Amphiphilic Cyclodextrin Carriers Embedding Porphyrins: Charge and

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J. Phys. Chem. B 2005, 109, 7258-7265

Amphiphilic Cyclodextrin Carriers Embedding Porphyrins: Charge and Size Modulation of Colloidal Stability in Heterotopic Aggregates Antonino Mazzaglia,*,†,‡ Nicola Angelini,§ Domenico Lombardo,§ Norberto Micali,*,§ Salvatore Patane´ ,| Valentina Villari,§ and Luigi Monsu` Scolaro‡ Istituto per lo Studio dei Materiali Nanostrutturati, ISMN-CNR, Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica, and Dipartimento di Fisica della Materia e Tecnologie Fisiche AVanzate, UniVersita` di Messina, Salita Sperone 31, 98166 Messina, Italy, and Istituto per i Processi Chimico Fisici, IPCF-CNR, Sezione di Messina, Via La Farina, 237, 98123 Messina, Italy ReceiVed: January 11, 2005; In Final Form: February 10, 2005

The interaction between the anionic 5,10,15,20-tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin (TPPS) and cationic vesicles formed by heptakis(2-ω-amino-O-oligo(ethylene oxide)-6-hexylthio)-β-cyclodextrin (SC6CDNH2) has been investigated in detail through a combination of elastic light scattering (ELS), quasi-elastic light scattering (QELS), zeta potential measurements, and time-resolved fluorescence anisotropy. ELS experiments provided the first structural characterization of these cationic vesicles both in the absence and in the presence of TPPS porphyrin, modeling the system as a spherical particle described by a single thin shell form factor. The structure of mixed hetero-aggregates is modulated by charge and size of the two components as function of different porphyrin/cyclodextrin (CD) molar ratios. At the limiting molar ratio studied, the absolute value of zeta potential (|ζ| ) 12.5 mV) seems to be a reference value for the formation of stable colloidal CD vesicular aggregates at thermodynamic equilibrium. New insights on the structure of these heterotopic colloids have been obtained by analysis of rotational correlation times at different molar ratios exploiting time-resolved fluorescence anisotropy experiments. At high porphyrin loads, the anisotropy decays behave as monoexponentials and the rotational correlation times (1-2 ns) together with the r0 values close to zero suggest the presence of small amounts of TPPS embedded in a hydrophobic environment either in monomeric or in aggregated form. At the lower porphyrin/CD molar ratios, the anisotropy decays exhibit a double-exponential behavior showing a predominant component with a slow rotational correlation time (20-25 ns) and limiting anisotropy values of ∼0.15. This component has been assigned to molecules that are more stabilized onto the CD vesicles, that is, porphyrins embedded into the oligo-ethylene “wall” of the CD vesicles. Scanning nearfield optical microscopy of the samples evaporated on glass surfaces gave further insights on the morphology and optical properties of these systems, confirming the embedding of TPPS on the vesicles and evidencing the role of the solvent.

1. Introduction The binding properties of complex systems formed by porphyrinoid sensitizers in colloidal microenvironments, such as lipid bilayer vesicles (liposomes), micelles, fluid clefts in monolayers, and emulsions, are actively investigated.1,2 Over the last twenty years, liposomes interacting with hydrophobic porphyrins and metalloporphyrins have been used to mimic membrane proteins3 and to study partitioning of sensitizers in different compartments1,4 to design more efficient systems for photodynamic therapy of tumors (PDT).1,5 Many investigations on porphyrins incorporated in neutral and charged micelles have * Corresponding authors. Dr. Antonino Mazzaglia, Istituto per lo Studio dei Materiali Nanostrutturati, ISMN-CNR, Unita` di Messina, c/o Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica, Universita` di Messina, Salita Sperone 31 Vill. S. Agata, 98166 Messina, Italy. Tel.: +39 090 6765427. Fax: +39 090 393756. E-mail: mazzaglia@ chem.unime.it. Dr. Norberto Micali, Istituto per i Processi Chimico Fisici, IPCF-CNR, Sezione di Messina, Via La Farina, 237, 98123 Messina, Italy. Tel.: +39 090 2939693. E-mail: [email protected]. † CNR-Istituto per lo Studio dei Materiali Nanostrutturati, Universita ` di Messina. ‡ Dipartimento di Chimica Inorganica, Universita ` di Messina. § CNR-Istituto per i Processi Chimico Fisici, Sezione di Messina. | Dipartimento di Fisica della Materia e Tecnologie Fisiche Avanzate, Universita` di Messina.

