelength. Overall, the results demonstrate that cell processing importantly reduces the differences between samples.

Therapeutic efficiency in cancer cells: extracellular versus intracellular situations

To confirm the therapeutic efficiency of the four selected nanoparticles, PC3 cancer cells were exposed to nanoparticles and treated with PTT for 10 minutes at safe laser power (0.3 W/cm²) with the three lasers at the set wavelengths of 680, 808 and 1064 nm. Cellular survival was assessed 24 hours upon PTT by flow cytometry in order to detect apoptotic and/or necrotic cells. Nanoparticles were either simply kept in the cell culture medium (extracellular condition, Figure 6A), situation close to the aqueous dispersion (in water), or internalized within the cells (intracellular condition, Figure 6B), same situation as the previous heating measurements in cells. For the extracellular condition, cells were detached, compacted on the form of small tumoral masses (100 million cells per mL), and the surrounding culture medium was supplemented with nanoparticles at [Au] = 0.5 mM during the 10 minutes of laser treatment. For the intracellular condition, nanoparticles were first internalized, and the cells nanoparticles-loaded were detached and treated in the form of small tumoral masses (100 million cells per mL). The final gold concentration of the cell sample was also equal to [Au] = 0.5 mM, adjusted through the initial incubation condition and the cell concentration in the pellets. Figures 6C and6D show typical flow cytometry analyses of the extracellular and intracellular conditions, respectively. Only the conditions with the most striking effect of cell internalization on the therapeutic efficiency of the nanoparticles are displayed. First, the nanospheres (here heated at 680 nm) induce very limited decrease in viability when dispersed outside the cancer cells (76% of cells are still viable, situated in the left-bottom quadrant in the cytometry plot), while once inside the cells, only 26% of the cells remain viable after treatment. Similarly, for the nanorods-1 and nanorods-2 exposed to lasers at 808 and 1064 nm respectively (away from the plasmon peak), the number of viable cells decreases from 38 % and 79 % in the extracellular situation to 11 % and 7 % in the intracellular situation. For the nanoraspberries, at 808 nm the impact of cell internalization is not pronounced (25% and 23 % viable cells before and after internalization), and at 1064 nm, while 52% of cells remained viable for the extracellular situation, it decreased to only 16% after internalization. All conditions are presented in Figure 6E-G, in terms of cells remaining viable after treatment. First, note that no toxicity was induced by laser treatment in the absence of nanoparticles (Figure 6E), confirming the absence of side effects on healthy tissues without nanoparticles within. It is also noticeable that nanoparticles themselves, without laser application, have reduced impact on cell viability, confirming their low toxicity (conditions no laser in Figures 6F and6G). For all nanoparticles, intracellular confinement was mostly beneficial for therapy, leading to an increase of the cellular death as predicted from temperature elevation curves of Figure 2. Nanospheres only induced mortality in extracellular environment when submitted to the laser 680 (but less than 25 % cell death) and no cell death was reached at 808 and 1064 nm. Nevertheless, after the cellular confinement of nanospheres, more than 75 and 25 % cell death were induced after PTT treatments at 680 and 808 nm respectively. Concerning the nanoraspberries, in extracellular conditions, PTT treatment induced a quasi-complete cell death at 680 nm and was also efficient after treatments at 808 and 1064 nm, with respectively 75 and 50 % of cancer cells death. They become even more promising in cells with a similar efficiency at 680 nm but a higher therapeutic result at 808 and 1064 nm, with more than 75% cell death in both cases. However, because the absorbance of the nanoraspberries is large and covers both the NIR-I and NIR-II windows, they were already very efficient in both windows when extracellular, so that the impact of cell confinement is noticeable mostly for the 1064 nm only. Nanorods-1 were highly efficient at wavelength close from their surface plasmon resonance, with an almost complete cell death after laser treatment at 680 nm both in extracellular and intracellular environment. They were also promising candidates after 808 nm laser treatment by inducing more than 50 % cell death when in the extracellular environment and an almost complete cell death after cellular internalization. In the NIR-II, they only became efficient when confined in cells, and induced 50 % cell death. For nanorods-2, when both extracellular and intracellular, laser treatment had a limited effect at 680 nm but induced a quasi-total cell death at 808 nm, where the absorbance was maximal. At 1064 nm, they induced no mortality of the cancer cells in the extracellular environment, whereas cellular confinement turned them into highly efficient nanoheaters, with an almost complete mortality. Figures S5-S7 finally show the percentage of apoptotic cells only (Figure S5, always below 5-10 %), of necrotic cells only (Figure S6, slightly higher, but never over 15 %), and the percentage of both apoptotic and necrotic cells (Figure S7, most common situation). Whatever the treatment, the cells damaged were found mostly both apoptotic and necrotic. Finally, Figure S8 shows another analysis of the photothermal treatment, by metabolic assay. The results are in agreement with cell death evaluated by flow cytometry, and even more marked, with an effective treatment generally leading to a collapse if not a total inhibition of the cells metabolic activity. Remarkably, the treatment was totally efficient for nanoraspberries, whatever the laser applied, and their cellular localization, demonstrating once again their unprecedented treatment versatility.

