Design, synthesis and characterization of the multifunctional Iren-AuSiO2_Aptamer nanoplatforms

Multifunctional light-responsive nanoplatforms were implemented by incorporating an Iridium-based metal complex, Iren, with photosensitizing and luminescent properties [41], within the polysiloxane matrix of gold-core/silica-shell nanoparticles (Iren-AuSiO2_COOH).

The preparation of Iren-AuSiO2_COOH was performed according to the well-known reverse microemulsion method [46] and a schematic representation of the synthetic procedure is illustrated in Fig. 1A. In a W/O microemulsion, water nanodroplets are stabilized by surfactant molecules and dispersed in a continuous oil phase. These reverse micelles act as nanoreactors, within which the homogenous and highly reproducible synthesis of nanoparticles takes place, minimizing the batch-to-batch variability. In the first step, the reduction of tetrachloroaurate(III) to Au0 leads to the formation of the gold core. Then, the addition of the silane precursors in alkaline environment gives rise, through hydrolysis and condensation processes, to the formation of the silica shell. The addition of the photosensitizing and luminescent Iren before the start of the polymerization process, ensures its physical incorporation into the polysiloxane matrix. Finally, the nanoparticles surface was functionalized with a coating agent containing a siloxy alkyl chain with a terminal carboxyl group. The full characterization of the Iren-AuSiO2_COOH nanoparticles, morphological features, surface charge, optical properties, photosensitizing and thermoplasmonic abilities, was reported in the Supplementary Information. TEM images revealed a homogeneous population of spherical gold-core/silica-shell particles, with an average size of 49.13 ± 4.28 nm and a gold core of 6.31 ± 0.81 nm (Supplementary Fig. 1A). Iren-AuSiO2_COOH are characterized by a hydrodynamic diameter of 85.51 ± 1.17 nm, Polydispersity Index (PDI) of 0.148 and a negative ζ-potential value of -23.5 ± 3.95 mV (Supplementary Fig. 1B). Similar ζ-potential values are reported for silica surfaces functionalized with terminal carboxyl groups in neutral aqueous medium [47, 48]. The successful loading of Iren within the nanoparticle silica shell was confirmed by UV–Vis spectroscopy (Supplementary Fig. 1C,D). In particular, the extinction spectrum of the colloidal solution of Iren-AuSiO2_COOH shows a broad band centered at 520 nm, corresponding to the characteristic Localized Surface Plasmon Resonance (LSPR) of the spherical gold core [49], and a more intense absorption band at lower wavelengths (250–300 nm), due to electronic transitions involving the Iren molecule [41]. Under light excitation, the emission spectrum of Iren-AuSiO2_COOH presents the characteristic band of the Iridium compound, peaked at 520 nm. The goodness of the emission spectrum was confirmed by the excitation one (Supplementary Fig. 1D).

Fig. 1

Schematic illustration of the protocol for the obtainment of the final nanoplatforms. A Iren-AuSiO2_COOH preparation by reverse microemulsion technique. Surfactant: 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol; Mesna: sodium 2-mercaptoethanesulfonate; APTES: (3-aminopropyl)triethoxysilane; TEOS: tetraethoxysilane; capping agents: 11-Triethoxysilylundecanoic acid and N-(3-triethoxysilyl) propylsuccinic anhydride. B Covalent crosslinking strategy to achieve aptamers-nanoplatforms conjugates (Iren-AuSiO2_Aptamer). EDC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; NHS: N-Hydroxysuccinimide. C Iren-AuSiO2_Aptamer samples prepared and tested in this study

In order to validate the photo-triggered properties of the obtained nanoplatforms, light-induced singlet oxygen generation and photothermal effects were assessed (see Supplementary Information for details). In particular, the singlet oxygen generation capability was evaluated by chemical method using 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) as detection probe [50]. The photooxidation of ABDA in presence of Iren-AuSiO2_COOH was monitored by measuring its absorbance at 378 nm; while the absorbance attenuation of ABDA was negligible for the control solution (see Supplementary Information and Supplementary Fig. 2A), in presence of Iren-AuSiO2_COOH, the absorption decreased significantly with the extension of the irradiation time (Supplementary Fig. 2B). For an immediate comparison, the ABDA absorbance values as function of the irradiation time were plotted (Supplementary Fig. 2C) and for both solutions (control and sample) a good linear relationship was observed.

