Metal-Enhanced Fluorescence Study in Aqueous Medium by Coupling Gold Nanoparticles and Fluorophores Using a Bilayer Vesicle Platform.

Gold nanoparticles (AuNPs) display excellent plasmonic properties, which are expected to assist fluorescence enhancement for dyes, and the phenomenon is known as "metal-enhanced fluorescence" (MEF). In this study, we demonstrate AuNP-induced MEF for a modified bipyridine-based construct 4-(pyridine-2-yl)-3H-pyrrolo[2,3-c]quinoline (PPQ) when it binds with biologically important Zn2+. Importantly, this phenomenon is observed under aqueous conditions in a biocompatible bilayer vesicle platform. When PPQ binds with Zn2+ to form the complex in the presence of appropriate AuNPs, MEF is evident once compared with the fluorescence intensity in the absence of AuNPs. Among the three different sizes of AuNPs used, the enhancement is observed with an average diameter of 33 nm, whereas 18 and 160 nm do not show any enhancement. A possible mechanism is ascribed to the radiating plasmons of the AuNPs, which can couple with the emission frequencies of the fluorophore under a critical distance-dependent arrangement. We witness that the enhancement in fluorescence is accompanied with a reduction in lifetime components. It is proposed that the mechanism may be predominantly derived from the enhancement of an intrinsic radiative decay rate and partly from the localized electric field effect. Overall, this work shows a rational approach to design fluorophore-metal configurations with the desired emissive properties and a basis for a useful nanophotonic technology under biological conditions.

Introduction

Recently, metal-enhanced fluorescence (MEF)[1−3] has been recognized as a popular method by the scientific community, which has led to new developments and applications by improving the photoluminescence intensity of variety of materials, such as quantum dots, dyes, lanthanide nanocrystals, carbon dots, and so forth.[4−6] It is a common belief that metals quench the fluorescence when the emitters are very close to the metals. However, in MEF, when the fluorophores or emitters are placed in close proximity but to an optimum distance from the metallic nanostructures, mainly silver or gold, they are benefitted by the additional plasmon-enhanced optical fields. The observed augmented fluorescence intensity is credited to the local field enhancement associated with the excitation of localized surface plasmon resonances (LSPRs) in the metal nanostructures. Most importantly, the coupling between the frequencies emitted by the fluorophore and the plasmon resonance of the metal particle causes the metal to radiate light (with enhanced intensity) at the same frequency. The plasmonic nanoparticle here serves as a transmitting optical antenna to transfer the near field to the far field at the fluorescence wavelength. Although many comprehensive works are available in the literature,[1−4] we still believe that the far-reaching capability of MEF is still untested. Because most of the studies and their applications are mainly accomplished using solid substrates such as metal films, periodic nanoparticle arrays, and so forth, the MEF studies in aqueous solutions are limited.[7]

Considering this, the current work exploits the gold nanoparticle (AuNP)-induced plasmonic enhancement of the fluorophore 4-(pyridine-2-yl)-3H-pyrrolo[2,3-c]quinoline (PPQ) and its Zn2+ complex in a carefully accomplished bilayer structure of a “niosome”. Niosome[8−14] is an important class of pharmaceutical nanocarriers made up of nonionic surfactants, which find many diverse applications in the area of nanomedicine especially in the transdermal drug delivery systems. They mimic synthetic biological membranes just like liposomes;[15] that is, it comprises a “bilayer” and a “water pool”, which can suitably entrap both hydrophobic and hydrophilic dyes, respectively, to our advantage. Here, we demonstrate that the fluorescence intensity of the PPQ–Zn2+–PPQ (PZP) complex is significantly enhanced to ∼3-fold, if they are confined in close proximity of AuNPs of appropriate size. Entrapment of PZP has been performed in Span 80 niosome. Meanwhile, in earlier reports from our group,[16,17] we have established that PPQ forms a 2:1 complex with Zn2+ (PZP) in an aqueous micellar and niosome environment.

