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Driving the Interactions between Organic Nanoparticles and Phospolipidic Membranes by an Easy Treatment of the Surface Stabilizer. Luigi Tarpani and L...
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Driving the Interactions between Organic Nanoparticles and Phospolipidic Membranes by an Easy Treatment of the Surface Stabilizer Luigi Tarpani and Loredana Latterini* Dipartimento di Chimica and Centro di Eccellenza sui Materiali Innovativi Nanostrutturati (CEMIN), Università di Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy S Supporting Information *

ABSTRACT: Polymer-stabilized perylene nanoparticles were prepared through a solvent exchange method. The formation of the nanostructures in aqueous solution was confirmed by the appearance of a red-shifted emission attributable to the formation of excimer-like aggregates. The behavior of organic nanostructures in the presence of lipid vesicles was investigated through steady-state and time-resolved fluorescence measurements. When no further surface treatment is applied to the nanoparticles, changes in the decay times and emission spectra demonstrate that inside the lipid bilayers the nanoparticles redissolve into the monomeric form with a rate and efficiency determined by the working temperature (above and below the transition temperature Tm of the phospholipid). On the other hand, when the stabilized shell is UV-cured to induce photo-crosslinking of the polymeric chains, the nanoparticle stability increases and their redissolution in the membrane is prevented. Confocal fluorescence images support the data obtained in bulk. The results indicate that the prepared nanostructures could be successfully used either as nanometric carriers for the delivery of poor water-soluble lipophilic compounds or as imaging tools depending on the rigidity/cross-linking degree of their polymeric stabilizer shell.

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INTRODUCTION Many of the known organic compounds which could find application as phototherapeutic agents, fluorescent markers, and drugs are limited in their use due to their poor water solubility. Aggregation of active compounds in aqueous media causes undesirable modifications of their properties and therapeutic effects. Therefore, most recent studies are focused on the preparation and characterization of new materials which can be used as delivery systems for hydrophobic compounds.1,2 Among others, nanostructured materials turn out to be excellent candidates for this purpose due to their reduced size, which allows an efficient uptake by the cells.3,4 Moreover, the surface of the nanoparticles can be processed to enhance their affinity toward specific biological targets.5,6 In the literature several data have been reported regarding the preparation of nanostructured materials which can potentially find application in drug delivery; examples are polymeric nanoparticles,7−10 solid lipid nanoparticles,11 ceramic nanomaterials,12−15 and magnetic16−18 and metal19−21 nanostructures. However, in many cases, postsynthesis methodologies are required to improve the biocompatibility and bioavailability of the nanocarriers, resulting in an increment of costs and preparation time. Organic nanoparticles (ONPs) can be described as core−shell nanoparticles in which the core is constituted by a hydrophobic fluorescent compound coated by a water-soluble stabilizer layer (usually a polymer22 or a surfactant23,24). These nanostructures usually exhibit optical © 2013 American Chemical Society

properties very different from those observed for the starting monomeric materials, and the modifications are strictly dependent on the preparation conditions.25 The solvent exchange method (also known as the reprecipitation method) is widely used to prepare ONPs because of its simplicity and versatility.26 By choosing appropriate experimental conditions (e.g., nature and concentration of the stabilizer, temperature), it is possible to prepare, through this method, water-soluble biocompatible nanoparticles of hydrophobic organic compounds. Poly(vinyl alcohol) (PVA) and poly(vinylpyrrolidone) (PVP) can act as stabilizers of the nanostructures to assist the formation of stable colloidal dispersions of ONPs.27−29 Both polymers present low toxicity and good biocompatibility30,31 and thus are excellent choices for biomedical application.32,33 The oligomer chains of PVA and PVP have the ability to polymerize upon irradiation in the UV region (UV-curing).34,35 When these polymers are used to stabilize the ONPs, their photoinduced polymerization (photo-cross-linking procedures) can lead to a stiffer capping shell around the particles, thus improving the stability and the optical properties of the colloids. The photo-cross-linking of a polymer on the surface of different kinds of nanostructures has already been demonstrated to be an efficient method to decrease the rate of Received: December 14, 2012 Revised: August 9, 2013 Published: August 16, 2013 11405

