Amphiphilic Cyclodextrins as Capping Agents for Gold Colloids: A

Apr 5, 2008 - Antonino Mazzaglia , Luigi Monsù Scolaro , Alessio Mezzi , Saulius Kaciulis , Tilde De Caro , Gabriel M. Ingo and Giuseppina Padeletti...
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J. Phys. Chem. C 2008, 112, 6764-6769

Amphiphilic Cyclodextrins as Capping Agents for Gold Colloids: A Spectroscopic Investigation with Perspectives in Photothermal Therapy Antonino Mazzaglia,*,† Mariachiara Trapani,‡ Valentina Villari,*,§ Norberto Micali,§ Francesca Marino Merlo,⊥ Daniela Zaccaria,⊥ Maria Teresa Sciortino,⊥ Francesco Previti,| Salvatore Patane` ,| and Luigi Monsu` Scolaro*,‡ Istituto per lo Studio dei Materiali Nanostrutturati, ISMN-CNR, c/o Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica, UniVersita` di Messina, Salita Sperone 31, 98166 Messina, Italy, Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica, UniVersita` di Messina, and CIRCMSB, Salita Sperone 31, 98166 Messina, Italy, Istituto per i Processi Chimico Fisici, IPCF-CNR, Salita Sperone, Contrada Papardo, 98158, Faro Superiore, Messina, Italy, Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, UniVersita` di Messina, Salita Sperone 31, 98166 Messina, Italy, and Dipartimento di Fisica della Materia e Tecnologie Fisiche AVanzate, UniVersita` di Messina, Contrada di Dio, S. Agata, 98166, Messina, Italy ReceiVed: December 21, 2007; In Final Form: February 8, 2008

Amphiphilic cyclodextrins (CDs) modified in the upper rim with thiohexyl groups and in the lower rim with oligoethylene amino (SC6NH2) or oligoethylene hydroxyl groups (SC6OH) can bind gold colloids, yielding Au/CD particles with an average hydrodynamic radius (RH) of 2 and 25 nm in water solution. The systems were investigated by UV-vis, quasi-elastic light scattering, and FTIR-ATR techniques. The concentration of amphiphiles was kept above the concentration of gold colloids to afford complete covering. In the case of SC6NH2, basic conditions (Et3N, pH 11) yield promptly the decoration of Au, which can be stabilized by linkage of CD amino and/or thioether groups. The critical aggregation concentration of SC6NH2 was measured (∼4 µM) by surface tension measurements, pointing out that about 50% of CDs are present in nonaggregated form. Whereas Au/SC6NH2 colloids were stable in size and morphology for at least one month, the size of the Au/SC6OH system increases remarkably, forming nanoaggregates of 20 and 80 nm in two hours. Under physiological conditions, the gold/amino amphiphiles system can internalize in HeLa cells, as shown by extinction spectra registered on the immobilized cells. The gold delivered by cyclodextrins can induce photothermal damage upon irradiation, doubling the cell mortality with respect to uncovered gold colloids. These findings can open useful perspectives to the application of these self-assembled systems in cancer photothermal therapy.

Introduction The increasing interest toward nanosystems for diagnostics, biosensing, and therapeutics is at the basis of a consistent research effort focused on multifunctional colloidal carriers.1 Recently, El Sayed et al. demonstrated that antibody-conjugated gold systems are useful probes for living cells because of the sensitivity of their peculiar plasmon resonance band in the visible near-IR region.2 Promising bioanalytical applications can be pointed out by labeling gold with different molecules as dendrimers,3a sugars,3b micellar copolymers,3c polymers,3d or amphiphiles.3e Small gold colloidal particles having an average diameter of 2-4 nm can successfully deliver photosensitizer drugs to cancer cells acting as carrier systems.4 Active targeting can be achieved by decorating gold nanoparticles with receptors to investigate their specific cellular uptake.2,5 Different tech* Corresponding authors. E-mail: [email protected]; [email protected]; [email protected]. † Istituto per lo Studio dei Materiali Nanostrutturati, ISMN-CNR, c/o Universita` di Messina. ‡ Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica, Universita` di Messina and CIRCMSB. § Istituto per i Processi Chimico Fisici, IPCF-CNR. | Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, Universita` di Messina. ⊥ Dipartimento di Fisica della Materia e Tecnologie Fisiche Avanzate, Universita` di Messina.

