teoría del desarrollo Cognitivo

For this approach, the key to successful incorporation of the dye into the NPs is to exploit the higher hydrophobicity of the NP core compared to the water/base solution. The selected dye was mixed with d-UPTES and co-sprayed into the water/base solution. The synthetic procedure was then continued following Method C (Fig. 6.10).

Figure 6.10. Schematic representation of the step-by-step procedure applied for dye incorporation via a non- covalent approach using Method C.

The dyes selected for this type of incorporation are py and C153 (Fig. 6.2a and b, respectively). Both fluorophores present photoluminescence properties dependent on the polarity of the surrounding environment and therefore their incorporation into the NPs can be followed using fluorescence spectroscopy.

Py is rather insoluble in water (solubility = 0.135 mg L-1) with a ΦPL of 65% in ethanol. 52,53

The sensitivity of py to the polarity of the local environment can be extrapolated from two main features: (i) the ratio between the emission intensities of the third and the first peak in the PL spectra (I3/I1 ratio) and (ii) the presence of a broad band centred at ~465 nm, which is indicative of

the formation of an excimer and is usually present when the dye aggregates (i.e. in confined environments).54, 55 Considering the structure of the Jeffamine, the polarity of the core of the ureasil NPs can be compared to that of ethanol. The emission properties of py in a mixture of EtOH/water at different ratios were therefore monitored to investigate the typical value of the I3/I1 ratio in the

two environments (Fig. 6.11). The emission spectrum of py in EtOH (Fig. 6.11a, red line) is characterised by an intense emission band ranging from 350 to 450 nm and consisting of four vibronic maxima. The corresponding emission spectrum at the same concentration in water presents the same band structure. In addition, a strong emission band centred at ~470 nm is

observed, which is typical of emission from the pyrene excimer and suggests that the py molecules are aggregated.54 This hypothesis is also confirmed by the appearance of the solution of py in pure water, which was cloudy compared to the other samples investigated.

Figure 6.11. Investigation of the PL properties of pyrene in environments with different polarity. (a) Emission spectra (λex = 335 nm) of a solution of py (2.5 × 10-5 mol L-1) in water (black line) and EtOH (red

line). The I1 and I3 emission lines are labelled. (b) I3/I1 intensity ratios for py in a water/ethanol mixture at

different volume percentage of water.

A series of solutions containing different water/EtOH volume ratios was prepared and upon addition of the same aliquots of py stock the I3/I1 ratio were calculated for each mixture. The

results are presented in Fig. 6.11b. The I3/I1 ratio decreases from 0.74 to 0.55 as the concentration

of the water is increased from 0 to 80%, respectively. The I3/I1 value for py in water, however, is

slightly higher than expected. This result, might be due to the presence of a contribution of the emission coming from the excimer which can overlap with that of Peak 3, leading to an increase in the intensity of the latter. Py-doped ureasil NPs were prepared following Method C, through addition of the same volume of a stock solution of py as that used for the calibration curve, to the d-UPTES/THF mixture. The NPs prepared with this method show Dh of 149±2 nm and PdI of

0.08±0.01. The emission spectrum of the py-ureasil NPs and the corresponding I3/I1 ratio were

monitored over the course of two days (Fig. 6.12). All the doped-NP samples present an emission spectra characterised by the typical features of the py monomer, with a well-structured emission band ranging between 350 and 450 nm. In addition, the emission of the excimer is also observed at ~470 nm. The intensity of this contribution, increases from t = 2 h to 6 h, and then decreases progressively with time (Fig. 6.12b). Accordingly, in the same time range, the corresponding I3/I1

ratio presents a main decrease from 0.68, which is higher than that of the pyrene in water, to 0.64 and then continues to decrease in time reaching 0.59 after 48 hours. A small increase in both the size and the PdI of the doped-NPs was also observed over the course of the 48 hours, from 149 to 177 nm and from 0.08 to 0.26, respectively.