The heat generation of the nanoplatforms under continuous illumination was investigated (Supplementary Information). As clearly demonstrated by the thermal images acquired after an irradiation time of 90 min (Supplementary Fig. 3), in the case of the control solution (see Supplementary Information for its description), a non-significant temperature variation was observed, whereas in the case of Iren-AuSiO2_COOH, a photothermal heating of the irradiated solution as well as the surrounding environment was highlighted. The observed thermoplasmonic effect can be explained as a result of a photothermal conversion of the energy absorbed by Iren molecules and partially transferred to the metal nanoparticle. In particular, the evidenced spectral overlap between the emission band of the Iridium compound and the LSPR of the gold core, allows donor–acceptor energy transfer processes, from the Iridium-based molecules to the gold core, which in turn converts the received energy into heat [51]. Therefore, using a single excitation wavelength in the Iren absorption region, Iren-AuSiO2_COOH nanoplatforms can act as luminescent probes, photosensitizing agents, and heat nanosources.

In order to specifically address Iren-AuSiO2_COOH to target tumor/stromal cells, conjugation with the EGFR CL4 (Iren-AuSiO2_CL4) or PDGFRβ Gint4.T (Iren-AuSiO2_Gint4.T) aptamers, and dual functionalization with both CL4 and Gint4.T (Iren-AuSiO2_CL4_Gint4.T) were performed. Iren-AuSiO2_Scr, decorated with a non-targeting scrambled aptamer, were used as a negative control. The schematic representation of the synthetic steps involved in the development of the aptamers-conjugated gold–silica nanoplatforms and a key illustration of all the prepared samples is shown in Fig. 1B,C. Specifically, to obtain aptamers-nanoplatforms conjugates (Iren-AuSiO2_Aptamer), the free –COOH groups of Iren-AuSiO2_COOH were activated—through EDC/NHS chemistry [52, 53]—to react with the 5' NH2-aptamers, with consequent formation of amide bonds (− CO–NH −). In particular, EDC reacts with a carboxylic group on nanoparticles surface, resulting in an amine-reactive O-acylisourea intermediate. The addition of NHS stabilizes the amine-reactive intermediate by converting it to an amine-reactive NHS ester. Then, through a nucleophilic attack the NHS ester is easily displaced by the amine group on the 5′-end of aptamer to yield a stable amide bond (Fig. 1B). Extinction spectra of Iren-AuSiO2_COOH/NHS and all conjugated samples are displayed in Fig. 2A. Where the spectral profile of Iren-AuSiO2_COOH/NHS results superimposable on the extinction spectrum of Iren-AuSiO2_COOH (Supplementary Fig. 1C), in the case of Iren-AuSiO2_CL4, Iren-AuSiO2_Scr, Iren-AuSiO2_Gint4.T and Iren-AuSiO2_CL4_Gint4.T, a shoulder at 260 nm—characteristic of the maximum absorption peak of RNA sequences [54]—was observed. This absorption feature proves the successful RNA functionalization. The stability of aptamers-conjugated nanoparticles was monitored by recording extinction spectra over 7, 14, 21 and 28 days. As shown in the Supplementary Fig. 4A-D, no significant variation of the spectral profiles – decrease in optical density or shift of absorption maxima—was observed over time, highlighting the stability of the nanoplatforms in the aqueous environment.