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source. The images were obtained with a 60×, 1.4 NA oil-immersion objective (512 × 512 pixels) with a 10 μm pinhole. Fluorescence decay time maps were obtained with a fluorescence lifetime imaging (FLIM) module (Nikon) using the same excitation source and a pixel dwell time of 10−20 μs (which resulted in a few seconds per frame). A homemade water circulating microscope stage was used to control the temperature during image acquisition. AFM Measurements. Images of the nanoparticles were recorded with an atomic force microscope (Solver-Pro P47H, NT-MDT) in semicontact conditions to minimize any interactions between the AFM tip and the substrate. Cantilevers with resonance frequencies in the 190−325 kHz range were used for all the experiments. The samples for AFM imaging were prepared by placing a drop of the water suspension on mica and spin-coating the substrate to spread the particles. A grain analysis was carried out to determine the height distribution of the particles from the images. The particle size distribution was obtained from the AFM images, analyzing at least 350−400 nanoparticles in each sample.

ripening and growth processes naturally occurring when weak stabilizers are employed in the preparation of the nanoparticles.36−38 Phospholipid bilayers are extensively used as models of biological membranes in the study of the uptake of different compounds of pharmacological interest, i.e., drugs, fluorescent probes, and phototherapeutic agents.39−41 In this paper we present the preparation of ONPs and their interaction with phospholipid bilayers to investigate if the organic compound becomes available at the molecular level or it is preserved in the aggregate form in biological membranes.

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EXPERIMENTAL SECTION

Materials and Reagents. Acetone, chloroform, and methanol from Sigma-Aldrich and nanopure water (≤18.0 MΩ) obtained with a Millipore Milli-Q gradient system were used as solvents. Perylene (Py), PVA (MW 89000−98000), PVP (MW 360000) and dipalmitoylphosphatidylcholine (DPPC) were purchased from Sigma-Aldrich and used without any further purification. Particle Preparation. PVA-stabilized Py nanoparticles were prepared by the reprecipitation method described in the literature.26 Briefly, 20 mg of PVA was slowly dispersed in 20 mL of Milli-Q water (0.1%, w/v, solution) under vigorous stirring and mild heating (T = 50 °C) to allow the complete dissolution of the polymer. Then 200 μL of a 2.0 × 10−3 M solution of Py in acetone was added dropwise to the PVA solution, keeping the temperature at 323 K for at least 30 min to eliminate the acetone by evaporation. PVP-stabilized Py nanoparticles were prepared through the same method described above by dissolving 80 mg of PVP in 20 mL of Milli-Q water (0.4%, w/v) to have a comparable molar concentration of stabilizer in solution. Particle Photo-Cross-Linking. A 3 mL volume of the aqueous colloidal solution of polymer-stabilized ONPs was irradiated at 254 nm for increasing times, up to 270 min, to induce the photopolymerization of the oligomer chains. A low-pressure arc mercury lamp (10 μW/ cm2), equipped with a 254 nm centered band-pass filter (Oriel model 56400), was used as the radiation source. The suspensions were magnetically stirred during irradiation. The effects of photoinduced polymerization were spectrofluorimetrically checked. Vesicle Preparation. Liposomes composed of DPPC were prepared following the lipid film rehydration procedure, which is described in the literature.42 A 14.6 mg sample of DPPC was dissolved in 15 mL of a 1:1 chloroform/methanol mixture, and the solution was kept under a nitrogen stream until the solvent was evaporated, leaving a lipid film in the bottom of the vial. The remaining traces of solvent were evaporated by placing the vial inside a vacuum desiccator overnight. Afterward, the lipid film was slowly rehydrated adding MilliQ water at a temperature T above the transition temperature Tm of the lipid (314 K for DPPC). The aqueous solution was put through different cycles of vortexing to allow the lipid film to rehydrate and form the vesicles. Through this process, multilamellar vesicles (MLVs) were prepared with a polydisperse size distribution. No further downsizing procedure was used. The final lipid concentration of the prepared MLV suspension was 2.0 mM. Optical Properties. Absorption spectra were recorded with a double-beam Perkin-Elmer Lambda 800 spectrophotometer. Corrected fluorescence emission and excitation spectra were acquired with a Fluorolog-2 fluorimeter (Spex, F112AI); the emission quantum yields were determined using anthracene in EtOH (ΦF = 0.27)43,44 and quinine sulfate in H2SO4 (0.5 M, ΦF = 0.55)43,44 as standards. A single photon counting detection system (Edinburgh Instrument 199S) was used to collect the fluorescence decay profiles using a 370 nm pulsed diode laser (HORIBA Jobin Yvon) as the excitation source and a long-pass filter at 420 nm to avoid the interference of the source emission on the signal. A water-circulating sample holder coupled to a thermal bath was used to control the temperature during the experiments. Fluorescence images were recorded through a laser scanning confocal microscope (Nikon, PCM2000) using a 400 nm pulsed diode laser (Picoquant) with a pulse width of ca. 70 ps as the excitation