Evidently, a key point in MEF is the molecule (emitter)–nanoparticle distance; by varying the distance, Novotny and co-workers showed a continuous transition from fluorescence enhancement to fluorescence quenching,[18] using a scanning probe microscope. On the other hand, Alivisatos et al. and Aslan et al. have evidenced “quenching” of the molecular emission, when a molecule was “directly” attached to the metal surface.[19,20] Riding on these ideas and with the developments in nanoparticle synthesis throughout the last decade, noble metal nanoparticles with different morphologies have been designed and used as flexible substrates in MEF.[6,21−29] Generally, core–shell structures are preferred to acquire a better MEF effect. They are composed of noble metal nanostructures such as a metal core or a shell, with a certain thickness of the dielectric layer including silica or a polymer and a luminescent species. To name a few, the core–shell technology was successfully implemented by Geddes and co-workers[20] and Halas group,[21,22] where they have evidenced enhanced molecular fluorescence of the dyes adsorbed on the silica layer (shell), few nanometers away from the Ag and Au surfaces (core). Meanwhile, Aroca and Guerrero introduced the shell-isolated nanoparticle in MEF, in which the isolated nanoparticle consists of a metal core and a compact dielectric shell.[26,27] However, most of these substrates require multiple synthetic steps with a highly accurate fabrication process in the nanoscale range. Moreover, most of them are not suitable for MEF applications under biological conditions. Therefore, biocompatible platforms for MEF will be of great demand. In the current study (Scheme ), we have implemented a strategy based on the core–shell technique to form an adduct between the PZP molecules in the bilayer while the AuNPs remain outside the bilayer. This is made possible through electrostatic interactions of positively and negatively charged surfaces of the niosome and AuNPs, respectively. However, AuNPs stay at an optimum distance from the fluorophore because of the presence of the few nanometer-thick organic bilayer. There are many reports available in the literature on MEF in biosensors, which are mostly done on two-dimensional planar, solid MEF platforms.[30] However, in this case, we demonstrate the MEF effect in the AuNP–niosome hybrid system, most importantly in bulk aqueous conditions.

Scheme 1

Schematic Diagram Demonstrating the Possible MEF between the Reporter Molecule PZP and the AuNPs in Span 80 Niosome

The observed MEF is a result of the coupling of the radiating plasmons (RP) of the AuNPs with the excited fluorophore complex PZP. It crucially also depends on the scattering profile for each metal, and scattering is a sensitive function of the shape, size, and environment of the nanostructure. From the three different average sizes of ∼18, 33, and 160 nm AuNPs, we have observed that the optimum size of the AuNPs to deliver the MEF effect is ∼33 nm. Furthermore, according to the associated reduction in lifetime components along with the enhancement, we propose that the enhanced fluorescence intensity is primarily derived from the intrinsic radiative decay rate enhancement of PZP and may be partly from the localized electric field effect.

Results and Discussion

FE-SEM Studies of the Niosomes and ET(30) Studies of the Fluorophores

The synthesized vesicles (niosomes) were first structurally characterized using the scanning electron microscopy (SEM) technique (Figure a). They were immediately formed, as the dry nonionic surfactant films swell in excess water under vigorous vortex mixing for 5–6 min. Further, the entrapment of a particular fluorophore in a niosome depends on the polarity of the molecule, such that it is partitioned between the hydrophobic bilayer and the interfacial water pool region. The bilayer structures of Span niosomes are clearly evident in Figure S9. Consequently, the location of the fluorophore in the niosome becomes absolutely crucial for the outcome of the MEF experiment, as an optimum distance between the metal and the fluorophore is the prerequisite to accomplish the process. Hence, we have investigated the location of the fluorophore by performing ET(30) studies.[11,31] The details can be found in the Experimental Section. Depending on the polarity of the dyes, ET(30) values are the indicators of the specific regions, where the dyes are present in the niosome. The determination of ET(30) values for PPQ and its complex PZP in amphiphilic surfactant medium assisted invaluably in understanding the location of the fluorophores in the niosome environment (Figure ). For free PPQ, the ET(30) value was found to be 50.14 kcal/mol, which indicated that PPQ probably resided in the core hydrophobic bilayer region of the niosome. On the contrary, the ET(30) value for PZP was found to be 61.32 kcal/mol, indicating that the complex formation mostly occurred in the air–water interface regions of the niosome, where the percentage of the water content was much more than that in the hydrophobic bilayer area. This was not completely unanticipated as there was probably no net electrostatic attraction for the zinc ions toward the core bilayer region of the neutral niosome, and as a result, most of the complex formation happens at the air–water interface of the vesicle, suggesting a higher ET(30) value.

