Commercial reagents 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane (PTA; 97%, Aldrich), 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane (DAPTA; 97%, Aldrich), Gold(III) chloride trihydrate (HAuCl₄·3H₂O, ≥99.9, Aldrich), trisodium citrate dihydrate (HOC(COONa)(CH₂COONa)₂·2H₂O, Aldrich), oleylamine (OLAm, 70%, Aldrich), oleic acid (OLAc, ≥99%, Aldrich), 1-octadecene (ODE, 90%, Aldrich), 2-Propanol (iPrOH, for HPLC, VWR Chemicals), and chloroform (CHCl₃, 99.2%, VWR Chemicals) have been used as received. Literature methods have been used to prepare [Au(C≡ C–C₅H₄N)(PTA)] (Gavara et al., 2013). and [Au(C≡ C–C₅H₄N)(DAPTA)] (Aguiló et al., 2013) compounds, that from now on will be named PTA Au(I) complex and DAPTA Au(I) complex, respectively, for the sake of simplicity.

The synthesis of hydrophilic Au NPs was based on that reported by Turkevish and coworkers (Turkevich et al., 1953). Briefly, 8.8 mg HAuCl₄·3H₂O (0.02 mmol) were dissolved in 60 mL of deionized water and the solution was heated to the boiling point. Once the temperature was reached, 5.2 mL of an aqueous solution of sodium citrate at 2% was added and let the reaction for 20 min at this temperature. The color of the solution changed from yellow to dark red as a result of the formation of gold nanoparticles.

The synthesis of hydrophobic Au NPs was adapted from that described by Yu and co-workers as follows: (Yu et al., 2005) A mixture of ODE (30 mL), OLAc (4.5 mL), and OLAm (5.5 mL) was stirred in a 100 mL three-necked flask for 20 min at 120°C under vacuum. Meanwhile, in a glovebox HAuCl₄·3H₂O (120 mg, 0.30 mmol) was dissolved in ODE (7.5 mL) and OLAm (1.5 mL) in an auxiliary vial. Once the purge was complete and the system under N₂, the solution of gold precursor was injected into the flask and temperature was fixed at 150°C. After 30 min of reaction, the heating was stopped, and the solution was washed with 2 equivalent volumes of iPrOH, and centrifuged at 4,500 rpm for 4 min. The product was redispersed in chloroform.

The functionalization of hydrophobic Au nanoparticles with DAPTA was performed by a simple reaction at room temperature in chloroform. 4.2 mg of DAPTA (7.94 × 10⁻³ mmol) were dissolved in 8 mL chloroform and mixed with a solution of hydrophobic Au nanoparticles dispersed in chloroform. The solution was shaken for 24 h. After this time, the solution was centrifuged and the resultant precipitate was redispersed in chloroform.

The functionalization of hydrophobic Au nanoparticles with PTA was adapted from the protocol described above but using 1.3 mg (2.85 × 10⁻³ mmol) of PTA instead of DAPTA.

The synthesis of Au-DAPTA (or -PTA) hydrophilic nanocomposites were done following the same procedure described for the nanocomposites in organic media but substituting the chloroform for deionized water.

Three different samples of each nanocomposite were synthesized using different amounts of Au NPs. The volumes of Au NPs solution used in each case are compiled in Table 1.

Au NPs were prepared for observation by TEM by dilution in chloroform/water followed by sonication. A droplet of the solution was then poured in holey carbon covered copper TEM grids. A JEOL 2000 FX II conventional TEM operating at an accelerating voltage of 80 kV was used.

Scanning electron microscopy (SEM) was carried out at 20 kV using J-7100F (Jeol) equipped with a thermal field electron source.

Dynamic Light Scattering (DLS) measurements were carried out in a Zetasizer NanoS Spectrometer. The samples were measured in quartz cuvettes.

IR spectra were recorded with a FTIR 520 Nicolet Spectrophotometer. For the measurements, a pellet of a mixture of the sample and KBr was used.