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RESULTS AND DISCUSSION Optical Properties of the Nanoparticles. Upon injection of the 2 mM stock solution of perylene in aqueous polymeric solutions, the formation of the organic nanoparticles is confirmed by fluorescence measurements (Figure 1). In-

Figure 1. Fluorescence excitation (dashed lines) and emission (solid lines) spectra of Py in acetone (red) and PVA-stabilized (black) and PVP-stabilized (light blue) Py nanoparticles in water (λexc = 400 nm).

dependently from the nature of the polymer used, the spectral properties of perylene are markedly different from those observed in homogeneous organic media; in particular, the aqueous suspensions present a broad, structureless emission band centered at about 590 nm, and it is red-shifted compared to the fluorescence of the compound in homogeneous solution. The appearance of this red-shifted emission resembles Py excimer fluorescence,45 confirming that the Py molecules are aggregated inside the nanoparticle core. A fluorescence quantum yield of 6% is determined for the aqueous samples, which is remarkably lower than the value of 69% measured for perylene in acetone. With both polymers, a low-intensity emission is still observable in the 400−500 nm range, where the monomer Py emits. This residual emission is likely due to the small amount of the active compound dissolved in the water phase. Considering the spectral integrals for the two systems, the amount of Py in molecular form is about 3-fold when PVP is used as the stabilizer compared to when PVA capping is used. Taking into account that the molar concentrations of the two polymers in solution are comparable, the relative contribution of the monomeric band suggests that the particle formation process is more efficient in PVA solutions. The fluorescence 11406

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nuclei, but also the effects of the two polymers on nucleation and growth processes of the particles and/or on the arrangement of Py in the particles, as suggested by the differences in the fluorescence decay times. Considering that similar concentrations of the polymers in solution have been used and the higher molecular weight of PVP compared to PVA, the viscosity of the PVP solution is at least 1 order of magnitude higher,46−49 thus reducing the diffusion coefficient of Py in the PVP aqueous solution, according to the Stokes− Einstein equation.50 The higher viscosity of the PVP solution makes it easier to drive the particle growth through a diffusioncontrolled mechanism and reduces the occurrence of particle coalescence, thus limiting the size increase of PVP-stabilized ONPs. Furthermore, the influence of polymer properties on the kinetics of the ONP nucleation process cannot be excluded at this stage, and deeper investigations are currently in progress. Interaction of Py Nanostructures with Phospholipidic Vesicles. The emission properties of the as-prepared ONPs are monitored in the presence of increasing volumes of a 2.0 mM solution of DPPC liposomes. Fluorescence spectra of the samples are recorded at 318 and 298 K (Figure 3; Figure S5,

decay of the Py nanoparticles is measured at two different emission wavelengths (423 and 600 nm) and compared with that recorded for Py in acetone. The decay profiles are all described, with good statistical parameters, with a monoexponential function. At 600 nm, fluorescence lifetimes of 15.1 and 8.8 ns are obtained respectively for PVA- and PVPstabilized NPs, which are about 3 and 2 times longer than the typical τF value of perylene in homogeneous solution, confirming the presence of excimer-like aggregates of Py as described in previous works.27,28 If the fluorescence decay is collected at 423 nm (where the fluorescence emission bands of the monomer are present), the obtained decay times (5.1 and 4.5 ns in PVA and PVP suspensions, respectively) correspond, within experimental error, to the Py decay time in polar environments, and no longer components can be detected. Furthermore, no modifications of the τF of the Py nanoparticles are observed in deaerated solution, thus indicating that the polymer shell limits oxygen diffusion into the perylene core. Morphological Characterization. AFM images are recorded from the same samples investigated by stationary and time-resolved fluorescence to confirm the formation of nanoparticles and to characterize their morphology. AFM height images show for both samples the presence of spherical particles having a smooth topography (Figure 2).