been also reported,6-10 as far as their equilibrium11 or their kinetic behavior are concerned.12 The mechanism of interaction between photodynamic drugs, such as hematoporphyrins, protoporphyrins,13 glycoporphyrins,14 and cytotoxyin,15 with liposomes has been widely investigated, and it has been established that photophysical and photochemical parameters are quite sensitive to the microenvironment. A combination of hydrophobic and electrostatic interactions influences the partition of dye in a lipid bilayer: hydrophobic guests are incorporated into the lipid region while hydrophilic sensitizers interact mainly with the aqueous interface at the hydrated internal core of the liposome. As a result of the localization of sensitizers inside the vesicles, monomers and self-aggregates of the chromophore are formed. The presence of these supramolecular oligomers is due to a high local concentration of sensitizer.6 After being incorporated in the bilayer, the collision process of excited states and the rotational and diffusional freedom of the sensitizer molecule are consistently slowed as a consequence of the increase of the microviscosity.1 Conventional and nonlinear optical spectroscopic methods16 can be conveniently exploited to investigate both structural and dynamical features of sensitizer/ bilayer systems. In this respect, chiral recognition of micelles versus a chiral functionalized porphyrin was described.17

10.1021/jp0501998 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/22/2005

Colloidal Stability in Heterotopic Aggregates Biopolymers,18-22 calixarenes,23-26 cyclodextrins (CDs),27-33 and calixarene-appended CDs34 constitute further microdomains where chromophores can be localized. CDs offer a hydrophobic microenvironment which excludes water molecules and includes various guests. Porphyrin/CD systems have been extensively studied as promising models for hemoproteins,28,30,35,36 and some of these investigations have been focused on the hydrophobic environments around metalloporphyrins,31,32 similarly to the microdomains of myoglobin and hemoglobin in which an ironcontaining coordination site is involved. Hydrophilic porphyrinβ-CD conjugates37 and porphyrins bearing four covalently bound permethylated-β-CD38 were synthesized and characterized as soluble hosts having the advantage of multiple interactions with different substrates. The interaction between CDs and green plant pigments39 and fluorescent ratiometric methods exploiting the coordinating properties of the metal in zinc(II)-porphyrin assembly on β-CD40 have been also described. Recently specially designed amphiphilic β-CD capable of forming small micelles and aggregates41,42 or bilayer vesicles41,43-45 have been reported as potentially less immunogenic (due to their oligo-ethylene oxide exterior)46 and more versatile guest encapsulators with respect to a single CD molecule.45 Such vesicles retain guest molecules even on dilution43-45 and can be targeted by using receptor-specific groups such as galactosyl moieties.47 Furthermore, cationic amphiphilic CDs can influence significantly the photochemistry of a photosensitizing drug by increasing its photostability.48 In analogy with polymeric micelles and dendritic core-shell architectures,49 in which terminal functional groups on the oligo-ethylene shell control the biocompatibility and incorporate possible targeting functionalities while the inner blocks entrap sensitizers, we have prepared and characterized novel heterotopic nanoaggregates of porphyrins entangled in CD vesicles,50 investigating also their intracellular delivery.51 Differently from polymeric micelles and dendrimers, these amphiphilic CDs display a relatively definite and low number of hydroxyl groups (seven terminal OH groups on the oligo-ethylene glycol chains)41,44,47 which are chemically versatile for an easy design of new systems. The aggregates of these molecules display multiple complexation in binding polymer guest molecules45 and proteins.47 The final challenge is to achieve targeted PDT, that is, to obtain the selective delivery of the photodynamic active drug directly to the action sites.15,52-55 In particular, in our previous paper51 we reported a detailed investigation on a colloidal system formed by an anionic water-soluble porphyrin (TPPS) and cationic amphiphilic CD (SC6CDNH2; Scheme 1), using a combination of UV/vis absorption, steady-state and time-resolved fluorescence, and quasielastic light scattering (QELS) at different relative molar concentrations of porphyrin and CD. Here we report an investigation on the colloidal stability of these complex systems in a wide range of temperatures, exploiting different complementary techniques, for example, laser Doppler electrophoresis. Time-resolved fluorescence anisotropy experiments afford useful insight into the dynamics of porphyrins embedded within the nanocarriers, at different sensitizer/CD molar ratios. We report also a scanning near-field optical microscopy (SNOM) characterization of these heterotopic colloids on glass surfaces giving further insight on their morphology and optical properties. 2. Experimental Methods and Data Analysis Materials. β-Cyclodextrin (Wacker) was crystallized from distilled water and dried under a vacuum (0.1 mmHg, 80 °C) for 4 h. Heptakis(2-ω-amino-O-oligo (ethylene oxide)-6-hexyl-