A Cary 100 Scan 388 Varian UV/Vis spectrophotometer was used with quartz cuvettes for optical characterization.

The compositions and concentrations of the nanoparticles solutions were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The measurements were performed with an Optima 3200 RL PerkinElmer spectrometer. For the measurements, 50 mL of the solutions was precipitated in MeOH and redispersed in CHCl₃. The solution was evaporated in an oven overnight at 90°C. Aqua regia (2.5 mL) and H₂O₂ (0.7 mL) were added to the precipitate before the vial was sealed and then heated to 90°C for 72 h. The resulting solution was transferred to a 25 mL volumetric flask and diluted with Milli-Q water.

¹H NMR [δ(TMS) = 0.0 ppm], ³¹P{¹H} NMR [δ(85% H₃PO₄) = 0.0 ppm] spectra have been obtained on a Varian Mercury 400 and Bruker 400.

SAXS was performed on the NCD-SWEET beamline at the ALBA Synchrotron at 12.4 keV, and the distance sample/detector was 6.2 m to cover the range of momentum transfer of 0.028 < q <2.56 nm⁻¹. The data were collected on a Pilatus3S 1M detector with a pixel size of 172.0 × 172.0 μm². The exposure time was 30 s. The q-axis calibration was obtained by measuring silver behenate (Huang et al., 1993). The program pyFAI was used to integrate the 2D SAXS data into 1D data (Kieffer and Karkoulis, 2013). The data were then subtracted by the background using PRIMUS software (Konarev et al., 2003). The maximum particle dimension Dmax and the pair distance distribution function P(r) were determined with GNOM (Svergun, 1992). The low-resolution structure of the aggregates was reconstructed ab initio from the initial portions of the scattering patterns using the program DAMM (Svergun, 1999; Krebs et al., 2004).

MDA-MB-231 breast adenocarcinoma and HT-29 colon carcinoma were maintained in DMEM high glucose (PAA) supplemented with 50 mg/L gentamycin and 10% (V/V) fetal calf serum (FCS) at 37°C under 5% CO₂ atmosphere and passaged every 7 days. Antiproliferative effects were determined as follows: a volume of 100 μL of a 38,000 cells/ml (HT-29) or 40000 cells/ml (MDA-MB-231) suspension were seeded into 96-well plates and incubated for 48 or 72 h at 37°C/5% CO₂. After the incubation period the cells of one individual plate were fixed by addition of 100 μL of a 10% glutaraldehyde solution per well. After 30 min. One hundred and Eighty Microliter PBS were added and the plate was stored at 4°C (t₀ plate) until further procedures (see below). Nanoparticle suspensions were diluted 1:100 with cell culture medium to the final test concentrations. In the remaining plates the medium was replaced by the medium containing the nanoparticles or solvent control (water or CHCl₃). Then the plates were incubated for 72 h (HT-29) or 96 h (MDA-MB-231) at 5% CO₂/37°C. The medium was removed and the cells were treated with 100 μl of a 10% glutaraldehyde solution. Afterwards the cells of all plates (including the t₀ plate) were washed with 180 μL PBS and stained with 100 μL of a 0.02% crystal violet solution for 30 min. The crystal violet solution was removed and the plates were washed with water and dried. A volume of 180 μL of ethanol 70% was added to each well and after 2–3 h of gentle shaking the absorbance was measured at 595 nm in a microplate reader (Victor X4, PerkinElmer). The mean absorbance value of the t₀ plate was subtracted from the absorbance values of all other absorbance values in order to correct for the initial cell biomass. The IC₅₀-values were calculated as the concentrations reducing the cellular proliferation in comparison with the solvent control by 50%.