Fig. 2

Characterization of Iren-AuSiO2_Aptamer nanoplatforms. A UV–Vis spectra, B hydrodynamic diameters, C ζ-potential values of Iren-AuSiO2_Aptamer nanoplatforms dispersed in water

The DLS results (Fig. 2B,C, Supplementary Table 1) reported that the Iren-AuSiO2_COOH/NHS nanoplatforms were characterized by a hydrodynamic diameter equal to 102.0 ± 0.15 nm (PDI of 0.172) and a negative ζ-potential value of -26.0 mV. Iren-AuSiO2_CL4 and Iren-AuSiO2_Scr were characterized by a hydrodynamic diameter of 102.1 ± 0.93 nm (PDI = 0.163) and 101.4 ± 0.20 nm (PDI = 0.162), respectively, and a negative ζ-potential value of -22.3 and -22.9 mV each. Iren-AuSiO2_Gint4.T and Iren-AuSiO2_CL4_Gint4.T were characterized by a hydrodynamic diameter equal to 104.4 ± 0.47 nm (PDI = 0.168) and 103.7 ± 0.82 nm (PDI = 0.162), respectively, and a negative ζ-potential value of -28.3 and -26.4 mV each. Therefore, Iren-AuSiO2_COOH/NHS as well as all Iren-AuSiO2_Aptamer samples have a hydrodynamic diameter very similar to each other. However, these values are larger than that of Iren-AuSiO2_COOH, plausibly due to variations in the surface coating and/or hydration sphere. Conversely, no significant changes were observed in the surface charge values of all the nanoplatforms (Iren-AuSiO2_COOH, Iren-AuSiO2_COOH/NHS, Iren-AuSiO2_Aptamer), as result in all cases of an extensive presence of free carboxyl groups on the surface of the nanoplatforms. The amount of each aptamer conjugated to the nanoplatforms was evaluated by RT-qPCR analysis on Iren-AuSiO2_Aptamer (Supplementary Fig. 5). We quantified approximately 3.0 pmol aptamer per 16.0 pmol of Iren-AuSiO2_ Aptamer nanoplatform and the efficiency of conjugation ranged between 2 and 6% with a mean ± SEM equal to 3.3 ± 0.7%.

2D cell imaging by Iren-AuSiO2_Aptamer nanoplatforms

To assess the cell targeting/uptake ability of nanoparticles conjugated to a single aptamer, CL4 or Gint4.T, or functionalized to both aptamers, we took advantage of different human cell lines expressing only EGFR, only PDGFRβ, both, or neither of the receptors. Specifically, human MES-TNBC MDA-MB-231 and BT-549 cells were chosen as double-positive cells as they express both EGFR and PDGFRβ ([23] and Supplementary Fig. 6) and, consequently, are specifically targeted by the two aptamers either when grown in classical 2D cultures and 3D Matrigel-embedded cultures, or implanted in mice [23, 30, 39]. Furthermore, we previously proved that CL4 strongly improves the uptake of drug-loaded and aptamer-decorated poly(lactic-co-glycolic)-poly ethylene glycol-based nanoparticles into both cell lines in vitro and in vivo [24]. As models of EGFR+/PDGFRβ− cell lines, we used breast cancer BT-474 and epidermoid carcinoma A431 cell lines, which express moderate and high levels of EGFR, respectively, without expressing PDGFRβ ([23, 44] and Supplementary Fig. 6), and are thus recognized by CL4 [19] but not Gint4.T [35, 39]. Moreover, stromal MSCs that express high levels of PDGFRβ were selected as EGFR−/PDGFRβ+ cells ([30, 55] and Supplementary Fig. 6). Importantly, we previously demonstrated that Gint4.T, by blocking PDGFRβ, efficiently inhibits MSCs recruitment into TNBC thus preventing their pro-metastatic function [30]. Finally, EGFR−/PDGFRβ− breast cancer MCF7 cells ([23] and Supplementary Fig. 6) were used as a negative control.