Figure 2. AFM images and histograms of size distributions (a, b) of PVA-stabilized Py nanoparticles (scale 4 μm) and (c, d) of PVPstabilized Py nanoparticles (scale 1 μm). Figure 3. Fluorescence emission spectra of PVA-stabilized Py nanoparticles (λexc = 380 nm) with increasing amounts of DPPC vesicles (0 to 4.6 × 10−4 M) at 318 K (a) and at 298 K (b).

When the particles are stabilized by PVA, the grain dimension analysis (Figure 2b) indicates that the particle diameter is on the order of tens of nanometers and presents a quite broad distribution which could be reproduced by two Gaussian functions centered at 75 and 92 nm with fwhm values of 38 and 19 nm, respectively. It has to be noted that the particle size is also affected by the presence of the PVA molecules acting as a stabilizer on the surface of the nanoparticles and the real diameter of the emitting nucleus is smaller. When PVP is used as a capping agent, the dimensions of the resultant ONPs are considerably smaller, being centered at 7.3 nm with a narrower size distribution (about 4.3 nm). Taking into consideration that the concentrations of perylene and the stabilizers are the same in the two systems, the different sizes of the resulting ONPs can be due to not only the different dimensions of the stabilizer shells on the surface of the Py

Supporting Information) to investigate the interactions between nanoparticles and the vesicles when the latter are in the gel or the liquid-crystalline phase; the vesicle phase is thermally controlled by setting the temperature respectively above and below the DPPC transition temperature (Tm = 314 K).51,52 Both the ONP and DPPC suspensions are thermally equilibrated at the temperature used in the experiments (T) by use of thermal baths. After each addition, the solution equilibrates for about 30 min before the fluorescence spectrum is recorded. When T > Tm (Figure 3a), the intensity of the band at 590 nm (nanoparticles) decreases upon addition of increasing concentrations of DPPC, while an increase of intensity in the 400−500 nm range (monomer) is detected, 11407

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(caused by their good affinity for the lipid bilayer), where the NPs experience a more constrained environment. To clarify the nature of the interactions between ONPs and the lipid vesicles, fluorescence intensity imaging is used to monitor the fluorescence distribution in the ONP−vesicle systems. The fluorescence images recorded at 298 K (Figure 4a) show a heterogeneous fluorescence distribution in the

suggesting an increase of the Py concentration in monomeric form (see below). These spectral changes occur progressively with an increase of the vesicle concentration. When the same experiment is carried out at T < Tm (Figure 3b), the emission intensity increases in the whole spectral range (400−700 nm) with an increase of the DPPC concentration. The same intensity changes are observed in the excitation spectra (Figures S1−S4, Supporting Information). The steady-state fluorescence data in the presence of increasing DPPC concentrations suggest that polymer-stabilized Py nanoparticles interact with lipophilic lipid bilayers, and the nature of the interactions depends on the liposome phase, which is controlled by the temperature. A similar behavior is also observed when the same amounts of DPPC vesicles are added below and above the Tm of the lipid to PVP-stabilized Py nanoparticles (Figure S5). To obtain deeper insight into the nature of the interactions detected between ONPs and the lipid vesicles, time-resolved fluorescence measurements are carried out in the presence of different DPPC concentrations and with control of the temperature above and below the phase transition temperature. The decay profiles are collected at 540 nm to detect the emission signals from both the NPs and the monomer. The fitting parameters of the decay curves are collected in Table 1.

Figure 4. Confocal fluorescence microscopy images (λexc = 400 nm) of DPPC vesicles/PVA-stabilized Py NPs (a) at 298 K (scale 5 μm) and (b) at 318 K (scale 10 μm).

membrane. Furthermore, accumulation of spherical structure at the water−bilayer interface can be observed. Thus, the intensity changes observed in the spectra and in the decay times recorded at 298 K are likely due to a higher scattering contribution and to environmental constraints imposed by ONP accumulation at the vesicle rim. On the other hand, the images recorded at 318 K (Figure 4b) show a more homogeneous fluorescence distribution in the lipid bilayer, confirming that, at T above the Tm of the lipid, the permeability of the membrane is higher. FLIM is performed on the DPPC liposomes showing emission from the Py nanoparticles to confirm the effective dissolution of the nanostructures inside the lipid bilayer. Spatial distribution images of the fluorescence lifetimes (Figure 5) reveal that, inside the membrane, perylene