J. Phys. Chem. B, Vol. 109, No. 15, 2005 7259 SCHEME 1

thio)-β-CD was synthesized according to general procedures.41,44 5,10,15,20-Tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin tetrasodium salt was purchased from Aldrich Chemical Co. The solvents used were purified and dried by standard techniques. All the other reagents were of the highest commercial grade available and were used as received or were purified by distillation or recrystallization when necessary. Sample Preparation. Preparation of CD vesicles and encapsulation of porphyrins were carried out by using conventional experimental procedures used for liposomes.56 CD vesicles were prepared from stock solutions of SC6CDNH2 in CHCl3 (180 µM), which were first slowly evaporated overnight to form thin films. These CD films were hydrated and sonicated for 20 min at 50 °C and equilibrated overnight. All the mixed solutions of porphyrin and CD were prepared in phosphate buffer (10 mM) at pH 7 diluted with pure microfiltered water and then consecutively equilibrated overnight at 4 °C, sonicated for 15 min, and equilibrated again at room temperature for 20 min, according to the published procedure.51 The investigated aggregates were studied at 1:1, 1:2, 1:5, 1:10, and 1:50 porphyrin/ CD ratios ([TPPS] ) 3 µM). Elastic and Quasielastic Light Scattering. Elastic light scattering (ELS) and QELS measurements were carried out using a computerized homemade goniometer and the 532 nm line of a duplicated Nd:YAG laser as exciting source. A Malvern 4700 correlator was used to obtain the intensity autocorrelation function in the time domain ranging from 10 µs up to 1 s. The investigated temperature range was 290 e T e 320 K, with an accuracy of (0.01 K. The measured scattering angle range was 20 e θ e 150°, corresponding to exchanged wavevector values of 5.5 e k e 30.5 µm-1 (being k ) [(4πn)/λ] sin(θ/2), where n is the refractive index of the sample and λ the wavelength of the incident light in the vacuum). Measurements at different angles showed a linear dependence of the relaxation rate on the square of the scattering wavevector thus indicating the diffusive nature of the detected relaxations.57-59 In a QELS experiment the measured intensity-intensity time correlation

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Mazzaglia et al.

function g(2)(t) is related to the electric field correlation function, g(1)(t), by the Siegert relation:57-59

g(2)(t) ) B(1 + f|g(1)(t)|2)

(1)

where B is the baseline and f is a spatial coherence factor. In the case of dilute solutions of monodispersed particles g(1)(t) ) exp(-k2Dt), where D is the translational diffusion coefficient. From the diffusion coefficient D, the mean hydrodynamic radius RH of diffusing particles can be calculated using the equation