Figure 1

(a) SEM image of Span 80 niosomes. The inset shows the magnified image, where the bilayers were easily seen in multilamellar vesicles. (b) Niosome after addition of 33 nm AuNPs. (c,d) Different parts of the same niosome, shown in (b). As shown in (d), the shaded regions in black are the approximate bilayer thickness, around 20–30 nm.

Figure 2

ET(30) values for (a) PPQ and (b) PZP complex, when the experiments were performed in Span 80 niosome. The chemical structures of the molecules were also shown in the inset.

Synthesis and Characterization of the AuNPs

Once the vesicles were synthesized and characterized, we prepared and characterized the nanoparticles before performing the MEF studies of PZP. Among noble metal nanostructures, Ag and Au are the popular choices because of their high plasmonic activity in the visible region.[32] We limit this report by mentioning the MEF produced by the AuNPs only. We prepared the AuNPs of three different average sizes, namely, 18, 33, and 160 nm. The size and morphology of the prepared AuNPs were characterized by SEM techniques (Figure ), while dynamic light scattering (DLS) reconfirmed the size distribution obtained from SEM. The extinction spectra (Figure a) of the AuNPs were also recorded using UV–visible spectrophotometry. Very importantly, it was observed that the plasmonic bands of the AuNPs shifted continually toward the red region and they were broadened, as the size of the AuNPs increased from 18 to 160 nm. This red shift of the plasmonic band also confirmed the progressive increase in the size of the AuNPs. The number densities of the produced AuNPs were 1.16 × 1013, 1.33 × 1012, and 1.17 × 1010 for 18, 33, and 160 nm, respectively (calculation S1 in the Supporting Information). The SEM image for the 160 nm AuNPs can be found in Figure S14.

Figure 3

(a) Extinction spectra of different AuNPs, while the inset shows the actual color of the suspensions. (b,c) SEM images for 33 and 18 nm AuNPs, respectively. The inset shows the size distribution. (d) DLS data of different AuNPs.

MEF Studies and Analysis

To demonstrate MEF, we recorded and compared the fluorescence emission spectra of PZP, encapsulated in niosome, with and without the addition of AuNPs. Upon excitation at 340 nm, PZP exhibited a fluorescence emission maximum at 450 nm (Figure ). However, the striking fact was significant ∼3-fold enhancement in emission intensity, followed by the addition of 33 nm AuNPs (Figure ), whereas we witnessed an unchanged intensity with 160 nm and quenched, lower fluorescence intensity with 18 nm AuNP additions. Similar fluorescence enhancement was observed when we repeated the same study by encapsulating PZP in Span 60 niosomes (Figure S6). Span 80 and Span 60 contain equal number of carbon atoms (17) in their hydrophobic chains except the fact that there is an unsaturation at the eighth carbon for Span 80, making the chain depart from linearity.[11] We suggest that the enhanced fluorescence with 33 nm AuNPs was definitely due to the MEF effect, enforced by the AuNPs under a suitable structural arrangement around the vesicle, as shown in Scheme . It is known that the core of the bilayer is hydrophobic in nature, but the interfacial part is hydrophilic because of water penetration. The fluorophore complex PZP probably remained very close to the membrane–water interface of the bilayer, and they formed the “core”, whereas AuNPs were adhered close to the outermost surface of the vesicle but separated by the head group “spacer” or “shell” of the bilayer.