Table 1. Fluorescence Decay Parameters of PVA-Stabilized Py NPs (λexc = 370 nm; λem = 540 nm) in the Presence of Increasing Concentrations of DPPC Vesicles T (K)

[DPPC] (mM)

τ1 (ns)

A1 (%)

τ2 (ns)

A2 (%)

χ2

318

0 0.11 0.25 0.46 0 0.18 0.30 0.46

5.5 5.5 5.5 5.5 5.1 5.3 5.3 5.5

43.6 46.7 53.7 59.8 41.2 41.4 43.0 46.4

15.3 15.3 15.3 15.3 15.1 16.0 16.6 17.2

56.4 53.3 46.3 40.2 58.8 58.6 57.0 53.6

0.99 1.04 1.10 1.06 1.01 1.06 1.05 1.06

298

In all cases, the fluorescence decay can be satisfactorily fitted by biexponential functions, confirming the presence of a longer and a shorter component assigned to the nanoparticles and the monomer, respectively. It has to be noted that the relative weights of the two contributions are not a measure of the species abundance, but they are affected by their fluorescence efficiencies. For the decay curves recorded at 318 K, the fluorescence decay time does not change upon increasing the DPPC concentration, but their relative weights are affected. In particular, upon increasing the lipid concentration, the contribution of the long component decreases, while the weight of the monomer increases. This behavior supports the hypothesis that the nanoparticles redissolve in the lipid bilayer to give molecular perylene. On the other hand, when the fluorescence decays are recorded at 298 K, the small changes of the pre-exponential factor are accompanied by changes in the decay time. In particular, a lengthening of the slow component together with a small reduction of its relative weight is observed upon addition of DPPC. Therefore, ONP dissolution and Py redistribution in the bilayers in molecular form cannot account for the fluorescence behavior observed at 298 K; as a matter of fact, at this temperature, the membrane has a higher rigidity/ viscosity,51,52 which can limit the ONP permeability. The increase of the longer lifetime can be explained by an accumulation of the nanoparticles on the vesicle surfaces

Figure 5. FLIM image (λexc = 400 nm) of DPPC vesicles/PVAstabilized Py NPs at 318 K (scale 0.5 μm).

has a decay time of 4.5−5.2 ns, which is in agreement, within the experimental error, with the bulk measurements; thus confirming that the fluorophore is in monomeric form. It can be noted that the Py decay time gradually decreases, moving from the center of the bilayer to the vesicle rim, likely for the changes in medium polarity, as already observed in the literature,53 or for the occurrence of new competitive processes, although the effects of specific interactions with ionic groups of 11408

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DPPC50 and/or of different oxygen contents cannot be excluded. Stabilization of Py Nanoparticles by Photo-CrossLinking. To stiffen the polymer shell and improve the nanoparticle stability, in cases where dissolution has to be avoided, the ONP suspensions have been photochemically treated to induce polymer photo-cross-linking. Both PVA- and PVP-stabilized ONP samples are irradiated at 254 nm for 270 min to induce the photopolymerization of the oligomer chains on the surface of the nanoparticles. Higher photo-cross-linking efficiency is observed for PVP suspensions, which show a viscosity increase during irradiation and changes in the fluorescence properties of the colloidal system. As shown in Figure 6, upon UV irradiation, the fluorescence emission attributed to the nanoparticles becomes narrower and

AFM images collected for the sample after photo-crosslinking of the polymer shell (Figure 7) show the presence of

Figure 7. AFM images (left) and histogram of the size distributions (right) of PVP-stabilized Py nanoparticles after irradiation at 254 nm (scale 3 μm).