niques including Rayleigh and Raman scattering, absorption, diffractometry, and microscopy (SNOM, TEM, etc.) have been exploited to locate gold in biological matrixes and understand the cellular components alteration following neoplastic diseases.2,6 In the field of therapeutic applications, gold colloids of different shapes (spheres, rods, and shells) can increase the sensitivity of cancer tissues to heat sources and can be used as drugs in photothermal therapy (PTT).7 PTT is a recently investigated treatment for cancer that is based on the properties of a light-absorbing species to convert efficiently the absorbed radiation into heat in the local environment. In noble metals nanoparticles, the electromagnetic radiation induces the coherent oscillations of electrons in the conduction band (plasmon absorption) of metal nanoparticles, with the consequent local heating of the matrix species (e.g., tissues, cells, etc.). PTT can be complementary to the photodynamic therapy (PDT), which could exhibit a decreased efficacy in poorly vascularized tissues because of insufficient oxygen levels to generate hyper-reactive oxygen species, responsible for photodynamic damage. Additionally, PTT can be used in more pigmented tumors where PDT is less efficient because of the higher absorption of visible light.8 In this respect, it seems relevant to design gold colloidal particles with high biocompatibility and high potentiality as active drugs in specific cellular sites. Our aim is to prepare self-

10.1021/jp7120033 CCC: $40.75 © 2008 American Chemical Society Published on Web 04/05/2008

Amphiphilic CDs as Capping Agents for Au Colloids SCHEME 1: Sketched Structure of the Investigated Amphiphilic CDs

organized multifunctional hybrid (inorganic and organic) systems having a controlled number of components, shape and size, and ability to deliver complexed drugs with different activity to their biological site of action. Supramolecular aggregates of amphiphilic cyclodextrins (CDs) are versatile systems for the encapsulation of hydrophobic or hydrophilic drugs.9 Furthermore, these CD carriers can be conveniently tailored by covalently appending receptor targeting glycosyl groups,10 which can increase selectivity toward specific cell lines. The cationic SC6NH2 is highly biocompatible and potentially less immunogenic because of the oligoethylene glycol chains, resembling the design of “stealth” liposomes. In this respect, porphyrins embedded in SC6NH2 nanoassemblies are effective agents for photodynamic damage in cancer cells.11a-c Multilayer films obtained by electrostatically assembling cationic CDs and anionic porphyrins behave as useful photoresponsive materials, yielding singlet oxygen upon irradiation.11d Promising multifunctional nanoassemblies of amphiphiles and porphyrins for simultaneous delivery of nitric oxide and singlet oxygen have also been recently reported.12 In line with the above-mentioned studies, here we present an investigation on a new water-soluble multifunctional colloidal binary system based on gold colloidal particles (Au) and both amino and hydroxyl-terminated amphiphilic CDs (Scheme 1) as carriers for cellular uptake of metal colloids. In this scenario, gold nanoparticles capped with amphiphilic CDs could also encapsulate drugs and therefore could be the starting point to develop effective systems for combined therapies (i.e., PTT and PDT). Our colloidal systems were studied by complementary techniques such as UV-vis, quasielastic light scattering (QELS), and FTIR-ATR, which furnished insights on the gold covering. Furthermore, the internalization process of gold colloids into immobilized cancer cells was revealed by the corresponding extinction spectra measured using optical microscopy in transmission mode. In perspective, the photothermal properties of this system were performed by irradiating cancer cells through a pulsed focused laser. Experimental Section Materials and Methods. Heptakis(2-ω-amino-oligo(ethylene glycol)-6-deoxy-6-hexyl-thio)-β-CD (SC6NH2) and heptakis(2oligo(ethylene glycol)-6-deoxy-6-hexyl-thio)-β-CD (SC6OH) were synthesized according to the general procedures.9b,c The solvents used were purified and dried by standard tecniques. 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. UV-vis spectra were obtained on a Hewlett-Packard model 8453 diode array spectrophotometer using 1-cm path length quartz cells. Preparation of Au/SC6NH2 and Au/SC6OH. Preparation of CD nanoaggregates was carried out by using conventional procedures.11 CDs were dissolved in CHCl3 (330 µM), and the