Both the emission spectra and the I3/I1 ratio values indicate that upon incorporation in the

NPs, py presents an intermediate optical behaviour between that observed for the dye in pure EtOH and in pure water (Fig. 6.12). From these results, it seems that the fluorophore is initially incorporated inside the NPs however, it spontaneously leaches out with time. It would be tempting to assume that the presence of the excimer emission for the py-doped NP samples, is on its own indicative of the confinement of py units into the NP cores. However, the aggregation of the dye is clearly observed in water as well so in this instance, the I3/I1 ratio is a preferred diagnostic tool to

investigate the local environment surrounding the dye, suggesting that pyrene leaches out spontaneously from the NPs with time. This might be due to the inherent polarity of the Jeffamine units, which is higher than that of pure aliphatic chains. The I3/I1 ratio, however, is still higher than

that recorded for pyrene in a 100%v/v water solution which represents a promising initial result to

start from for the optimisation of the non-covalent dye-encapsulation approach.

Figure 6.12. In time evolution of the PL properties of the py-doped NPs. (a) Normalised emission spectra of the py-doped NPs in time (λex = 335 nm) and py in water and in EtOH (dashed lines) and (b) I3/I1 intensity

ratio (black dots) and normalised PL maxima of the excimer emission (red dots). The lines serve only to guide the eyes.

The second dye that was encapsulated through the non-covalent approach is C153. The emission of this fluorophore is known undergo a blue-shift when the polarity of the environment is decreased.56, 57 C153 was incorporated into the NPs using the same process described above. The

obtained NPs present a Dh of 117±2 nm, with a PdI of 0.14±0.03. The emission spectrum of C153

in water (Fig. 6.13a) consists of a broad band between 430 and 680 nm, with a maximum centred at ~549 nm. The PL spectrum of C153-doped NPs presents comparable features but is slightly blue-shifted (2 nm), suggesting a change in the polarity of the local environment of the dye. The corresponding excitation spectra for both samples present very similar features; a broad band from 330 to 500 nm with the maxima centred at 411 nm. From these results, it seems that the properties of both samples are dominated by that of the dye dissolved in a water solution. The main spectroscopic differences between the two samples are observed in the absorption spectra (solid lines), whose maximum is red-shifted for the solution of C153 in water compared to that of the C153-doped NPs (429 nm vs 418 nm, respectively), indicating that the dye might be surrounded by a more apolar environment upon incorporation into the NPs.58 To test this hypothesis, the solution of NPs was dialysed for 24 hours against a NH4OH/water solution (pH = 9) and the emission

spectrum was measured again (Fig. 6.13b).

Figure 6.13. PL properties of C153 in water, upon incorporation into ureasil core-shell NPs and upon dialysis. (a) Normalised absorption (solid lines), emission (λex= 420 nm, dashed lines) and excitation (λem=

550 nm, dashed-dot lines) spectra of C153 in water (green) and in ureasil NPs (blue) (3.6 × 10-5 mol L-1). (b) Normalised emission spectrum (λex= 390 nm) of C153 in water (green) and in the NPs (blue) after dialysis.

When comparing the normalised emission spectra of C153 in water and of C153-doped NPs after dialysis, it is clear that the dye is present in two very different local environments, as demonstrated by the dramatic blue-shift in the emission maximum of the C153-doped NPs sample after dialysis (from 547 to 468 nm). Although this value is an indication that the dye is surrounded by the a more apolar environment compared to water, it is hard to strictly correlate the polarity of this surrounding to that of the core of the NPs. Models, such as the Lippert-Mataga relationship,59

can be of aid when trying to predict the entity of the Stokes’ shift of a fluorophore as a function of the polarity of the environment. However, it is extremely difficult to model the polarity of the nanoparticle interior and exterior environments, which includes contributions from the poly(propylene) chains, the silica shell, the encapsulated water and the ammonia.