spherical nanoparticles with a size distribution centered at 2.4 nm, indicating a significant reduction of the mean size of the colloids; from these measurements a distinction between the contribution of the stabilizer shell and the perylene cores to this effect cannot be made. It has to be noted that if core processes are operative, they do not lead to an increase in particle size,54,55 suggesting that in the present systems the particle surface energy is strongly reduced by the polymer capping. The observed reduction in size for the photo-cross-linked ONPs can be attributable to shrinkage of the PVP shell in a tighter conformation, which induces modifications in the arrangement of perylene units either in monomeric or in aggregate form, in agreement with steady-state and time-resolved fluorescence measurements. Interaction of Photo-Cross-Linked Py Nanostructures with Phospholipid Vesicles. Steady-state fluorescence spectra of photo-cross-linked ONPs in the presence of increasing DPPC vesicles (Figure 8) are recorded in the same experimental conditions used for non-irradiated samples (PVA- and PVP-stabilized Py nanoparticles) above and below the transition temperature of the phospholipid. Differently from what was observed for the untreated nanoparticles, the emission spectra at the two temperatures follow the same trend upon addition of the vesicles, when the nanoparticles are photo-cross-linked; the emission intensity clearly increases in the entire spectral range and slightly shifts toward shorter wavelengths. The absence of a vibrationally structured emission spectrum typical of the Py monomer after addition of the liposomes clearly excludes a redissolution of the cross-linked nanoparticles inside the hydrophobic lipid bilayer. The spectral changes, although similar, appear to be affected by temperature since the effect is smaller at 298 K when the vesicles are in the gel phase and present a lower permeability, while the intensity increase is more evident when the phospholipids are in the liquid-crystalline phase. In Table 2 the fitting parameters of the fluorescence decays recorded on photo-cross-linked PVP NPs in the presence of increasing DPPC concentrations below and above the trasition temperature are reported. The decay profiles were recorded at 540 nm to make the comparison easier. Below the transition temperature of the lipid, the addition of the vesicles does not affect the two decay times, while at 318 K both the components show a shortening (about 35% and below 10% decrease for the decay time of the short and long components, respectively). In both cases, the change in the pre-exponential factors is quite small.

Figure 6. Excitation (dashed lines) and emission (solid lines) fluorescence spectra of PVP-stabilized ONPs before (red) and after (black) irradiation at 254 nm.

is shifted in the blue region with a maximum at about 460 nm; modifications in the excitation spectra are observable with the appearance of an additional shoulder at about 330 nm. It is important to underline that the spectral features are not affected by dilution, indicating that the particle redissolution is inhibited in the photo-cross-linked suspension. A fluorescence quantum yield of 9% is estimated for the nanoparticles after photo-cross-linking; this value has been determined assuming that during photo-cross-linking process, the fraction of light absorbed by the particles did not change and that no changes in the refractive index of the medium occurred. Fluorescence decay profiles are also recorded at 540 nm for the PVPstabilized ONPs after UV irradiation. The decays can be fitted by a biexponential function, resulting in two components of 8.8 and 2.0 ns. In particular, the cross-linking process did not affect the long decay component, while a decrease of the short component decay time from 4.5 to 2.0 ns is observed, which could be due to a medium effect.53 These decay time values can be regarded as average descriptions of the emitters’ populations, which could be become quite complex due to stiffening of the polymer. The ipsochromic shift of the emission spectrum and the changes in the fluorescence decays observed after UV irradiation suggest that the curing process, which stiffens the polymer matrix, alters the arrangement of the fluorescent molecules, thus modifying their relaxation path. UV irradiation in the same experimental conditions is also applied to PVAstabilized ONPs, but in this case no modifications in the viscosity and/or fluorescence properties of the suspension are observed, demonstrating a lower efficiency of photo-crosslinking for this polymer, likely for the lower absorption coefficient of PVA at the irradiation wavelength. 11409

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Figure 9. Confocal fluorescence microscopy image of DPPC vesicles/ cross-linked Py NPs collected after 30 min of contact (scale 15 μm).

luminescence intensity and localization are mantained after 30 min, thus demonstrating the higher photostability of the photocross-linked nanoparticles (Figure S6, Supporting Information). Therefore, these data confirm that the UV treatment of the polymeric layer on the surface of the ONPs prevents the redissolution of the perylene cores as monomers inside the hydrophobic lipid bilayer, different from what was observed for the untreated suspensions, thus proving two distinct types of interaction depending on the rigidity of the stabilizer shell.