D)

kBT 6πηRH

(2)

where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. In general, g(1)(t) may be expressed as the Laplace transform of a continuous distribution G(Γ) of decay times (relaxation rates Γ).57-59 The effective diffusion coefficient D(k) ) [Γ(k)/k2] can be obtained from the standard second-order cumulant analysis of the autocorrelation functions.60,61 Electrophoretic Mobility Measurements. The electrophoretic mobility µE was measured using principles of phase analysis light scattering (PALS).62,63 For that purpose, a Zeta PALS Brookhaven instrument equipped with a diode laser at a power P ) 30 mW and with λ ) 661 nm was used in the temperature range of 290 e T e 320 K. Under the influence of an electric field E, the moving particles (in the measurement zone through the fringes) cause frequency shifts of scattered light proportional to their velocity V. The measured electrophoretic mobility µE for the investigated systems allows for the calculation of the particles zeta potential, ζ, by means of the Henry equation:62,63

µE )

2ζ 3η

f(κa)

(3)

where  is the dielectric constant of the solvent while f(κa) is a correction function, which depends on the Debye-Hu¨ckel constant κ and the particle dimension a. The Debye-Hu¨ckel length κ-1, which characterizes the length scale of the double layer around a charged particle in an electrolyte solution, is given by

κ-1 )

x

kBT e2I

furnishes a relationship between zeta potential ζ and particle surface charge Qeff:62,63

(4)

with kBT being the thermal energy and I ) 1/2(∑zi2ni) the ionic strength, which depends on the valence zi and the concentration ni of the ions of type i present in solution. For low-potential values of ζ (i.e., eζ/kBT , 1) and in the limit of extended double layer (i.e., for κa . 1), the so-called Smoluchowsky limit is valid, for which we have f(κa) ) 1.5.62,63 The previous conditions are fulfilled in our investigated systems, giving values of κa ranging between 40 and 180. In such a way measurement of the migration rate (i.e., velocity V) of dispersed particles under the influence of an electric field allows for an estimation of the electrophoretic mobility µE of the particles (i.e., µE ) V/E). Once µE is determined, the particle zeta potential ζ can be calculated using eq 3.62,63 Further insights about the interaction process can be obtained by the analysis of the particle surface charge. For low values of the potential, the solution of the linearized PoissonBoltzmann equation (which relates the mean particle electrostatic potentials, ψ(z), to the ion density distribution, F(z), in solution)

ζ)

Qeff

(5)

4πa(1 + κa)

Time-Resolved Fluorescence Anisotropy. Time-resolved fluorescence anisotropy was measured by a time-correlatedsingle-photon-counting64 homemade apparatus. The excitation source was a synchronously mode-locked rhodamine 6G dye laser (Spectra Physics 375B) which provided excitation pulses of about 2 ps full width at half-maximum at a repetition rate of 82 MHz. An excitation wavelength of 570 nm was used. The fluorescence pulses were detected with a microchannel-plate photomultiplier (Hamamatsu R1645U-01, about 200-ps rising time), and the decay profiles were collected with a computercontrolled multichannel analyzer card (EG&G Ortec Trump8k/2k). The fluorescence anisotropy data were then analyzed using the nonlinear least-squares iterative reconvolution procedures based on the Marquardt algorithmn.65 In this way, the instrumental resolution (corresponding to the minimum measurable time value) was about 50 ps. Fluorescence anisotropy, r(t), is defined using the following expression:66

r(t) )

IVV(t) - IVH(t) IVV(t) + 2IVH(t)

)

D(t) S(t)

(6)

where the sum data S(t) must be equal to the total intensity I(t). In the first step S(t) was analyzed using a reconvolution procedure based on the following multiexponential model:

S(t) ) I(t) ) I0

∑i Ri exp(-t/τi)

(7)

to obtain the parameters describing the intensity decay (Ri and τi). In the second step, holding constant the parameters obtained from the first step, the difference D(t) was analyzed considering a multiexponential decay of the anisotropy:66

D(t) ) S(t) r(t) ) S(t)r0 gj exp(-t/θj) ) S(t)

∑j

∑j r0j exp(-t/θj)

(8)

where the parameters r0 ) ∑jr0j, θj, and gj represent respectively the limiting anisotropy in the absence of rotational diffusion, the individual rotational correlation times, and the associated fractional amplitudes in the anisotropy decay (∑jgj ) 1). In the simple case of spherical molecules, each θj is related to the volume (Vj) of the rotating unit by the following equation:66

θj )

ηVj kBT

(9)

where η is the microviscosity, T is the temperature in K, and kB is the Boltzmann constant. Finally, the decays of the parallel (IVV) and perpendicular (IVH) components of the emission were reconstructed using all the parameters obtained by the first two steps, on the basis of the following expressions

1 IVV ) I(t)[1 + 2r(t)] 3

(10)

1 IVH ) I(t)[1 - r(t)] 3

(11)

and, finally, superimposed with the experimental data.