J. Phys. Chem. C, Vol. 112, No. 17, 2008 6765 solutions were first slowly evaporated overnight to form thin films. These CD films were hydrated, sonicated for 20 min by a micropoint sonicator (SonoPlus MS-73) in an ice bath, and equilibrated overnight. Gold colloids were prepared in water by reducing a solution of tetrachloroaurate(III) tetraethyl ammonium salt [Et4N][AuCl4] (1.4 mM) with citric acid by a slight modification of the typical procedure.13 The obtained gold colloidal solution (1 mL) was mixed with the amphiphilic CD solution (8 µM, 25 µL of stock solution). All the solutions were studied at pH 3 and 11 (Et3N, 0.1%). The molar concentration of gold colloidal particles (∼0.6 µM) was evaluated through the size distribution function obtained by QELS, by considering the initial concentration of gold atoms in the salt and the density of gold. The samples for FTIR analysis were obtained by freeze-dried solutions at pH 11 of Au, SC6NH2, and Au/SC6NH2, separately. Quasi-Elastic Light Scattering. Quasi-elastic light scattering experiments were performed by using the photon correlation spectroscopy technique and a He-Ne laser source (λ ) 632.8 nm) at a power of 10 mW, linearly polarized orthogonally to the scattering plane. A homemade computer-controlled goniometric apparatus collected the scattered light in a pseudo-crosscorrelation mode (through two cooled R943-02 photomultipliers at the same scattering angle). The scattered light, collected in a self-beating mode, was analyzed using a MALVERN 4700 correlator, which builds up the normalized intensity autocorrelation function:14a,b

g2(Q,t) ) 〈I(Q,0)I(Q,t)〉/〈I(Q,0)〉2

(1)

where the exchanged wave vector Q ) (4πn/λ) sin(θ/2) (n is the refractive index of the solvent, λ is the wavelength of the incident light in the vacuum, and θ is the scattering angle). For scattered electric fields obeying Gaussian statistics, the following Siegert’s relation holds:

g2(Q,t) ) 1 + a|g1(Q,t)|2

(2)

where g1(Q,t) ) 〈E*(Q,0)E(Q,t)〉/〈I(Q,0)〉 the normalized scattered electric field autocorrelation function and a is a constant that depends on the experimental setup. In the case of diffusing monodisperse spherical scatterers, the intensity correlation function decays exponentially, according to g2(Q,t) ) 1 + a exp[-2Γ(Q)t]; the relaxation rate, Γ(Q), is related to the translational diffusion coefficient of particles through Γ(Q) ) DQ2. From the diffusion coefficient D, the mean hydrodynamic radius RH of diffusing particles can be calculated by using the Einstein-Stokes relation, RH ) kBT/(6πηD), where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. In the case of polydisperse samples, the correlation function is the result of a superposition of exponential decays with different decay rates: g1(t) ) ∫τA(τ) exp(-t/τ) d(ln τ). The relaxation time distribution is obtained by using the Laplace inversion through the algorithm REPES.14c Tensiometry. The surface tension of aqueous solutions of SC6NH2 was measured at pH 6.5 and 11 (Et3N, 0.1%) with a homemade tensiometer based on a PC-interfaced Mettler Toledo AL 204 balance. Different solutions of CDs, in the concentration range 0.5-8.5 µM, were prepared in chloroform and slowly evaporated. The CD films were hydrated with water (Romil Super-Purity) and then sonicated. The reported surface tension is the mean value of at least 10 different measurements for each CD solution. The critical aggregation concentration (cac) of SC6NH2 at pH 6.5 is ∼5 µM (surface tension ) 55 ( 3 dyn cm-1), while at pH 11 it is ∼4 µM (surface tension ) 56 ( 3 dyn cm-1).