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CONCLUSIONS The preparation of Py nanoparticles was successfully achieved through the reprecipitation method using two different polymers as stabilizers. The optical properties of the nanostructures were drastically different from those related to the compound in the molecular form. A broad and nonstructured emission centered at 590 nm was attributed to the formation of ordered Py aggregates in the core of the nanoparticles. When PVP-stabilized Py nanoparticles were irradiated at 254 nm, photo-cross-linking of the stabilizer shell occurred, bringing about a modification of the optical properties of the colloid and a considerable size reduction of the nanoparticles. In this case, the connection between the oligomers caused a loss of π−π stacking order of the Py cores, and a blue-shifted emission from the smaller organic aggregates became predominant. The ability of PVA-stabilized ONPs to redissolve and return to the molecular state once a suitable lipophilic environment was reached was proven using lipid vesicles as models of biological targets. When the stabilizer shell was not subjected to UV curing, a decrease in the emission of the nanoparticles together with an increase of the Py fluorescence in the monomeric form was detected when increasing concentrations of DPPC vesicles at a temperature above the Tm of the lipid were added to the NP solution, thus demonstrating the redissolution of Py inside the lipid bilayer. The same experiment ran at T < Tm showed that the mechanism of interaction was different when the liposomes were in the gel phase, leading to the accumulation of the nanoparticles on the surface of the lipid bilayer. The data were confirmed by the images of the spatial distribution of fluorescence collected with the confocal microscope. In addition, FLIM images showed a continuous lifetime distribution of the redissolved Py, going from 5 ns inside the hydrophobic lipid bilayer to about 2 ns at the more hydrophilic interface between the vesicles and the water. On the other hand, when the PVP oligomer chains of the stabilizing shell were photo-cross-linked together and the resulting NPs put in contact with phospholipidic vesicles, no redissolution of pyrene

Figure 8. Fluorescence emission spectra of photo-cross-linked Py nanoparticles (λexc = 380 nm) with increasing amounts of DPPC vesicles (0 to 4.6 × 10−4 M) at 318 K (a) and at 298 K (b).

Table 2. Fluorescence Decay Parameters of Photo-CrossLinked PVP-Stabilized Py NPs (λexc = 370 nm; λem = 540 nm) in the Presence of Increasing Concentrations of DPPC Vesicles T (K)

[DPPC] (mM)

τ1 (ns)

A1 (%)

τ2 (ns)

A2 (%)

χ2

318

0 0.11 0.25 0.46 0 0.11 0.25 0.46

2.2 1.8 1.5 1.5 2.2 2.0 1.8 2.0

42.3 41.0 42.0 43.6 40.2 41.2 44.2 44.9

8.8 8.5 8.2 8.2 8.8 8.7 8.7 8.7

57.7 59.0 58.0 56.4 59.8 58.8 55.8 55.1

0.87 1.14 1.29 1.20 1.08 0.89 0.86 1.00

298

Fluorescence imaging is carried out to better clarify the interactions between photo-cross-linked ONPs and the phospholipid vesicles. A sample is prepared by adding 1 mL of a solution of DPPC liposomes (2.0 mM) to 4 mL of the aqueous colloidal solution of photo-cross-linked Py nanoparticles. A drop of this mixture is placed on the top of a glass slide and promptly visualized with a confocal laser scanning microscope setup. Fluorescence distribution images are collected after 30 min of contact between the photo-crosslinked ONPs and lipid vesicles (Figure 9). The fluorescence is not homogeneously distributed inside the vesicles, different from what was observed for the untreated nanoparticles (Figure 4). Indeed, Figure 9 shows that an intense fluorescence emission is localized in defined bright spots (indicated by the arrows), which can likely be attributed to Py nanoparticles or to clusters of them, while the lipid bilayer shows a lower background signal (blue regions). Confocal images collected in the same conditions at different contact times show the 11410

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molecules inside the lipid bilayer was observed and the nanoparticles could be visualized without optical and morphological modifications taking place over time. In summary, this work demonstrated that the postpreparation treatment of the polymer shell stabilizing the organic nanoparticles is a critical process to tune the interaction between the synthesized nanostructures and model membranes. When no UV irradiation is applied after the synthesis, the ONP formation and redissolution can be successfully used to deliver hydrophobic compounds, thus overcoming the limitations due to the poor solubility in an aqueous medium. On the other hand, when a photo-cross-linked shell is formed around the nanoparticles, the redissolution is prevented and ONPs can be successfully visualized over time due to their increased optical and morphological stability, thus making them efficient nanomaterials for imaging applications.

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ASSOCIATED CONTENT

S Supporting Information *

Excitation and emission spectra of nanoparticles in the presence of vesicles and fluorescence images of nanoparticles in the vesicles (Figures S1−S6). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39-0755855636. Fax: +39-0755855598. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Università di Perugia and the Ministero per l’Università e la Ricerca Scientifica e Tecnologica (Rome).

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