Colloidal Stability in Heterotopic Aggregates

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Figure 1. Form factor of SC6CDNH2 vesicles (15 µM) in phosphate buffer (10 mM, pH 7) in the absence (0) and in the presence of TPPS at a 1:5 molar ratio (4). The continuous lines are the best fits according to eq 12.

Microscopy. Porphyrins/CD aggregates for the microscopy analyses were deposited on glass cover slides from samples at different molar ratios and dried under air. Information on morphology and optical properties of the sample were obtained by means of SNOM. This technique is a true scanning probe method which provides simultaneously optical and topographic information on the features of the sample surface, overcoming the diffraction limit with a lateral optical resolution well below 100 nm.67,68 SNOM measurements were performed by means of a “homemade” instrument working in illumination-transmission mode configuration.69-71 The microscope was operating in constant gapwidth mode, stabilizing the distance between the sample and the tip through a nonoptical tuning-fork-based mechanism.72 Fluorescence in transmission mode, at the nanometer scale, was obtained by exciting the sample with the 457-nm Ar line coupled to a commercially available tapered optical fiber (Nanonix) with a nominal aperture of 50 nm and collecting the optical data through a 40× epi-plan objective (Nikon). The emitted light, collected by the objective, was then filtered through a high-quality long-pass optical filter (cutoff 550 nm), almost eliminating the laser photons. Finally, the signal was detected by a miniaturized phototube (Hamamatsu RU5000) directly connected below the objective and the filter. To improve the signal-to-noise ratio we used a dual phase lock-in amplifier (SR 830 Stanford), detecting the signal from the phototube and modulating the exciting light at 1 kHz by a mechanical chopper placed between the fiber coupler and the laser source. Owing to the features of the technique, namely, super-resolution and room condition working capability, the sample was observed without any pretreatment, preserving, in such a way, its topographic and optical properties. Measurements were performed with a scan rate of about 12 nm/min and a sampling of 128 points/row. Optical and topographic data were simultaneously collected during the scans and stored in a personal computer for further analysis. 3. Results and Discussion Charge and Size Modulation in the Heterotopic Aggregates. The ELS profiles of SC6CDNH2 vesicles and of the system TPPS/SC6CDNH2 at a 1:5 molar ratio are reported in Figure 1. The data were fitted using the model which approximates spherical vesicles with radius R by a single thin shell form factor73,74 and introducing a size distribution with a given polydispersity, according to the relation

P(k) )

∫

[

]

sin[kR(x)] 2 f(x) dx kR(x)

where f(x) is the Gaussian distribution around R.

(12)

Figure 2. Hydrodynamic radii of vesicles versus SC6CDNH2 concentration in the absence (0) and in the presence of TPPS (3 µM, 4) at different porphyrin/CD molar ratios.