6766 J. Phys. Chem. C, Vol. 112, No. 17, 2008 FTIR-ATR Spectroscopy. FTIR-ATR spectra were recorded, at room temperature (298 ( 0.01 K), using a Bomem DA8 Fourier transform spectrometer, operating with a Globar source, in combination with a KBr beamsplitter and a DTGS/KBr detector. The powders were contained in a Golden Gate diamond ATR system, based on the attenuated total reflectance (ATR) technique and covering a wavelength range from 600 to 4000 cm-1. In this configuration, the evanescent wave will be attenuated in regions of the IR spectrum where the sample absorbs energy. A property of the evanescent wave that makes ATR a powerful technique is that the intensity of the wave decays exponentially with a distance from the surface of the ATR. This distance is of the order of micrometers and makes ATR generally insensitive to sample thickness, allowing for the analysis of thick or strongly absorbing samples. The spectra were recorded with a resolution of 2 cm-1, averaging 100 scans to achieve a good signal-to-noise ratio and highly reproducible spectra. All the IR spectra were normalized taking into account the effective number of absorbers. No smoothing was applied, and spectroscopic manipulation such as baseline adjustment and normalization was performed using the Spectracalc software package GRAMS (Galactic Industries). Optical Microscopy. HeLa cells (American Type Culture Collection) were propagated in RPMI 1640 medium (GIBCO) supplemented with 100 units/mL of penicillin, 10% fetal bovine serum (Sigma), and 2 mM L-glutamine (pH 7.4). They were incubated overnight in the same medium containing Au and Au/SC6NH2 system. After incubation, the cells were trypsinized, washed three times with phosphate buffer saline (PBS, pH 7.4), and transferred to a multiwell slide pretreated with 0.01% polyL-lysine P6282 (Sigma Aldrich), which promotes cell adhesion to solid substrates. The cells were then washed and covered with a cover plate for spectroscopic analysis. Images of immobilized HeLa cells were collected through an inverted Zeiss microscope (Axiovert S100) with an objective 50× and equipped with a DVC camera. By using an optical fiber positioned on the image plane equivalent to that of the camera, the spectral information was obtained on a defined portion of the image (4-µm sized). The extinction spectra were measured by using the transmission mode of the microscope and a diode array spectrometer (Ocean Optics model S2000) as detecting device. The apparatus was calibrated at zero-absorbance outside the cells. Photothermal Damage. To evaluate the photothermal damage on cell lines induced by the Au/SC6NH2 system, HeLa cells were seeded in a six-well plate for 24 h and treated separately with Au, SC6NH2, and Au/SC6NH2. After overnight incubation, the cells were trypsinized, washed three times with PBS, placed in a cuvette, and exposed to focused laser impulses (Nd:YAG 532 nm, 11 kHz, 1 ns/impulse) with an energy of 1 µJ/impulse. Mock (not treated HeLa cells) was not photodamaged after 15 min of irradiation. After 20 min of irradiation, mock presented a high percentage of death cells. Treated cells underwent three successive irradiation treatments (5 min each). The time interval between each irradiation was 2 min. Following laser irradiation, cells were collected and cytotoxicity was evaluated by trypan blue exclusion standard assay.15 Percentage of death cells was evaluated in a Burker chamber by using inverted microscopy. Results and Discussion UV-Vis, FTIR-ATR, and QELS Investigations on Gold/ Amphiphilic Cyclodextrin Systems. Gold colloids were prepared in water by reducing a solution of [Et4N][AuCl4] with

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Figure 1. UV-vis spectra of Au (∼0.6 µM, pH 11): (a) free and (b) in the presence of SC6NH2 (8 µM), (c) Au (∼0.6 µM, pH 3) in the presence of SC6NH2 (8 µM). The inset shows an enlarged portion of the spectra.

Figure 2. Scattered electric field autocorrelation function at θ ) 90° for Au (∼0.6 µM, pH 11): (a) free and (b) in the presence of SC6NH2 (8 µM). The inset shows the corresponding RH distribution.

citrate (pH ) 3) by using a slightly modified literature procedure.13 The interaction of the amphiphile with Au has been monitored through UV-vis absorption spectroscopy and QELS. Figure 1 shows the plasmon resonance centered at 530 nm, typical for gold colloids stabilized with citrate. This band remains unaltered by changing pH from acid (not shown) to basic conditions by adding Et3N (Figure 1, trace a). In the presence of base, uncharged SC6NH2 can rapidly interact with Au, as evidenced by a moderate bathochromic shift of the plasmon resonance to 538 nm (Figure 1, trace b). This system remains stable for at least one month. At pH ) 3, the addition of CD to gold particles gave rise to a quite small bathochromic shift (∼3 nm), accompanied by a slight decrease of the absorbance (Figure 1, trace c). This could be due to the electrostatic interaction between the negative citrate stabilizing Au and the cationic charges of the CDs. QELS evidences a visible change in the autocorrelation functions relative to Au in the absence and in the presence of CD (Figure 2). Two families of particles having RH of 1 and 25 nm are present in Au solution after the reduction step (Figure 2, trace a). In the presence of uncharged SC6NH2, an increase in hydrodynamic radius up to 2 nm was detected for the smallest particles as shown in Figure 2 (trace b). The inset of Figure 2 reports the hydrodynamic radii distribution obtained by using the inversion method. The distributions indicate that the largest nanoparticles (RH ) 25 nm) constitute less than about 0.1% of the total mass of gold colloidal particles. After the amphiphile is added, these particles seem to slightly increase in size, as also evidenced by the bathochromic shift in