Discussion

The influence of the biological environment on nanoparticles-mediated thermal therapy has been rarely explored. Nanoparticle efficiencies are generally measured in aqueous dispersion rather than directly in the relevant cellular medium. Yet, it is mandatory to take into account all bio-nano-interactions to develop effective medical nanoparticles. The challenge is then to identify thermal fingerprints of nanoparticles in the cellular environment, and address the question of how the nanoparticles-mediated heating may evolve when tested from the suspension to the cancer cells. Once inside the cells, nanoparticles are confined within endosomes in very close contact with one another, and such confinement is likely to occur in the final therapeutic applications. Notably, for the other well-known magnetic nanoparticlesmediated heating modality, magnetic hyperthermia, the intracellular confinement is already known to dramatically decrease the heat generation. [49][50][51] In the case of plasmonic heating, will plasmon resonance, a surface phenomenon, be affected by nanoparticle confinement?

Herein, the variety of the four nanoparticles investigated together with the original measurements in water and in cells using the exact same methodology were some major assets to resolve the fundamental question of the impact of nanoparticles endosomal confinement on heat generation. Overall, it appeared that this dense intracellular proximity is beneficial to the heating in the near infrared (NIR). Practically, nanoparticles heating in the visible range became efficient in the NIR first windows (NIR-I, 750-980 nm) after cell internalization, while nanoparticles initially designed for the first NIR-I biological window became efficient in the second window (NIR-II, 1000-1400 nm). This was due to a shift of their plasmons towards the infrared wavelengths. Nanoparticles might thus induce completely different temperature elevations according to their environment, aqueous or cellular, emphasizing the need of in situ cellular measurements of their therapeutic potential.

Nanoparticles-mediated PTT is an emergent and promising cancer treatment, already in clinical trials, [START_REF] Rastinehad | Proc. Natl. Acad. Sci. U.S.A[END_REF] with the advantage of an ultra-focal therapy minimizing side effects. It is known that irreversible damages after exposures comprised between 4 and 6 minutes are induced when the tumor is submitted to a temperature over 48 °C (corresponding to a 11°C temperature elevation). [22] This study clearly evidences all nanoparticles, exposed with laser power as low as 0.3 W/cm², are able to generate this temperature increase. However, it also shows that the main difference between the selected nanoparticles is not coming from the temperature increase they are able to provide, but rather from the concentration needed to reach it. Indeed, at high concentrations, the heating saturates at similar temperature elevations of about 20 °C. Nanoparticles reaching this maximal temperature increase for the lowest gold concentrations must then be considered best candidates for PTT applications. Indeed, it means smaller quantities will be necessary to induce a therapeutic effect that is beneficial for in vivo use. From the results, this candidate varies depending on the laser wavelength used; maximal heating at lowest concentration is reached with the nanoraspberries and nanorods-1 at 680 nm, and with the nanorods-2 at 808 nm. In this NIR-I window, such maximal temperature is reached for gold concentrations as low as 0.5 mM. In the NIR-II window, none of the nanoparticles attain such heating even for concentrations as high as 2 mM; however, when internalized in cells the heating efficiency increases and the nanoraspberries reach the maximal temperature at 1 mM followed by the nanorods-1 and -2 that approach it around 2 mM. It is also worth noticing, that the nanospheres are the most affected by endosomal confinement, transitioning from very limited heating when in water to competitive with the other designs once in cells. With their small size, they engender a quite spectacular cancer cell death of 75% at 680 nm.