The intrinsic Iridium-compound associated fluorescence emitted (Em = 520 nm) from the unconjugated Iren-AuSiO2_COOH/NHS or Iren-AuSiO2_Aptamer nanoplatforms (5 μM Iren concentration) was collected by confocal microscopy after incubation of the cells with the nanoparticles at 37 °C in the presence of yeast tRNA and salmon sperm DNA competitors to hinder any non-specific interactions. As shown (Fig. 3A), the signal associated with Iren-AuSiO2_Aptamer (Iren-AuSiO2_CL4, Iren-AuSiO2_Gint4.T and Iren-AuSiO2_CL4_Gint4.T) nanoparticles was clearly visible in the cytoplasm of MDA-MB-231 cells at 30 min and further increased at 60 min of incubation. Conversely, an almost undetectable signal was obtained with unconjugated Iren-AuSiO2_COOH/NHS or scrambled decorated Iren-AuSiO2_Scr nanoparticles at the two incubation times. Importantly, MDA-MB-231 cells treated with the dual-targeting EGFR/PDGFRβ nanoparticles had significantly higher fluorescence intensity than that treated with single-targeting EGFR or PDGFRβ nanoparticles (Fig. 3A), indicating that CL4 and Gint4.T, simultaneously attached to the nanoparticles, confer improved cellular uptake. Similar results were observed on EGFR+/PDGFRβ+ BT-549 cells (Fig. 3A). Labeling cells with WGA to visualize cell membrane confirmed that the CL4 and Gint4.T aptamers properly drive the nanoparticles in the cytoplasm (Supplementary Fig. 7A). As expected, no signal was observed in double negative MCF7 control cells (Fig. 3A).

Fig. 3

Selective cell uptake of CL4 and/or Gint4.T-decorated Iren-AuSiO2_Aptamer nanoplatforms in 2D cultures. Representative confocal images of A MDA-MB-231 and BT-549 (EGFR+/PDGFRβ+), and MCF7 (EGFR−/PDGFRβ−) cells, B A431 (EGFR+/PDGFRβ−) cells, and C MSCs (EGFR−/PDGFRβ+) cells, incubated with Iren-AuSiO2_Aptamer or unconjugated Iren-AuSiO2_COOH/NHS nanoparticles at 37 °C for the indicated times. After washing and fixation, cells were labeled with NucRed (blue) to stain nuclei, and nanoparticles are displayed in red. Magnification: 63 × , 1.0 × digital zoom, scale bar = 10 μm. All digital images were captured under the same settings to enable a direct comparison of staining patterns. A-C Mean fluorescence intensity (MFI) was quantified using Zeiss software on at least ten separate images for each condition. Bars depict means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 relative to Iren-AuSiO2_Scr; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001

As a next step, the nanoparticle’ formulations were incubated for 60 min onto only EGFR+ A431 (Fig. 3B) and BT-474 (Supplementary Fig. 7B) cell lines, and only PDGFRβ+ MSCs (Fig. 3C), and confocal microscopy analyses revealed that the internalization ability of the nanoparticles strictly depends on the aptamer present on the nanoparticle’s surface and the expression of its receptor partner on the target cell.

Overall, these results show efficient capability of Iren-AuSiO2_Aptamer nanoparticles to selectively enter into the cell through EGFR and/or PDGFRβ recognition and indicate that the carriers’ parameters, including composition, size, shape and surface chemistry, do not affect the interactions of CL4 and Gint4.T aptamers to their proper receptor on membranes of target cells.

Uptake of Iren-AuSiO2_Aptamer nanoplatforms in 3D multicellular tumor spheroids

Next, we wondered whether multifunctional nanoparticles retain their targeting ability in more relevant in vitro cancer models by using 3D multicellular spheroids obtained by co-culturing tumor and stromal cells on non-adhesive culture dishes, which resemble the organization and properties of a native tumor, as a key factor of translational medicine [56, 57]. Even if these models have been successfully used to study tumor-MSC interaction [56, 57], to date a still limited number of studies have employed 3D tumor spheroids for evaluating the functionality of nanomedicine [58].