Figure 4

Emission spectra of PZP encapsulated inside the Span 80 niosome in the presence of 160, 33, and 18 nm AuNPs, when excited at 340 nm.

The adhesion of AuNPs to the vesicle membrane was apparent as can be seen in Figure b–d, as they preferred to stay in the air–water interface area because of their larger size. Also, Figure S13 evidently confirms the adhesion of the AuNPs on the niosome surface, when SEM images of niosomes were collected and compared with and without the addition of AuNPs. Possibly, the governing factor in this case was the electrostatic attraction between the negatively charged citrate-capped AuNPs (negative zeta potential value) and the positively charged surface of the niosomes (positive zeta potential value). See Figure S5 for more details. Moreover, this observed MEF effect in solution phase is even more significant, as it has to overturn the unfavorable effect caused by random orientations of PZP as these random orientations of the molecules in solution make it difficult to account for the proper interactions with nanoparticles at any given time. To confirm, we performed a steady-state fluorescence anisotropy measurement for both PPQ and PZP complexes inside niosomes (Figure S8) and no significant anisotropy (r) values suggested a completely random molecular motion in the bilayer. Also, a similar enhancement pattern was observed for both Span 60 and Span 80 niosomes across different excitation wavelengths, when we performed an excitation wavelength-dependent study (Figure S7).

As revealed by the earlier reports, the distance between the fluorophore and metal surface played the key role here. To reinforce, Novotny and co-workers showed previously that for molecule–gold distances shorter than 5 nm, the molecular fluorescence was quenched.[18] Also, there are several reports which show that monolayers of molecules on gold and silver surfaces showed surface-enhanced Raman scattering with complete quenching of fluorescence.[33−36] It is already mentioned that core–shell structures are common and preferred to acquire a better MEF effect. Certainly, the thickness (distance) between the metal and the fluorophore determines whether a strategy will be a successful one or not. In this study, the thickness for the bilayer of the niosomes was found to be around ∼20 nm, as can be understood from Figures d and S10. Consequently, the optimum distance was <10 nm between the metal and the fluorophore, as can be postulated from Scheme . The repeated reproducible observations (Figure S3) confirmed the fact that this distance turned out to be optimum for MEF for this above-mentioned system. Hence, the observed findings are legitimate, as if the fluorophore complex inside niosome and AuNPs outside mimic the core–shell strategy, by maintaining an optimum distance to demonstrate the MEF effect. Another study,[37] under aqueous conditions from our group, demonstrated a similar 2- to 3-fold enhancement in fluorescence intensity for the negatively charged carbon dots, when coupled with ∼40 nm positively charged AuNPs with a silica coating of ∼8 nm thickness. As expected, the free PPQ molecule (without complexation with Zn2+) did not show any fluorescence enhancement (Figure S1) in the presence of the same set of AuNPs. This indicated that the distance was possibly large between them for any substantial MEF to take place, as PPQ stayed in the core region of the hydrophobic bilayer (Scheme ). Also, in the absence of AuNPs, the PZP complex did not show any fluorescent enhancement (Figure S2). At the same time, we also failed to get any fluorescence enhancement after repeating the same experiment by replacing the niosome solution with neutral tween micellar solution (Figure S4). This was probably expected, as the size of the micelles was much smaller (less than 10 nm) to replicate the same structural requirement to induce the MEF effect for this system. Moreover, as mentioned earlier, the exact same study performed with 18 and 160 nm AuNPs did not show any enhancement, highlighting the significant effect of the size for the scatterers in MEF. It is important to note that the MEF studies were repeated several times, and the statistical information about the reproducibility of the MEF studies is shown in Figure S3.