Overall, the nanoraspberries are promising candidates that reach high heating efficiency in both water and upon internalization within cells due to their large plasmon absorbance spectra covering the NIR-I and -II regions. They reach maximal heating (20 °C) when irradiated with the 680 and 808 nm lasers and are the most efficient design at the 1064 nm wavelength.

Additionally, when compared to the 25 nm photothermal state-of-the-art gold nanostars, these nanoraspberries have a comparable size as well as a high heating efficiency in the two NIR windows, while the 25 nm nanostars are efficient at the beginning of the NIR-I window. [35] This NIR-II heating efficiency certainly is an advantage as NIR-II is recognized to offer more efficient tissue penetration when considering absorption and scattering effects in tissue.

Conclusion

Herein, we demonstrated that the efficiency of gold nanoparticles for PTT depends on the nanoparticles' design as well as the inter-nanoparticles interactions. A switch in the heating profile from visible or NIR-I values to the NIR-I or NIR-II windows respectively has clearly been determined when the nanoparticles are internalized in the endosomes of cells, where they are in close proximity to each other. The nanoraspberries revealed as a very versatile antitumoral nanotool since they are less impacted by the cellular internalization due to their large plasmon and their surprising heating efficiency in the two NIR windows.

Experimental Section

Gold nanoparticles

Gold nanorods: Two types of gold nanorods differing by their size that results in varying peak absorbance, being at 680 nm for the nanorods-1 and at 800 nm for the nanorods-2, were purchased from nanoComposix (product number GRCH660 and GRCH800, respectively).

The dimensions (length x diameter) specified by the manufacturer of the nanorods-1 are 40 nm ± 15 nm x 15 nm ± 5 nm and the ones of the nanorods-2 are 55 nm ± 18 nm x 15 nm ± 5 nm, these dimensions have here been confirmed via TEM analysis (48±5 nm x 16±1 nm for the nanorods-1 and 53±6 nm x 11±1 nm for the nanorods-2).

Gold nanospheres:

The synthesis of the nanospheres consisted in two steps and was adapted from Aufaure et al. [START_REF] Aufaure | [END_REF] First, HMBPene were produced by mixing 4-pentenoyl chloride (5 mmol) frozen in a roundbottom flask with 5 mL of tris-trimethylsilylphosphite. The mixture was then evaporated under vacuum at 70 °C (0.1 Torr) for 20 minutes and hydrolyzed by adding 20 mL of MeOH during 4h. The solvent was further evaporated and the remaining yellow oil was crystallized at pH 2.3 in a MeOH-H2O 9:1 system. The resulting solid was finally filtered on Büchner. Second, the single core nanoparticles were synthesised. Briefly, tetrachloroauric acid in water (2.8 mM, V=36 mL) was heated until ebullition. Then HMBPene at pH adjusted to 9.2 (50 mM, V=4 mL) was quickly added and mixed during 10 minutes. Unreacted products are removed with Amicon® Ultra centrifugal filters (30 K). All reagents were purchased from Sigma-Aldrich and Acros Organics.

Gold nanoraspberries:

The synthesis of the gold nanoraspberries was adapted from Plan Sangnier et al. [41] The first step consisted in producing HMBP-S-PEG45-COOH. To do so, 3 mL of ω-HS-PEG-carboxylic acid dissolved in dimethylformamide (83 mM) were mixed to 3 mL HMBPene dissolved in water (53 mM). A small amount of 1-hydroxycyclohexylphenyl ketone dissolved in dimethylformamide was then added to the mixture, the vial was sealed, and the mixture was stirred during 5 hours under UV light. Ultrapure water (8 mL) was added and the resulting solution was washed 3 times with dichloromethane and diethyl ether. All solvents were degassed prior use. The second step was the production of the nanoraspberries; 63 µL of tetrachloroauric acid (20 mM) were added to 2.5 mL of HMBP-S-PEG45-COOH in water adjusted at pH=4 (2.1 mM) and manually homogenized. When the mixture became light yellow, 19 µl of ascorbic acid (100 mM) were added. Finally, 30 µl of single-core NPs (0.65 mM) was added and manually homogenized. Unreacted products were removed with Amicon® Ultra centrifugal filters (100 KDa). All reagents were purchased from Sigma-Aldrich and Acros Organics.