The slight discrepancy between the fit and the data can be attributed to the possible occurrence of multilamellar vesicles. ELS analysis using eq 12 gave insights into the structure of SC6CDNH2 aggregates and pointed out that cationic amphiphilic CDs form vesicles with R ) 290 nm, and a polydispersity of 30%, by short sonication and equilibration time (1520 min). On increasing sonication time, smaller aggregates occurred, as already described in previous reports.43,44 In these latter studies, we detected smaller SC6CDNH2 nanoparticles by using transmission electron microscopy, even if a deeper investigation on the aggregate structure was not performed. On the other hand, in the presence of TPPS (at a 1:5 porphyrin/CD molar ratio), the vesicles exhibit a decrease of both R (140 nm) and polydispersity (20%). Figure 2 reports a comparison of the dependence of the hydrodynamic radii RH for CD vesicles and for the porphyrin/ CD system as function of CD concentration. RH was calculated according to eq 2, and the error bars are related to the polydispersity of the aggregates, which has been also evidenced by the presence of a continuous distribution of decay rates in the intensity correlation functions. In the absence of porphyrin, the mean vesicle hydrodynamic radius increases from 120 up to 1000 nm when the CD concentration is changed in the range 1 × 10-6-6 × 10-4 M. Moreover, a sensitive increase of the mean vesicle hydrodynamic radius RH is detected on adding porphyrin at high values of the porphyrin/CD ratio. As previously reported51 the increase of aggregate size can be related to the electrostatic nature of the interactions between positively charged CDs and negatively charged porphyrins. To get a deeper insight into the main features of the charge interaction process, the zeta potential (ζ) of the vesicles has been analyzed through laser Doppler electrophoresis experiments. Figure 3 displays the calculated zeta potential (eq 3) for the vesicles as a function of CD concentration in the absence (Figure 3A) and in the presence (Figure 3B) of porphyrin. For the sake of comparison, the corresponding hydrodynamic radii RH are also reported. In the absence of added porphyrin, the vesicle surface exhibits a positive zeta potential of ζ ≈ 12.5 mV, which remains approximately constant as a function of concentration. This observation can be explained by protonation effects occurring at pH ) 7 on the surface of CD vesicles due to the presence of the terminal amino groups. Therefore, the effective charge of CD macro-ions is positive as expected by the shielding effect of counterions and by the solvation of species at thermodynamic equilibrium. A quite different trend is observed on adding the negatively charged TPPS to the CD colloidal system. At lower porphyrin loads (i.e., at molar ratios of 1:5, 1:10, and 1:50) most of the negative TPPS molecules are solubilized in the vesicular phase, because they interact statistically with more amphiphilic CDs, which constitute the positive colloidal surroundings.

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Figure 3. Zeta potentials (ζ) of the vesicles as a function of SC6CDNH2 concentration in the absence (A, 9) and in the presence of TPPS at different porphyrin/CD molar ratios (B, 2). For comparison the corresponding RH in the absence (A, 0) and in the presence of TPPS (B, 4) are also reported.

Figure 4. Calculated effective charge (Qeff) on the vesicles as a function of SC6CDNH2 concentration in the absence (A, [) and in the presence of TPPS at different porphyrin/CD molar ratio (B, 1). Hydrodynamic radii RH of vesicles in absence (A, 0) and in the presence of porphyrin (B, 4) are also reported as reference.

On the other hand, at higher porphyrin loads (i.e., at molar ratios of 1:1 and 1:2) the porphyrin saturates charges on the vesicle surface by electrostatic interaction with cationic amphiphilic CDs. Figure 4 reports the charge calculated according to eq 5 for the investigated systems, in the presence (Figure 4A) and in the absence (Figure 4B) of porphyrin. The data clearly indicate that TPPS adsorption onto vesicle surface causes some structural changes in the system. This reorganization is probably induced by the electrostatic interaction process due to partial charge compensation.

Mazzaglia et al.

Figure 5. Zeta potential (ζ) on the vesicles as a function of temperature at different TPPS/SC6CDNH2 molar ratios (A). The corresponding hydrodynamic radii are reported for comparison (B).