Mechanism of MEF

At present, MEF is thought to be consisting of two mechanisms: (1) local electric field effect and (2) intrinsic radiative decay rate effect.[4] However, the local field of the nanostructure also may or may not increase the radiative decay rate (kr) of the fluorophore. In the first mechanism, the metal surface or particles produce an LSPR effect which can strengthen the local electric field. It is noteworthy that the local electric field effect alone does not alter the lifetime and quantum yield (QY) of fluorophores. The other mechanism is that the nanoparticles can strengthen the intrinsic radiative decay rate of the fluorophore. Unlike the local electric field effect, when fluorescence intensity is enhanced by the intrinsic radiative decay rate effect, the QY increases, but the lifetime decreases. To better understand this mechanism, one can point out the differences in the Jablonski diagram (Figure ). Hence, fluorescence lifetime is considered as an important characterization method, where a substantial decrease in lifetime with associated enhancement will confirm the MEF mechanism. Therefore, it turns out that in the absence of metals, Q0 = (Γ/Γ + knr + kq) and τ0 = (Γ + knr + kq) –1, where kq is the rate constant of the quenching process, if any, whereas in the presence of metals, Qm = (Γ + Γm/Γ + Γm + knr + kq) and τm = (Γ + Γm + knr + kq)−1.

Figure 5

Simplified Jablonski diagram demonstrating MEF using radiative decay rate mechanism. E: rate of excitation, E + Em: increase in rate of excitation in the presence of metal, Γ: radiative decay, knr: nonradiative decay, Γm: radiative decay in the presence of metal, km: nonradiative decay in the presence of metal.

Subsequently, as can be seen in Figure and Table , we noticed a decrease in the faster component of the lifetime of the fluorophore PZP from 0.1 to 0.06 ns followed by the addition of 33 nm AuNPs. However, the average lifetimes were found to be similar with no significant change, whereas the QY increased from 2.1 × 10–3 to only 6.7 × 10–3. The average lifetime of the fluorophores was calculated using the following equation:where τ is the lifetime of the particular component, α is the contribution of that particular component, and τf is the average lifetime. The findings definitely indicated that the possible mechanism to be predominantly derived from the enhancement of the intrinsic radiative decay rate rather than the local electric field effect in our study, as suggested by the decrease in the fluorophore lifetime and increase in the QY. To substantiate our claim, we have also performed both steady-state and lifetime titration experiments for the PZP molecule with an increasing amount of 33 nm AuNPs (Figure S11). We observed that the faster component was shortened with an increasing amount of particles, while steady-state fluorescence intensity increased continuously. The average lifetime, however, remained indifferent with the addition of the particles. This observation indeed suggested about the radiation enhancement mechanism for 33 nm AuNPs. When the same titration studies were performed with the addition of 18 nm AuNPs, we noticed an opposite trend (Figure S12). With the increasing amount of particles, both the faster component and the average lifetime were lengthened to some extent, while steady-state fluorescence intensity decreased continuously. This observation ruled out the possibility that the decrease in fluorescence intensity was due to a nonchalant attachment of smaller 18 nm particles in more number on the niosome surface, in which case, the lifetime would have decreased as well.

Figure 6

Time-resolved fluorescence decay rate of PZP (10–4 M) in Span 80 niosome in the presence of AuNPs. IRF: instrument response function. Excitation: 340 nm, temperature: 298 K.

Table 1

Lifetime Components and Average Lifetime at 298 K

sample	τ1a (ns)	τ2a (ns)	τ3a (ns)	α1b	α2b	α3b	χ2	τf (ns)
PPQ	0.093	1.247	7.90	53.55	41.79	4.66	1.2	0.93
PZP	0.124	1.537	3.79	32.86	40.52	26.62	1.1	1.67
PZP + 33 nm AuNPs	0.0575	1.464	4.573	10.16	75.93	13.92	1.08	1.75

Lifetime of a particular component.

Contribution of each component.