Amphiphilic CDs as Capping Agents for Au Colloids

Figure 3. Surface tension of SC6NH2 at pH 11 (b) and 298 K. Error bars represent the dispersion of data as resulting from 10 repeated measurements.

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Figure 5. Scattered electric field autocorrelation function at θ ) 90° for Au (∼0.6 µM, pH 3) (curve a) and in the presence of SC6OH (8 µM) at t ) 1 min (curve b), 15 min (curve c), 45 min (curve d), and 120 min (curve e).

Figure 4. UV-vis spectra of Au (∼0.6 µM, pH ) 3): (a) free, (b) in the presence of SC6OH (8 µM) at t ) 0, and (c) after 20 min.

their absorption spectra (Figure 1, trace b), which is more sensitive to this change. For the sake of comparison, no changes in the scattering profiles were detected on adding Et3N to Au in the absence of CD (data not shown). Surface tension measurements on SC6NH2 aqueous solutions (Figure 3) show a cac of ∼4 µM, suggesting that even at the investigated concentration (8 µM) small aggregates of uncharged CDs coexist with monomers (about 50%). Therefore, in line with UV-vis absorption, QELS, and tensiometry measurements, both monomers (1.5 nm) and small CD aggregates9f could be responsible for Au covering. An estimation of the minimum amount of amphiphilic CD able to decorate the smallest nanoparticles (RH )1 nm) was obtained by the ratio between the total surface area of the gold cluster and the contact area of the amphiphilic molecule (∼2.3 nm2) on the gold. In our experiments, an excess of CD carrier (about 14-fold with respect to gold particles concentration) was used. Assuming a polyhedral cluster of 1 nm, these simple considerations suggest an average covering of eight cyclodextrin units per cluster.16,17 To investigate the role of the terminal groups in the preparation of water-soluble gold/carrier systems, a neutral amphiphilic CD bearing non-ionizable hydroxyl terminal groups (SC6OH) was also mixed with gold nanoparticles. UV-vis spectra (Figure 4) show that the plasmon band is remarkably affected by the presence of this non-ionic CD in citrate buffer (pH 3): the plasmonic band becomes much broader and shifts to longer wavelengths, suggesting an incipient interparticle aggregation followed by eventual precipitation within 24 h. QELS revealed that gold colloids undergo a progressive aggregation process that starts just after the addition of SC6OH to the solution (Figure 5). The size distribution of Au is still constituted of two main peaks during the aggregation, both shifting toward larger mean hydrodynamic radii of 20 and 80

Figure 6. Time evolution of the RH distribution after adding SC6OH (8 µM) to a Au solution (∼0.6 µM, pH 3): Au (curve a); Au/SC6OH: t ) 1 min (curve b), 15 min (curve c), 45 min (curve d), and 120 min (curve e).

nm, respectively (Figure 6). An analysis of these distributions indicates that the mass percentage of the largest nanoparticles increases with time after the addition of SC6OH to the solution starting from a value of ∼0.1% (1 min), ∼0.4% (15 min), ∼0.5% (45 min), up to a final value of ∼5% (120 min). These results suggest that (oligo)-ethylene glycol chains and hydroxyl terminal groups of SC6OH could play a key role in the capping process and in the following stabilization of the gold colloids.7a,18,19 To get more insights on the cyclodextrin/gold interaction, FTIR-ATR spectra were acquired on freeze-dried samples. A comparison of the spectra of Au, SC6NH2, and Au/SC6NH2 is reported in Figure 7a-c. The spectrum of gold/cyclodextrin system (Figure 7c, solid line) shows differences in the broad band at 3100-3600 cm-1 because of N-H and O-H stretching modes with respect to the weighted sum of the relative components (Figure 7c, dashed line). This difference could be ascribed to the involvement of amino groups in binding with gold surface. The observed changes in the C-H (∼2950 cm-1)

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Figure 8. Optical microscope images of immobilized HeLa cells treated with Au (A) and Au/SC6NH2 (B). UV-vis extinction spectra of (A) and (B) at the marked point are respectively reported in (C) and (D).