In this respect, the value of |ζ| ) 12.5 mV for the zeta potential seems to be a reference value for the formation of colloidal stable CD vesicular aggregates at thermodynamic equilibrium. At different porphyrin/CD ratios, the basic components of this system (SC6CDNH2 and TPPS) reorganize themselves as a consequence of charge and size modulation (i.e., zeta potential). It is important to point out that, on changing stocks solutions, sonication, and equilibration times, slightly different absolute values are measured, even if the observed trends still remain unaltered. Experiments carried out on changing the temperature (290 e T e 320 K), on different data sets, pointed out negligible changes of the hydrodynamic radii and quite small changes of the zeta potential (Figure 5A), which can be mainly ascribed to variation of the medium properties. The observed zeta potential values are in line with the corresponding hydrodynamic radii (Figure 5B) which, at 1:5 and 1:10 porphyrin/CD molar ratios, seem to be sensitively influenced by early sample settling. Our results indicated that the mentioned physicochemical parameters were not strongly affected by storing samples for 2-5 days at T ) 4 °C. For longer aging time, settling effects such as formation of giant micrometer-sized hetero-aggregates, which eventually precipitate, were evident. These effects are currently under investigation in our laboratories. Evidence of Porphyrin Embedding in CD Vesicles. To focus on the interaction between porphyrin and CDs, timeresolved fluorescence anisotropy experiments at different sensitizer/CD were carried out. These investigations provide detailed information on porphyrin structural changes in the colloidal microenvironment. In Figure 6 IVV and IVH decay curves are reported for TPPS in buffered solution (A) and the TPPS/CD system at a 1:5 molar ratio (B). In this latter case the experimental curves are in good agreement with the decay curves reconstructed following the method reported in the experimental section by means of eqs 10-11. Figure 7 collects a series of anisotropy decay curves for TPPS in buffered solution and for the TPPS/SC6CDNH2 system at different molar ratios. As it is evident from Figures 6A and 7a TPPS alone shows a completely depolarized fluorescence emission. The size of this

Colloidal Stability in Heterotopic Aggregates

Figure 6. Time-resolved fluorescence decays for both IVV and IVH of TPPS (A) and the TPPS/SC6CDNH2 system at a 1:5 molar ratio (B). The lower part of the figure shows the typical weighted residuals (w.res.) with χ2 ≈ 1.

Figure 7. Time-resolved fluorescence anisotropy [r(t)] of TPPS (a) and TPPS/SC6CDNH2 system at 1:1 (b), 1:2 (c), 1:5 (d), and 1:50 (e) porphyrin/CD molar ratios.

molecule is large enough (radius ≈ 10 Å) to have a rotational correlation time of about 0.9 ns, in an aqueous environment (from eq 9). Differently from previous literature data obtained under different excitation conditions,6,75 our experimental value for the initial anisotropy is zero, suggesting that the relative angle between absorption and emission dipoles is close to the magic angle (54.7°). In the presence of CDs, the fluorescence emission of TPPS becomes more polarized, revealing the interaction between the chromophore and the vesicles. The different behavior of the initial fluorescence anisotropy of TPPS, with and without CD, could be explained taking into account that, under our experimental conditions, the absorbing dipoles are different in the two cases. It is worth noting that the used excitation wavelength (570 nm) is localized in the hollow between the second and third Q bands, both for TPPS and for TPPS/SC6CDNH2. Porphyrin/CD interactions would change the limiting anisotropy r0 value in response to environmental effects

J. Phys. Chem. B, Vol. 109, No. 15, 2005 7263

Figure 8. Wide topography (A), topography (B), and relative optical SNOM images (C) on a typical sample of a TPPS/SC6CDNH2 aggregate (1:10 porphyrin/CD molar ratio, transmission mode, λexc ) 457 nm).

that could alter the geometry of the fluorophore as expected even by slight distortion of the porphyrin plane (saddling or ruffling).76 At low CD concentrations (1:1 and 1:2 molar ratio, Figure 7, traces b and c, respectively), the anisotropy decay curves are single-exponential with a “fast” rotational correlation time θ of about 1-2 ns and r0 values of 0.06-0.08. This experimental evidence is fully compatible with the previously reported results: 51 under these conditions most of the porphyrin molecules remain free and solvated in solution (with zero r0 value), and the measured anisotropy decay can be safely assigned to the small percentage of TPPS molecules that are in different hydrophobic compartments of the CD system and/or form supramolecular aggregates of the chromophore.51 In contrast, on increasing CD concentrations (1:5, Figure 7 trace d; 1:10, not shown and 1:50, Figure 7, trace e), more than 90% of the total fluorescence emission is due to TPPS molecules solubilized in the vesicular phase. Under these conditions, the fluorophores are less free to rotate and actually stabilize onto the vesicle surface: indeed, the anisotropy decays evidence a double-exponential behavior, in which the predominant “slow” components exhibit rotational correlation times in the range 2025 ns and r0 values of about 0.15. Referring to eq 9, such values of rotational diffusion times (∼20-25 ns) are too short for the simple CD vesicles (whose radii are larger than 1000 Å). A likely explanation is that TPPS molecules are embedded on the wall of the vesicles and the larger rotational correlation times are related to the slow complete rotation of the porphyrins (i.e., an alteration of the microviscosity) and/or to local deformation of the colloidal wall.66 Morphology and Optical Properties of Heterotopic Vesicles on the Glass Surface. Figure 8A reports a wide topography on a sample obtained by evaporating a small volume of solution containing the CD vesicles in the presence of TPPS (1:10 porphyrin/CD molar ratio). The image evidences a population of vesicles with dimensions ranging from a few tenths to some hundreds of nanometers. Interestingly, performing various measurements on different samples at the same molar ratio, only the larger vesicles (typically greater than some hundreds of nanometers) evidence a clear fluorescence signal, suggesting