Analysis on the Basis of RP Model

To understand the role of a particular metallic nanoparticle in the observed MEF, we used the RP model introduced by Lakowicz, in 2005.[38] According to RP model, the emission from the excited fluorophore couples with the surface plasmon of the metal, and as a result, surface plasmon-coupled emission is observed with higher intensity, at the same wavelength as the free fluorophore. Very importantly, for AuNPs, it was proposed that the induced plasmons will radiate whenever the scattering cross section of the nanoparticle would be dominant compared to the absorption cross section of the colloid. Hence, the size of the metallic nanoparticles is very important for the observed MEF, as scattering efficiencies are directly proportional to the diameter of the particle. We have calculated (Figure ) the extinction, absorption, and scattering spectra of the AuNPs, using MiePlot4613 software.[39] We observed that the fluorescence spectrum of PZP has moderate overlapping with the scattering bands of all the three AuNPs (Figure d). However, it is known[38] and can also be seen in Figure a that the absorption component is very high for AuNPs around or less than 20 nm, whereas the scattering component is very less. Possibly because of high absorption coefficient, we have observed in fact a quenching effect instead of MEF, for 18 nm AuNPs (Figures and S13). To our delight, we observed the desired MEF, only with 33 nm, which has higher scattering cross section and lower absorption factor (Figure b). As we further increase the size of the AuNPs, we expect that the scattering component should increase with low absorption component, increasing the probability of successful MEF as explained in the RP model. However, the result is counterintuitive as observed with 160 nm AuNPs, showing absolutely no evidence of MEF (Figure ). At this point, we should also keep this in mind that the line width of the extinction band is directly related to their ability to concentrate and enhance the incident electromagnetic field.[40] Therefore, if a line width for a plasmonic band is found very broad, it becomes difficult for that particular nanoparticle to enhance the fluorescence, although theoretically scattering efficiencies are still high. This is probably the reason that we do not see any change in the fluorescence outcome with 160 nm AuNPs, as the plasmonic band is very broad (Figures and 7c), as well as the extinction coefficient is also less, compared to 18 and 33 nm AuNPs.

Figure 7

(a–c) Extinction, scattering, and absorption spectra of 18, 33, and 160 nm AuNPs. (d) Overlap of scattering spectra of 18, 33, and 160 nm AuNPs with the emission spectra of PZP.

Conclusions

To the best of our knowledge, first-time bilayer vesicle platforms have been used to study MEF under aqueous conditions. With the addition of suitable AuNPs, the fluorescence intensity of the reporter fluorophore molecule PZP was found to be increased ∼3-fold. The observations were independent of the excitation wavelength and the composition of the vesicle. The optimum distance between the metal and the fluorophore was postulated to be <10 nm, associated with the enhancement. The possible mechanism for the MEF phenomena was ascribed mainly because of the enhancement of intrinsic radiative decay rate, as suggested by the decrease in the fluorophore lifetime along with the increase in the QY. Among different AuNPs, MEF was achieved only with an average diameter of 33 nm, whereas 18 and 160 nm particles did not show any enhancement. These observations followed by theoretical calculation of the scattering cross sections of the nanoparticles substantiated the role of the size of the AuNPs in MEF. In addition, the obtained results also indicated the importance of the sharper line widths and higher scattering cross sections of the AuNPs in MEF. Further works are underway using different systems for a possible increase in the degree of enhancement.

Experimental Section

Materials

Span 80, cholesterol, HAuCl4, and trisodium citrate were purchased from Sigma Aldrich and used without further purification.

AuNPs Preparation

The AuNP synthesis comprised reduction of gold solution with trisodium citrate (Turkevich method). This method was chosen for the following two reasons: first, Turkevich method is known to produce spherical AuNPs. Second, the surface of the AuNPs will be stabilized by citrate and thereby rendering it negatively charged. In this reduction process, first 20 mL of 0.25 mM HAuCl4 solution was heated to boiling and then a requisite amount of aqueous solution of 1% trisodium citrate was added to a beaker containing HAuCl4 solution, under vigorous stirring. The boiling was continued till the solution changes its color. To produce larger particles, less trisodium citrate was added as shown in Table because reducing the amount of trisodium citrate reduced the amount of the citrate ions available for stabilizing the nanoparticles, causing the small particles to aggregate into bigger ones.