Figure 7. FTIR-ATR spectra on freeze-dried samples (from solutions at pH 11) of Au stabilized with citrate (A), neat SC6NH2 (B), and Au/ SC6NH2 (C). In (C) the spectra of the Au/SC6NH2 system (solid trace) is compared with the weighted sum of spectra A and B (dashed line). An enlarged portion relative to C-H and O-H stretching for Au (trace a) and Au/SC6NH2 (trace c) is shown in the inset.

and C-O (∼1065 cm-1) stretching bands could be attributed to the vibrational modes of the oligoethylene oxide chains of CD which would be affected by gold clusters. Moreover, Au spectra display COO- symmetric and antisymmetric stretching bands centered at 1391 and 1581 cm-1, respectively, broader than those in the Au/CD system, probably assignable to the citrate anion bound to gold.20 This spectral change is tentatively attributed to the partial displacement of the citrate by the amphiphile on the metal surface. Therefore, differences between spectra of Au and Au/SC6NH2 are mainly evident in the high frequency region (OH, NH, and CH stretching; see inset in Figure 7a), in the C-O stretching mode, and in the low frequency region (500-800 cm-1). It is noteworthy that in this region the bands relative to the S-C vibrational mode could be present. All these discrepancies could be attributed to differences in the conformation of amphiphilic cyclodextrins upon binding to Au. UV-vis and QELS measurements evidence that, in the case of protonated amino groups, the covering of the metal clusters is disfavored, while the uncharged SC6NH2 can promptly bind the gold surface. On the other hand, binding of gold clusters with CDs, exhibiting a larger tendency to aggregate (SC6OH, cac 570 nm. In the case of HeLa cells treated with Au/SC6NH2, a plasmon band centered at 550 nm can be detected into the cells (Figure 8b,d). The plasmon band was also detected in close proximity of the cellular membrane inside the cytoplasm and in the external portion of membrane (data not shown). This evidence demonstrates the intracellular uptake of gold colloids decorated with the amphiphilic CD (4 and 50 nm in diameter), similarly to previously reported systems based on gold/cationic micelles.22a It is important to note that, in our experimental conditions, the small citrate-stabilized gold nanoparticles could undergo degradation and subsequent elimination from the cells.22b Preliminary experiments were performed on the treated cells to investigate the photothermal effect. Figure 9 shows the survival percentage of HeLa cells after treatment with Au, SC6NH2, and Au/SC6NH2, respectively, and subsequent light irradiation by a focused pulsed laser. The carrier concentration was increased to 25 µM (about 3 times more than in the standard preparation) to afford a more efficient delivery.23 After treatment and in absence of irradiation, relatively low cytotoxicity was registered (in the range 3-7%). Despite that the covering efficiency is lower at pH 7.4, upon irradiating the cells treated with the Au/SC6NH2 system, a higher mortality (∼14%) was observed with respect to the cells treated with neat Au and free CD, respectively. Therefore, a photothermal effect upon irradiation is operative and the amphiphilic cationic CD could exert a protective role toward the gold nanoparticles against degradative cellular processes, resembling