In order to distinguish cancer cells from stromal cells we established tumor spheroids consisting of GFP-labeled BT-549 and unlabeled MSCs. In agreement with previous findings, both BT-549 cells [59, 60] and MSCs [61, 62] were able to form spheroids alone in non-attached culture (Supplementary Fig. 8A). When cancer cells were co-cultured with stromal cells at a 1:4 ratio, respectively [63], consistent heterotypic spheroids (BT-549 + MSCs), were obtained, reaching approximately 180 µm in diameter, in 13 days (Supplementary Fig. 8A and Fig. 4A). In order to better visualize formed spheroids, they were embedded in Matrigel and observed by confocal microscopy (Fig. 4B). NucRed 647 nuclear stain was used to visualize both cancer and stromal cells, while the GFP to visualize cancer cells. As shown (Fig. 4B), the presence of NucRed 647 nuclear stain (visualized in blue) either associated to the GFP signal (BT-549) or not (MSCs) (see arrows in the merged image), indicates the mixed composition of the spheroids. Moreover, immunofluorescence with α-SMA and FAP markers confirmed the presence of tumor-associated MSCs in the heterotypic spheroids (Supplementary Fig. 8B). To examine the penetration of the different nanoformulations into the spheroids, BT-549/MSCs spheroids were exposed to Iren-AuSiO2_Aptamer and unconjugated nanoparticles, at an Iren concentration of 5 μM, for 24 h at 37 °C and visualized by confocal microscopy (Fig. 4C). Importantly, the presence of the EGFR and PDGFRβ targeting aptamers on the surface of the nanoparticles, either single-targeted (Iren-AuSiO2_CL4 and Iren-AuSiO2_Gint4.T) or dual-targeted (Iren-AuSiO2_CL4_Gint4.T), allowed them to penetrate the mixed tumor/stromal spheroids as qualitatively displayed by the nanoplatform-associated fluorescent signal (visualized in red) that was visible throughout the spheroids. 3D images clearly indicated the accumulation of the aptamer-functionalized nanoparticles inside the spheroid mass. Conversely, no signal was detected with unconjugated Iren-AuSiO2 or Iren-AuSiO2_Scr negative control (Fig. 4C), thus indicating that passive infiltration of the spheroids by untargeted nanoparticles could not occur at least under the experimental conditions used.

Fig. 4

Selective uptake of CL4 and/or Gint4.T-decorated Iren-AuSiO2_Aptamer nanoplatforms in 3D heterotypic spheroids. A (left) Growth kinetic of BT-549-GFP + MSC spheroids represented in spheroid diameter over 13 days. The representative phase-contrast microscopy images of spheroids formation over the course of seven days are reported in Supplementary Fig. 8A. Data are presented as the mean ± SD (n = 3); (right) representative phase-contrast microscopy image of the spheroids grown at day 13. Magnification: 10 × , scale bar = 100 μm. B Representative confocal image of the heterotypic spheroid at day 13. BT-549-GFP cells are visualized in green and nuclei, stained with NucRed 647, in blue. White arrows in the merged images highlight the mixed composition of the spheroid (MSC, blue; BT-549-GFP, blue light). C Representative confocal images of BT-549-GFP/MSC spheroids grown at day 13 and then incubated with Iren-AuSiO2_CL4, Iren-AuSiO2_Gint4.T, Iren-AuSiO2_CL4_Gint4.T, Iren-AuSiO2_Scr or unconjugated Iren-AuSiO2_ COOH/NHS for 24 h at 37 °C. Nanoparticles, BT-549-GFP cells and nuclei are displayed in red, green and blue, respectively. 3D images are shown. B,C Magnification: 10 × , 1.0 × digital zoom, scale bar = 100 μm. All digital images were captured under the same settings to enable a direct comparison of staining patterns

Anticancer photokilling activity of Iren-AuSiO2_Aptamer nanoplatforms in 3D multicellular tumor spheroids

We wondered whether the nanoparticles conjugated to CL4, Gint4.T or both the aptamers, once penetrated into the spheroids, kill cancer and stromal cells upon light irradiation. Thus, we first validated that AuSiO2_COOH/NHS, AuSiO2_Scr, AuSiO2_CL4 or AuSiO2_Gint4.T nanoparticles, which had no load of photosensitizing and luminescent molecule Iren, when incubated for 24 h at 37 °C on MDA-MB-231 and BT-549 cells have no adverse effects on cell viability (Supplementary Fig. 9A,B).