ICP analysis

Gold concentration in the aqueous samples and in the cells was determined by elemental analysis using Inductively Couple Plasma Atomic Emission Spectrometry (ICP-AES) (iCAP6200 duo THERMOFISHER). First, samples were digested in aqua regia (5 mL), then the acid solution was evaporated. Finally, they were solubilized in a 1% HCl solution for the analysis.

TEM in water

Diluted suspensions of gold nanoparticles were dropped onto copper grids covered by a carbon film. Grids were observed with a Philips Tecnai 12 transmission electron microscope.

UV-visible spectra in water

UV-vis spectra were recorded on a Varian Cary 50 Scan UV-vis spectrophotometer. Gold solutions (0.1 M) were prepared by dissolving nanoparticles in water.

Photothermia in water

Aqueous measurements were made in 1.5 mL Eppendorf tubes containing 100 µl of nanoparticles solubilized in water. Concentrations were ranging from [Au] = 0.125 mM to [Au] = 2 mM. Each sample was illuminated with a 680 nm laser (Laser Components S.A.S France) positioned 4.5 cm above at 0.3 W/cm² (0.6 A), a laser 808 nm (Laser Components S.A.S France) positioned 4.5 cm above at 0.3 W/cm² (1.82 A) and a laser 1064 nm (Laser Components S.A.S France) positioned 4.5 cm above at 0.3 W/cm² (0.33 A). Temperature elevation was recorded with an infrared thermal imaging camera (FLIR SC7000) in real time and processed with the ALTAIR software. Heating was quantified both with the plateau temperature reached after 5 minutes of laser treatment and the specific absorption rate (SAR), meaning the power dissipated per unit mass of gold (W.g -1 ). SAR was determined using the following equation:

𝑆𝐴𝑅 = 𝐶.𝑚 𝑠 𝑚 𝐴𝑢 . 𝑑𝑇 𝑑𝑡 (2)
with C is the specific heat capacity of the sample (Cwater = 4.185 J/g/K), mAu is the total mass of gold in the sample (g), ms is the total mass of the sample (g) and dT/dt is the temperature increase at the initial linear slope (30 s).

Cell culture and nanoparticle internalization

Human prostate cancer (PC3) cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) at 37°C with 5% CO2 until they reach 90% confluence. Gold nanoparticles were dispersed in RPMI-1640 medium. Cells were then incubated with 30 minutes with the nanospheres or 24 hours with the nanoraspberries, nanorods-1 and nanorods-2. The incubation concentrations were ranging from 1.5 to 25 µM for the nanospheres and the nanoraspberries. For the nanorods-1 and -2, they were ranging from 3 to 50 µM. Cells were then washed twice with RPMI-1640 medium then left 2 hours in PS and FBS supplemented DMEM for a chase period.

Photothermia in cells

A T75 flask was prepared for each condition. PC3 cells were incubated with gold nanoparticles in order to get a panel of concentrations ranging from 0.2 to 4.4 pg/cell. Cells were detached, washed with cacodylate buffer, fixed with 2% glutaraldehyde in 0.1 M cacodylate and suspended in 40 µl in order to get solutions with concentration comprised between 0.125 and 2 mM. If the cells were used for viability assessment, they were covered with a sterile cover-slip (not fixed). Each sample was illuminated with lasers 680, 808 and 1064 nm positioned 4.5 cm above and set at 0.3 W/cm². PTT measurements were made in 1.5 mL Eppendorf tubes. The temperature was recorded with an infrared thermal imaging camera (FLIR SC7000) in real time and was processed with ALTAIR software. Heating was characterized as in water with the plateau temperature after 5 minutes of heating and the SAR.