7264 J. Phys. Chem. B, Vol. 109, No. 15, 2005

Mazzaglia et al. mobility of the colloidal particles, respectively. The different light scattering techniques pointed out the structural rearrangements of the vesicles in the presence of porphyrin. At the limiting molar ratio studied, the absolute value of the zeta potential (|ζ| ) 12.5 mV) seems to be a reference value for the formation of stable colloidal CD vesicular aggregates at thermodynamic equilibrium. In particular, at the highest concentration of TPPS, colloidal stable negative vesicles with increased size (ζ ) -12.5 mV) were detected. At intermediate concentrations of TPPS, the positive charge on the vesicle surface is saturated, confirming the expected collapse of the vesicles (ζ ∼ 0). At the lowest porphyrin concentration, vesicles constitute a positive microdomain where the sensitizer statistically interacts by promoting a reduction of size of the colloidal particles. Under these latter conditions, TPPS is embedded on the “wall” of vesicles and the rotational correlation times of about 20-25 ns can be probably attributed to microviscosity effects. Measurements carried out at different temperatures showed negligible changes in the structural parameters, suggesting a potential applicability of these nanocarriers. The exact knowledge of their thermal and colloidal stability is a prerequisite for the design of efficient second-generation photosensitizers. Acknowledgment. We thank Dr. Ruth Donohue and Dr. Raphael Darcy, National University of Ireland, for their collaboration in the synthetic work. MIUR (PRIN-Cofin 20022003) and CNR provided financial support. References and Notes

Figure 9. SNOM topography (upper) and fluorescence (bottom) on a sample of TPPS/SC6CDNH2 aggregates (1:10 porphyrin/CD molar ratio; transmission mode, λexc ) 457 nm).

the interaction of the porphyrins with the vesicles (Figure 8, parts B and C). The lack of fluorescence from all of the vesicles could be explained either by (i) a limit in the sensitivity of the technique imposed by the throughput of the optical nanosource or (ii) by desolvation and aggregation phenomena due to the sample preparation, with concomitant reduction of the fluorescence lifetimes. This latter explanation seems more reasonable, even because samples obtained from solutions containing nanoaggregates at higher porphyrin/CD molar ratios do not display any detectable fluorescence signal, in agreement with the experimental results obtained in solution. Further support comes from a comparison between topographic and optical data on a more resolved scan (Figure 9). These images indicate that the porphyrin fluorescence is mainly localized in the inner part of the vesicle. Excluding topographic artifacts due to the transmission mode configuration, which should lead to a less intense signal at the center of the vesicle, this experimental evidence could be ascribed to a total or partial loss of solvent from the external surface, leading to quenching of the fluorescence emission. 4. Concluding Remarks In summary we have focused on the structural features of heterotopic colloids formed by anionic porphyrin and cationic vesicles, through a variety of techniques. Our experimental evidences support previous findings and provide new insight on structural and dynamical properties of such colloids. Furthermore, the combination of time-resolved fluorescence anisotropy and zeta potential measurements appears to be a valuable tool for gaining detailed information on the local environment of the embedded chromophores and the mesoscopic

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