Next, before moving on to more complex 3D cell systems, we verified the therapeutic efficacy of Iren-loaded nanoformulations in 2D cell cultures. To this aim, BT-549 and MDA-MB-231, positive for EGFR and PDGFRβ, BT-474 and A431, positive for EGFR, MSCs, positive for PDGFRβ, and MCF7 cells, negative for both receptors, were incubated with the different nanoformulations containing Iren (5 µM) and decorated or not with the aptamers, for 1 h, given the rapid cell uptake of aptamer-decorated nanoparticles, washed to remove not-internalized nanoparticles, exposed to 1-h light irradiation and analyzed for their viability after 24 h. As shown (Supplementary Fig. 9C-H), Iren-AuSiO2_Aptamer nanoplatforms demonstrated efficient capability to selectively kill the cells, through EGFR and/or PDGFRβ recognition, in comparison with untargeted NPs (unconjugated Iren-AuSiO2_ COOH/NHS or scrambled-conjugated Iren-AuSiO2_Scr). No toxicity of aptamer-decorated nanoplatforms was observed on each cell line under dark conditions, thus indicating their safety behavior at least in the concentrations tested in the PDT experiments. We verified that free Iren is nontoxic and does not contribute to the photoinduced killing activity effects, at least at the concentration and exposure time used in the encapsulated form (Supplementary Fig. 9C-H).

Importantly, when incubated for 24 h with heterotypic BT-549 + MSCs spheroids, Iren-AuSiO2_CL4, Iren-AuSiO2_Gint4.T and Iren-AuSiO2_CL4_Gint4.T (5 µM Iren concentration; 1-h light irradiation), disrupted spheroid structure (Fig. 5A,B) and inhibited cell viability (Fig. 5C), as determined by spheroids number counting and CellTiter-Glo 3D cell viability assay, respectively, with the dual targeted nanoparticles being more effective than the single-targeted ones (approximately 80% inhibition, Iren-AuSiO2_CL4_Gint4.T vs 40% inhibition, Iren-AuSiO2_CL4, and 50% inhibition, Iren-AuSiO2_Gint4.T). Conversely, no effect was observed on controls consisting of untreated or treated with scrambled-decorated Iren-AuSiO2 nanoformulations spheroids (Fig. 5A-C). Similarly, a higher effect on cell viability inhibition was observed upon treatment of heterotypic MDA-MB-231 + MSCs spheroids with Iren-AuSiO2_CL4_Gint4.T compared to single-targeted nanoparticles (Fig. 5D,E).

Fig. 5

Anticancer activity of Iren-AuSiO2_Aptamer nanoplatforms on 3D spheroids of EGFR+/PDGFRβ+ cancer cells and MSC. A (left) Representative phase-contrast microscopy images of BT-549/MSC spheroids treated with Iren-AuSiO2_CL4, Iren-AuSiO2_Gint4.T, Iren-AuSiO2_CL4_Gint4.T or untargeted Iren-AuSiO2_Scr. Spheroids treatment with specific aptamer-decorated nanoplatforms, but not with Iren-AuSiO2_Scr, inhibits both the B number of spheroids and C cell viability, expressed as percentage of viable treated cells with respect to untreated spheroids. D Representative phase-contrast microscopy image of MDA-MB-231/MSC spheroids grown at day 13. E Cell viability assay on MDA-MB-231/MSC spheroids treated as in A. A,D Magnification: 10 × , scale bar = 100 μm. B,C,E Bars depict mean ± SD (n = 3). ***p < 0.001, ****p < 0.0001 relative to Iren-AuSiO2_Scr; #p < 0.05, ##p < 0.01, ###p < 0.001. No statistically significant variations among Iren-AuSiO2_Scr and untreated were obtained