Flow cytometry

Effect of PTT on the cells when nanoparticles are only in the media (extracellular condition) or when they are internalized within the cells (intracellular condition) was assessed via flow cytometry. For the extracellular condition, cells (not labelled with nanoparticles) were detached, resuspended in 1.5 ml Eppendorf tubes and centrifuged such as forming small tumoral masses (100 million cells per mL). The surrounding culture medium was then supplemented with nanoparticles during the 10 minutes of laser treatment only, at a concentration of [Au] = 0.5 mM. For the intracellular condition, nanoparticles were first internalized in the cells via a 30 minutes incubation for the nanospheres and 24 hours incubation for the nanorods and nanoraspberries followed by 2 hours chase. The cells loaded with nanoparticles were then detached and centrifuged such as forming small tumoral masses similarly to the extracellular condition (100 million cells per mL) and the final gold concentration of the cell sample was also equal to [Au] = 0.5 mM, adjusted through the initial incubation condition and the cell concentration in the pellets. The small tumoral masses were then exposed to PTT for 10 minutes and directly after laser treatment, cells were seeded into 6 wells plates. 24 hours later, they were labeled with APC Annexin V Apoptosis Detection Kit with Propidium Iodide from Biolegend. They were detached, washed twice with PBS and suspended in 100 µl Annexin V binding buffer. 5 µl of Annexin V and 10 %l of propidium iodide were added. Cells were analyzed with a Cyan ADP 9C flow cytometer (Beckman Coulter, Imagoseine platform, Institute Jacques Monod, Paris). Each analysis is based on a minimum of 50,000 events. Each condition corresponds to three analyses on three independent cell samples.

TEM in cells

For TEM imaging of cellular samples, cells were detached after nanoparticle incubation, washed with cacodylate buffer, fixed with 2% glutaraldehyde in 0.1M cacodylate buffer.

Oolong tea extract (0.5%) in cacodylate buffer was used to contrast the cells that were then post fixed with 1% osmium tetroxide. Finally, they were included in Epon after dehydration and ultrasectioned (70 nm). Sections were deposited onto cooper grids for observation with a Hitachi HT7700 electron microscope operated at 80 kV (Elexience -France), and images were acquired with a chargecoupled device camera (AMT). This work has benefited from the facilities and expertise of MIMA2 MET -GABI, INRA, Agroparistech, 78,352 Jouy-en-Josas, France.

UV-visible spectra in cells

Cells were cultured on 0.5 mm diameter glass coverslips. Culture medium was supplemented with the nanoparticles at highest gold concentrations of 50 µM. After incubation, cells were washed with DMEM supplemented with 10 % SVF and 1 % PS then 6 glass coverslips were overlaid in 96-well plates and the spectra were measured with an EnSpire Multimode Plate

Table of contents entry

A set of four plasmonic gold nanoparticles of multiple size and shape, spheres, rasbperrieslike multicores and rods, are investigated for their photothermal therapeutic heat generation within cancer cells. All provide a better therapy once inside the cells, due to an absorbance shift towards the infra-red wavelengths, for some with a remarkable increase of the heat generated intracellularly.

Keyword: gold nanoparticles-based photothermal therapy

Figure 1 .

 1 Figure 1. Panel of gold nanoparticles. A. TEM images of the nanoparticles dispersed in water, from top to bottom: single-core nanospheres, multi-core nanoraspberries, nanorods-1 and nanorods-2. B. Corresponding absorbance spectra for each nanoparticle type, dispersed in water, and obtained at a concentration [Au] = 0.12 mM.

Figure 2 :

 2 Figure 2: Internalization of the gold nanoparticles (nanospheres nanoraspberries, nanorods-1 and nanorods-2) in cancer cells. A) The dose of internalized nanoparticles is assessed in function of the incubation concentration. B) Absorbance spectra of the nanoparticles when internalized in cells at high doses (2-6 pg of gold/cell). C) TEM images of the four types of nanoparticles right after internalization in cancer cells, for high (2-4 pg/cell) and low (0.1-0.4 pg/cell) doses. The nanoparticles are confined in the endosomal compartments of the cells. Higher doses lead to increased amount of both the endosomes and the nanoparticles within.

Figure 3 :

 3 Figure 3: Nanoparticles in aqueous dispersion (in water situation) and cells containing nanoparticles (in cells situation) are irradiated with the three lasers, with wavelengths at 680, 808, and 1064 nm, always at 0.3 W/cm². A) Typical IR images for all nanoparticles (nanospheres, nanoraspberries, nanorods-1 and nanorods-2, from left to right) are displayed for both in water and in cells situations, at global sample concentration [Au] = 0.5 mM. B) Representative temperature curves showing the progressive heating and illustrating the plateau temperature T are displayed for each nanoparticle, in conditions where the changes between the in water and in cells situations are the most pronounced (680 nm laser for the nanospheres, 808 nm laser for the nanorods-1, and 1064 nm laser for the nanoraspberries and nanorods-2).