Amphiphilic CDs as Capping Agents for Au Colloids the serum proteins.22b Our supramolecular approach (inorganic particles/amphiphiles) could be promising to design novel hybrid multifunctional carriers with improved stability at physiological pH and efficiency to increase the targeting of inorganic particles and other drugs for combined therapies. Conclusions In summary, we have reported the first example of watersoluble gold/amphiphilic CD nanoassemblies by focusing on the relationship between their physicochemical properties and their activity as gold carrier. Our experimental procedure afforded citrate-stabilized gold colloids exhibiting a bimodal distribution with RH of 1 and 25 nm. QELS measurements indicated that the smallest particle size increases in the presence of uncharged SC6NH2, leading to nanoassemblies having RH ) 2 nm. This result suggests coverage of the metal particles by monomers or small CD aggregates. The interaction of Au with an analogous CD bearing hydroxyl terminal groups (SC6OH) induces interparticle aggregation leading to clusters with RH ) 20 and 80 nm. UV-vis absorption spectra show a moderate shift of the plasmon band in the case of Au/SC6NH2, probably because of the displacement of citrate by CDs. The binding process is probably driven by the interaction between deprotonated amino groups and gold. In the case of Au/SC6OH, the UV-vis bathochromic shift of the plasmon band is fairly significant. In both cases, we could not rule out the role of thioether groups or oligoethylene glycol chains in the stabilization of gold colloids, the former being more available when CDs are present as monomers. A deeper investigation on the nature of the binding is in due course. Spectroscopic and biological data show the intracellular delivery of metal nanoparticles mediated by amphiphilic CDs and the photothermal damage following light irradiation. Using this approach, we could easily prepare binary aggregates, combining the versatility of amphiphilic CDs modified with receptor targeting groups,10 and their high efficacy in photosensitizer internalization11 for application in complementary PDT and PTT therapies. Acknowledgment. We are grateful to Dr. V. Venuti, Prof. V. Crupi, and Prof. D. Majolino (Dept. of Physics, University of Messina) for FTIR-ATR measurements. This research was supported by CNR (FUSINT project) and MUR (PRIN 2006, No. 2006031909). References and Notes (1) Eullis, L. E.; DuPont, J. A.; DeSimone, J. M. In Nanobiotechnology II: More Concept and Applications; Mirkin, C. A., Niemeyer, C. M., Eds.; Wiley-VCH: Weinheim, 2007; p 285. (2) (a) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5, 829. (b) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115. (3) (a) Oh, E.; Hong, M.-Y.; Lee, D.; Nam, S.-H.; Yoon, H. C.; Kim, H.-S. J. Am. Chem. Soc. 2005, 127, 3270. (b) Liu, J.; Qin, G.; Raveendran, P.; Ikushima, Y. Chem.-Eur. J. 2006, 12, 2131. (c) Soo, P. L.; Sidorov, S. N.; Mui, J.; Bronstein, L. M.; Vali, H.; Eisemberg, A.; Maysinger, D. Langmuir 2007, 23, 4830. (d) Merican, Z.; Schiller, T. L.; Hawker, C. J.; Fredericks, P. M.; Blakey, I. Langmuir 2007, 23, 10539. (e) Zubarev, E. R.; Xu, J.; Sayyad, A.; Gibson, D. J. Am. Chem. Soc. 2006, 128, 4958. (4) Wieder, M. E.; Hone, D. C.; Cook, M. J.; Handsley, M. M.; Gavrilovic, J.; Russel, D. A. Photochem. Photobiol. Sci. 2006, 5, 727. (5) (a) Ackerson, C. J.; Jadzinsky, P. D.; Jensen, G. J.; Kornberg, R. D. J. Am. Chem. Soc. 2006, 128, 2635. (b) Yang, P.-H.; Sun, X.; Chiu, J.-F.; Sun, H.; He, Q.-Y. Bioconjugate Chem. 2005, 16, 494. (6) (a) Shamsaie, A.; Jonczyk, M.; Sturgis, J.; Robinson, J. P.; Irudayaraj, J. J. Biomed. Opt. 2007, 12, 020502-1. (b) Venkataran, A. J. S.; Subramaniam, C.; Kumar, R. R.; Priya, S.; Kumar, T. R. S.; Omkumar, R. V.; John, A.; Pradeep, T. Langmuir 2005, 21, 11562. (7) (a) Haba, Y.; Kojima, C.; Harada, A.; Ura, T.; Horinaka, H.; Kono, K. Langmuir 2007, 23, 5243. (b) Kim, J.; Park, S.; Lee, J. E.; Jin, S. M.;

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(23) At basic pH, the spectral features of Au/SC6NH2 (in the presence of CD excess) are unchanged with respect to those registered at low concentration of carrier. This fact suggests that a moderate excess of SC6NH2 did not induce any interparticle aggregation. At neutral pH and in 10 mM PBS (before cell treatment), Au/SC6NH2 shows incipient precipitation. Therefore, the nanoassemblies were prepared according to the established procedure (by adding base) and were incubated with the cells at physiological conditions. The controls did not show any relevant mortality. In analogy to previously reported systems,11 the excess of charged CDs favors the internalization of the gold particles.