Our synthetic strategy started by preparing two samples of Au NPs capped by hydrophilic and hydrophobic stabilizing ligands separately, i.e., citrate anions and oleylamine molecules, respectively (see Materials and Methods section). TEM micrographs of the as-prepared samples allowed to evaluate the morphological and size distribution of the NPs in the two samples. The hydrophilic sample consists of a collection of nanoparticles with a significant distribution of shapes including spherical (the most abundant), triangular, and elongated nanoparticles of low aspect ratio as shown in Figure 1a. On the contrary, Figure 1b is a TEM representative image of the hydrophobic sample in which only spherical faceted nanoparticles are observed. The size distribution was also much narrower in the hydrophobic sample, even though both samples showed an average diameter of ca. 14 nm and their deposition on the TEM grid suggests that the colloids are well-dispersed in solution without evidences of aggregation.

The correct formation of the resulting nanocomposites was evidenced by SEM micrographs as depicted in Figures 2, 3 corresponding to DNO_1 and PNA_1 samples, made of DAPTA Au(I) complex in chloroform and PTA Au(I) complex in water respectively, and both with the highest amount of Au NPs in the reaction medium. The images suggest that the samples are mainly constituted by aggregates of Au NPs, with sizes ranging between 100 and 300 nm approximately that are well-interconnected between them forming a kind of sponge-like 3D network. The individual aggregates are likely surrounded by a shell of the organometallic complex. Indeed, a quantitative shrinkage of the aggregates is evident when comparing topographic images in panels (a) and (c) in the figures, recorded with secondary electrons, with the corresponding Z-sensitive images in panels (b) and (d), recorded with backscattered electrons. The significant reduction in volume of the clusters between the two related images is indicative of the presence of an organic shell around the metallic NPs. Besides the presence of the organometallic-NP composites, few long fibers are also observed at low magnifications (Figures 2c,d, 3c,d). The vanishing of these fibers in images recorded using backscattered electrons confirms their organic origin, suggesting that they are formed as a result of the supramolecular interactions between free molecules of the organometallic moiety, as it was observed in previous studies of these organometallic complexes (Aguiló et al., 2013), and that they do not incorporate Au NPs in their structure. Generally speaking, the observed trend with amount of Au NPs was the same, independently of the organometallic molecule and solvent used for the synthesis of the nanocomposites: the larger the amount of Au NPs used for the synthesis, the higher the number of composite aggregates encountered, while few or none nanocomposites were found in samples containing the lowest amount of Au NPs (samples XXX_3 of each group), as shown in Figures S1–S4 in the SI. Some large aggregates of approximately 20 μm can also be appreciated by SEM, although in a much lower frequency compared with the previous smaller ones. DLS measurements performed on these samples mainly confirm the average size of the aggregates, this being of a few hundreds of nanometers as observed by SEM, as well as the presence of a small population of microscale aggregates as indicated by SEM too (see Figure S5 in the SI). Noteworthy, DLS data evidence the presence of smaller aggregates with sizes ranging from 20 to 60 nm approximately depending on the sample, which cannot be discerned from the larger ones by previous SEM characterization.