Table 2

Summary of the Physical and Optical Properties of AuNPs

HAuCl4 (mL)	1% trisodium citrate (mL)	particle average size (nm)	SPR absorption (nm)
20	0.5	33 ± 6	548
20	2	18 ± 2	528
20	0.312	160 ± 10	578

Niosome Preparation

Niosomes were prepared using a standard method, known as a thin layer evaporation method.[41,42] Nonionic surfactant Span 80 and cholesterol were taken in the molar ratio (mM) of 1:1. Later, these were dissolved in a 2:1 ratio of chloroform/methanol mixture. The solvents were evaporated in a rotary evaporator under a vacuum of 20 Hg at 30 °C and 100 rpm until a thin film is formed in the round-bottom flask, which was then hydrated with Millipore water.[43] The suspension was vortexed for 5 min and then sonicated for 30 min to obtain the final niosomal suspension.

DLS Technique

The particle size and zeta potential of the samples were analyzed by the DLS technique using a Malvern particle size analyzer (zetasizer nano-ZS).

SEM Studies

SEM images were collected using a JEOL JSM-7600F field emission scanning electron microscope. Before imaging, the solution was drop-cast on a silicon wafer and kept it for a long time in the presence of vacuum to make sure that it was completely dried. All the images were taken in the secondary electron imaging mode.

Sample Preparations for MEF Studies

While adding PPQ into niosome solution, first a small aliquot of 0.1 mL from a stock solution containing 10–2 mol of PPQ in DMF was added to 9.9 mL of aqueous niosome solution and mixed well by vortexing for 2 min. The resultant PPQ concentration was 10–4 M in all cases and the DMF content was always low (∼1% v/v). Afterward, 500 μl of 10–3 M Zn2+ solution was added to form the complex PZP. Nanoparticle suspension (500 μl) was added for each of the MEF experiments in 2.5 mL of PZP–niosome solution.

Absorption and Fluorescence Studies

Steady-state fluorescence measurements of samples containing PZP in niosome, with and without the nanoparticles, were performed with a Hitachi spectrofluorometer (F7000) using a 1 cm path length quartz cuvette. For all of the fluorescence data presented in this manuscript, all sample preparations and experiments were performed at 25 °C. Fluorescence lifetimes were measured from time-resolved intensity decay rate by the method of time-correlated single-photon counting using a Horiba Deltaflex Modular fluorescence lifetime system with the following instrumental settings: 340 nm NanoLED excitation, peak preset 10 000 counts, and emission wavelength was set at 450 nm. Quartz cuvettes were used for the measurements. For collecting the extinction spectra of the nanoparticle samples, we have used a Shimadzu UV-3600 Plus UV–visible spectrophotometer.

ET(30) Studies of the Fluorophores

ET(30) studies were performed to determine the location of PPQ and PZP in a niosome environment. The study was carried out by measuring the fluorescence maxima of the fluorophores by changing the solvent polarity, as fluorescence maxima of the molecules heavily depend on the micropolarity of the surroundings. First, solutions of different percentages of dioxane–water mixture were prepared, and then the same amount of PPQ and PZP in each of the solution was dissolved. The standard ET30 parameters are known from the literature for each of the dioxane–water mixture with varying percentages (see Table S2). Next, emission spectra of each of the mixture containing the fluorophores was recorded, and a correlation plot was obtained from the emission maxima of both PPQ and PZP in different compositions of dioxane–water mixture against the solvent polarity parameter (ET30). Then, the samples (PPQ and PZP) were entrapped in Span niosomes and their fluorescence maxima were recorded and compared with the calibration curve values obtained already. Accordingly, the location of the fluorophores PPQ and PZP in the niosome environment was confirmed. The ET30 values of PPQ and PZP can be found in Table S1.