Figure 4 .

 4 Figure 4. Average temperature elevation T compared in water (black curves) and in cells (red curves) for nanospheres, nanoraspberries, nanorods-1 and nanorods-2 (from top to bottom), upon irradiation with the three lasers at 680, 808 and 1064 nm (from left to right) and 0.3 W/cm². Gold concentrations are ranging from 0.125 to 2 mM.

Figure 5 :

 5 Figure 5: SAR values compared in water and in cells for (from top to bottom) nanospheres, nanoraspberries, nanorods-1 and nanorods-2 irradiated with the three lasers at 0.3 W/cm² and at the wavelengths (from left to right) 680, 808 and 1064 nm. Concentrations are ranging from 0.125 to 2 mM.

Figure 6 .

 6 Figure 6. Photothermal treatment of cancer cells with gold nanoparticles. The nanoparticles are A) added to the culture medium for the 10 minutes of PTT treatment only (extracellular condition) or B) internalized in the cancer cells prior to PTT. C-D) Cancer cells were exposed

AFigure S2 .

 S2 Figure S2. Photothermal ligh-to-heat conversion coefficient parameter (η) calculated for each laser at 680, 808 and 1064 nm wavelengths for all nanoparticles. The error bars represent the standard deviation between all measurements.

Figure S3 .

 S3 Figure S3. Theoretical temperature increase after laser submission of (from top to bottom) nanospheres, nanoraspberries, nanorods-1 and nanorods-2 when submitted at different lasers (680, 808 and 1064 nm) for concentrations ranging from A. 0 to 2 mM at 0.3 W/cm² and B. 0 to 20 mM.

Figure S4 .Figure S6 .Figure S7 .

 S4S6S7 Figure S4. Theoretical SAR for nanospheres, nanoraspberries, nanorods-1 and nanorods-2when submitted at different lasers (680, 808 and 1064 nm) for concentrations ranging from 0 to 3 mM at 0.3 W/cm²

Figure S8 .

 S8 Figure S8. Impact of all conditions of photothermal treatments, measured via the resulting metabolic activity of the cells 24 hours after the treatment. Nanospheres, nanoraspberries and nanorods-1 and -2 were applied to the cells extracellularly or intracellularly, upon the exact same protocol as used for flow cytometry analysis of induced cell death (compare with results in Figure6E-G)

Acknowledgements

This work was supported by the Sorbonne Paris Cité program (CardioNanoStem) and the European Union (ERC-2014-CoG project MaTissE #648779). We thank technical support from CNanoMat-University Paris 13 for physico-chemical characterization and IPGP multidisciplinary program PARI, Paris-IdF region SESAME Grant no. 12015908, the European Union (FEDER), the Région Haut de France, ESCOM and UTC within the framework of the chair: "Green Chemistry and Processes", the France BioImaging infrastructure from the ImagoSeine core facility (ANR-10-INBS-04).

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Reader Perkin Elmer. Baseline corresponding to the absorbance of cells cultured in the same conditions and not loaded with nanoparticles was removed from all measurements.

Theoretical calculation of the light-to-heat conversion coefficient, ΔT and SAR

The light-to-heat conversion coefficient was calculated as previously described in the literature. [54] η

With ɳ the light-to-heat conversion efficiency coefficient, mW the mass of the sample approximed to the mass of water, Cw the specific heat capacity of water, P0 the incident laser power and A the absorbance of the sample. B is the constant rate of heat dissipation to the external environment and is given by the decreasing temperature profile when the laser is turned off:

Considering ɳ and B constant (experimentally calculated equal to 65 % and 9.7.10-3 s-1) a fitting of the temperature increase is possible thanks to the equation (1). SAR can also be fitted: [50] ∆T = η *

Statistical Analysis

All values are presented as mean ± standard error of the mean (SEM) and the number of independent measurements was systematically superior to 3 (n > 3).

Supporting Information

Supporting Information is available from the Wiley Online Library.

Supporting Information

Endosomal confinement of gold nanospheres, nanorods and nanoraspberries governs their photothermal identity and is beneficial for cancer cells therapy

Anouchka Plan Sangnier, Aurore Van de Walle, Romain Aufaure, Magali Fradet, Laurence Motte, Erwann Guenin, Yoann Lalatonne*, Claire Wilhelm*