The size and shape of the aggregates were measured by SAXS for those samples prepared with the highest (XXX_1) and medium (XXX_2) amount of Au NP. Samples with less amount of Au NPs were not measured since previous SEM studies revealed the presence of no composites in the solutions. Each of the eight considered samples (DNO_1/2; DNA_1/2; PNO_1/2 and PNA_1/2) was measured as initially prepared and also with two additional diluted concentrations (i.e., with DNO_1_1 being the as-prepared sample, and DNO_1_2 and DNO_1_3 the samples derived from 1/500 and 1/1000 dilution), at different temperatures (from 20 to 40°C in a 5°C gradient, for better analysis of the aggregates' formation) and 1 week after their preparation to favor aggregation. The low-resolution structures were reconstructed ab initio from the scattering patterns using the DAMMIN program (see Materials and Methods section). The resulting size and morphologies profile are depicted in Figures 4 and Figures S6–S18 for all compounds that display significant aggregation. No significant aggregation was detected for the rest of the cases. Generally speaking, aggregates of sizes between 10 and 30 nm are measured by SAXS depending on the sample, which hardly agrees with the size of the aggregates observed and measured by analysis of SEM micrographs in Figures 2, 3, but on the opposite their sizes roughly coincide with the values observed for the smallest aggregates detected by DLS measurements and shown in Figure S5. The values obtained by DLS and SAXS point out the possibility that the relatively large aggregates (100–300 nm) observed by SEM might be formed by smaller subunits (10–60 nm), linked by organometallic domains, that are only appreciable by light and X-ray scattering experiments. From our experimental data it can be concluded that Au NPs are mainly wrapped within a layer of organometallic complex forming small metallic clusters of a few nanoparticles. Additionally, larger 3D aggregates based on non-covalent interactions are formed, which are responsible for the sponge-like structure observed. In general, it can be seen that temperature does not affect substantially the size of the aggregates. Only in the case of DNO2, a significant increase on the size is clearly detected in both diluted samples, DNO2_1 and DNO2_2. This can be ascribed to the higher contribution of hydrophobic interactions at higher temperatures, being DAPTA moieties (more soluble in water) avoiding the contact with organic solvent. Within each set of samples measured at different concentrations, the collected data indicates that no aggregation occurs for the most diluted samples (XXX_X_3), while appreciable degrees of aggregation were measured, in general, for the other two dilutions. This data could indicate the existence of a critical concentration of aggregates below which they lose their stability. Some other trends can be observed looking at this data regarding now the amount of Au NPs used in the reaction. That is, in organic medium, a light increase of size was detected for PNO_2_X samples containing a smaller amount of Au NPs or aggregates compared to analogous PNO_1_X samples. On the contrary, DNO_1_X samples give rise to the formation of larger structures compared to the DNO_2_X analogs. These observations could be rationalized based on the lower solubility of DAPTA (in DNO complexes) in organic medium with respect to PTA (in PNO derivatives). Following this reason, it makes sense to understand the contrary effect in water: smaller aggregates for samples PNA_2_X containing a smaller amount of Au NPs or aggregates, and larger aggregates for samples DNA_2_X containing also a smaller amount of Au NPs or aggregates were observed, since the higher solubility in water of DAPTA Au(I) complex makes the formation of aggregates more difficult in this solvent.

The infrared spectra of the nanocomposites display the expected bands both from the organometallic complex and from the stabilizing ligands of the NPs. The resulting broadening of the alkynyl moieties are indicative of Au···π interaction between the NPs' surface and the complex.

The UV/vis absorption spectra of the hybrid system present two different signals, as shown in Figure 5 for the hydrophilic nanocomposites with DAPTA and PTA. On the one hand, there is the band around 260 nm attributed to the Au(I) complex. On the other hand, the signal at 500–600 nm is assigned to the characteristic LSPR band of Au NPs. To better study the effect of the quantity of gold NPs added to the formation of hybrid system, absorption spectra of three different systems per organometallic molecule were recorded (DNA_1-3 and PNA_1-3). There is a change in the profile of the more energetically part of the absorption spectra, when the amount of nanoparticles used in the synthesis of the hybrid system increases, there is a loss of the vibrational shape in the band as an evidence of interaction between NPs and the corresponding organometallic complex (Ferrer et al., 2008).

The biological evaluation was focused on determining the effects against tumor cell growth in HT-29 colon carcinoma and MDA-MB-231 breast cancer (Table 2). Inspection of Table 2 let us retrieve some important conclusions: (i) Hybrid organic systems present higher cytotoxicity than the corresponding aqueous systems; (ii) hybrid systems present higher cytotoxicity than their respective counterparts, both organometallic complexes and NPs, being a nice cooperative effect than makes promising this encapsulation process regarding the study of their biological activity. However, the toxicity of the drug free organic NPs and the role of CHCl₃ as solvent for redispersion has to be taken into account in future studies.

Experiments with non-tumor cells were not performed as tumor selectivity in the used assay could not be expected. By theory, nanoparticles would be enriched in tumor tissue by the EPR effect. This effect is not present in static tissue culture models, and thus tissue selectivity of nanoparticles can not be evaluated in this assay (Nakamura et al., 2016).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.