Because MES-TNBC cells express both EGFR and PDGFRβ, in order to confirm the efficacy of the dual-targeting nanovectors on both cancer and stromal cells, we generated tumor spheroids consisting of cancer BT-474 cells (only EGFR+) and MSCs (only PDGFRβ+) that grew as colonies with approximately a 150 μm diameter after 13 days in culture (Supplementary Fig. 10A and Fig. 6A). As shown, uptake into BT-474/MSCs spheroids of either single-targeted Iren-AuSiO2_CL4 and Iren-AuSiO2_Gint4.T, as well as dual-targeted Iren-AuSiO2_CL4_Gint4.T nanoparticles after 24 h of incubation was clearly detected by confocal microscopy (Fig. 6B) and the cytotoxic effect of Iren-AuSiO2_CL4_Gint4.T nanoparticles on both the spheroid disruption (Fig. 6C,D) and cell viability inhibition (Fig. 6E) was higher than that of either Iren-AuSiO2_CL4 or Iren-AuSiO2_Gint4.T (approximately 70% inhibition for Iren-AuSiO2_CL4_Gint4.T vs 40% inhibition for Iren-AuSiO2_CL4 and Iren-AuSiO2_Gint4.T, relative to untreated cultures), thus confirming the ability of dual-decorated nanoparticles to kill both cancer and stromal cells through EGFR and PDGFRβ targeting, respectively.

Fig. 6

Anticancer activity of Iren-AuSiO2_Aptamer nanoplatforms on 3D spheroids of EGFR+/PDGFRβ− cancer cells and MSC. A Growth kinetic of BT-474 + MSC spheroids represented in spheroid diameter over 13 days. The representative phase-contrast microscopy images of spheroids formation over the course of thirteen days are reported in Supplementary Fig. 10A. B Representative confocal images of BT-474/MSC spheroids grown at day 13 and then incubated with Iren-AuSiO2_CL4, Iren-AuSiO2_Gint4.T, Iren-AuSiO2_CL4_Gint4.T, or untargeted Iren-AuSiO2_Scr for 24 h at 37°C. Nanoparticles and nuclei are displayed in red and blue, respectively. 3D image (Iren-AuSiO2_CL4_Gint4.T) is shown. Magnification: 10 × , 1.0 × digital zoom, scale bar = 100 μm. All digital images were captured under the same settings to enable a direct comparison of staining patterns. C Representative phase-contrast microscopy images of BT-474/MSC spheroids treated as indicated. Spheroids treatment with specific aptamer-decorated nanoplatforms, but not with Iren-AuSiO2_Scr, inhibits both the D number of spheroids and E cell viability, expressed as percentage of viable treated cells with respect to untreated spheroids. F Growth kinetic of A431 + MSC spheroids represented in spheroid diameter over 13 days. The representative phase-contrast microscopy images of spheroids formation over the course thirteen days are reported in Supplementary Fig. 10B. G Cell viability assay on A431 + MSC spheroids treated as in C. A,D,E,F,G Data are presented as the mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001 relative to Iren-AuSiO2_Scr; #p < 0.05, ###p < 0.001, ####p < 0.0001. No statistically significant variations among Iren-AuSiO2_Scr and untreated were obtained

Similarly, tumor spheroids consisting of A431 cells (only EGFR+) grown with MSCs (only PDGFRβ+) up to 13 days (Supplementary Fig. 10B and Fig. 6F) were higher affected by EGFR/PDGFRβ bispecific nanoformulations than those single-targeted, with a reduction of cell viability of approximately 90% for Iren-AuSiO2_CL4_Gint4.T, 40% for Iren-AuSiO2_CL4 and 30% for Iren-AuSiO2_Gint4.T relative to untreated cultures (Fig. 6G).

Anticancer photokilling activity of Iren-AuSiO2_Aptamer nanoplatforms on 3D patient-derived cancer organoids

To further prove the targeting efficacy and the photoinduced killing activity of dual aptamer-decorated nanoparticles, through selective recognition of EGFR-positive tumor cells and PDGFRβ-positive stromal component in the entire tumor bulk, we employed 3D patient organoids from human surgical specimens collected from three patients with diagnosis of breast cancer. Tumor samples, henceforth named M23, M41 and M43, were chosen for the presence of EGFR only in tumor cells (Fig. 7A, blue arrows) and PDGFRβ only in the stromal component (Fig. 7B), specifically in vascular endothelial cells (green arrows) and/or mesenchymal stromal cells (orange arrows), as assessed by immunohistochemical analyses.

Fig. 7