Aggregation Phenomena of Host and Guest upon the Loading of

Article pubs.acs.org/Macromolecules

Aggregation Phenomena of Host and Guest upon the Loading of Dendritic Core-Multishell Nanoparticles with Solvatochromic Dyes Emanuel Fleige,† Benjamin Ziem,† Markus Grabolle,‡ Rainer Haag,† and Ute Resch-Genger‡,* †

Institut für Organische Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany Division 1.10, BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Straße 11, 12489 Berlin, Germany

‡

S Supporting Information *

ABSTRACT: We systematically assessed the loading behavior of coremultishell nanoparticles (CMS NPs) for the solvatochromic dyes Coumarin 153 and Nile Red and studied the influence of the guest and its concentration on CMS NP aggregation using steady state absorption and fluorescence spectroscopy and dynamic light scattering (DLS). These measurements revealed the strong fluorescence of dye-loaded CMS NPs and formation of nonemissive dye aggregates in the outer CMS layer at higher loading concentrations of Nile Red, whereas in the case of Coumarin 153, a new species with red-shifted absorption and blue-shifted emission appeared. Moreover, dye loading triggers an aggregation of CMS NPs which have a hydrodynamic radius of 8 nm, thereby leading to CMS aggregates with a radius of 100−120 nm. These results underline the need for systematic studies of the influence of the guest and its loading concentration on CMS NP size for cellular uptake and in vivo imaging studies and the rational design of CMS NPs with improved transport and targeting abilities.

1. INTRODUCTION Nowadays, polymers have found new applications in the field of medicine as so-called polymer therapeutics.1−3 Especially dendrimers or hyperbranched polymers,4 which are often referred to as dendritic polymers, have a bright future as host systems for the encapsulation of different guests ranging from drugs to magnetic, radioactive, and fluorescent species that can act as reporters for imaging modalities such as magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescence imaging techniques.5−9 The advantages of these polymers compared to similar linear polymers are their high number of functional groups which allow various modifications, their high solubility,10,11 as well as a low intrinsic viscosity.12,13 One especially promising class of compounds can be derived from hyperbranched or dendritic polyglycerol (hPG) that can be obtained with controlled molar masses, different degrees of branching, and low polydispersities (1.2− 1.9).14,15 Furthermore, hPG showed similar or even better biocompatibility in several in vitro and in vivo tests than the widely used and FDA-approved poly(ethylene glycol)s (PEG).16 For these reasons, hPG-based systems have been widely exploited for different biomedical applications.17 hPGs were used for example to inhibit the proliferation of KU-7-luc bladder cancer cells with cis-platin,18 to transport fatty acids, pyrene, and paclitaxel,19 and deliver ibuprofene to human lung epithelial carcinoma cells (A549).20 Our group successfully exploited dendritic polyglycerols as biocompatible nanocarrier platform for drug/dye conjugation and encapsulation.6,17,21 The construction principle of core-multishell architectures was initially inspired by the polarity gradient of liposomes, ranging from a polar interior with a hydrophobic lipid bilayer to an © 2012 American Chemical Society

polar exterior. These universal nanocarrier systems have a high potential for drug delivery and diagnostic applications.22 CMSNPs are highly soluble in a wide variety of solvents and are able to solubilize hydrophilic as well as hydrophobic guests in their respective nonsolvents. Interestingly, the transport of guest molecules did not occur via a unimolecular mechanism but instead is ascribed to the formation of aggregates of the nanocarrier system itself. Because of their unique properties, these CMS-NPs found already various biomedical applications like the in vivo targeting of a F9 teratocarcinoma tumor and the modulation of the copper level in eukaryotic cells.23,24 Furthermore, we were able to show that CMS nanotransporters also benefit from the enhanced permeation and retention effect (EPR effect) and deliver their payload more selectively to tumor tissue.23 With the help of CMS nanotranporters, the skin penetration ability of the hydrophilic dye rhodamine B and the hydrophobic dye Nile Red could be improved in comparison to solid lipid nanoparticles and normal cream formulations, whereby the exact nature of the transport mechanism has not yet been fully understood.25,26 As for topological applications of such drug delivery systems, like the penetration of skin as well as for the cellular uptake by, e.g., cells from pathological tissue, the size of the carrier system plays an important role and affects the biodistribution and excretion pathways.27−29 Accordingly, the rational design of such systems requires a deeper understanding of the influence of the guest on CMS nanotransporter size and aggregation Received: September 20, 2012 Revised: November 16, 2012 Published: November 28, 2012 9452

dx.doi.org/10.1021/ma301977r | Macromolecules 2012, 45, 9452−9459

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of the acid were added. The reaction mixture was concentrated in vacuo, kept at 8 °C for 12 h, and then the precipitated urea derivative was filtered off. The reaction product was dissolved in methanol without further purification and reacted with hyperbranched polyglycerolamine (Mn = 10 kDa, degree of amination =70%, hPG10 kDa(−NH2)0.7).34 The crude CMS NP was purified by dialysis in methanol (molecular weight cutoff 1000 Da). The concentrated CMS solution was diluted with water and freeze-dried to yield the final product. The different mPEG350-water mixtures were prepared by a simple mixing of defined volumes of both solvents. The samples used for the assignment of the polarity of the dye environment were obtained from 0.001 mM stock solutions of the dyes in THF. The 10 μL samples of these solutions (0.01 nM dye) were filled in small sample vials. The solvent was evaporated and 2 mL of the different mPEG350-water solutions were added and the samples subsequently stirred at 1200 rpm for 24 h. Two stock solutions of the CMS nanotransporters were prepared by dissolving the freeze-dried CMS polymer in Milli-Q water, one with a CMS concentration of 1 mg/mL and the other with a CMS concentration of 5 mg/mL. The dye-loaded CMS solutions were prepared by the film uptake method. For this purpose, the corresponding amounts of the dye stock solution in THF were filled into sample vials. The THF was evaporated and 2 mL of an aqueous CMS nanocarrier solution containing either 1 or 5 mg/mL of CMS was added to the dye film. The samples were subsequently stirred at 1200 rpm for 24 h and filtered through 0.2 μm regenerated cellulose syringe filters to remove undissolved dye. The absorption spectra were recorded with a Cary 5000 UV−vis− NIR spectrophotometer from Varian Inc. (Australia). Fluorescence measurements were performed with a calibrated Spectronics Instruments 8100 spectrofluorometer (Aminco Bowmann, USA) equipped with Glan Thompson polarizers in the excitation and emission channel in a 0°/90° standard measurement geometry. The excitation polarizer was set to 0° and the emission polarizer to 54.7°. All emission spectra were corrected for the instrument-specific spectral responsivity of the fluorometer’s emission channel.35 The fluorescence quantum yields of the dyes in the CMS nanocarriers and the mPEG-water mixtures were determined relative to Nile Red in ethanol (fluorescence quantum yield of 0.64 as measured by us absolutely with a custom built calibrated integrating sphere setup36) and Coumarin 153 in ethanol (fluorescence quantum yield of 0.5337) following a previously described procedure.37 All spectroscopic measurements were performed with 1 cm quartz cells at 25 °C. The dynamic light scattering measurements for the size determination were performed on a Malvern Zetasizer Nano-ZS ZEN 3600 equipped with a He−Ne laser (633 nm) using backscattering mode (detector angle 173°). The samples were filtered through 0.2 μm regenerated cellulose syringe filters prior to the DLS measurement and left for 24 h to equilibrate. Then, 100 μL of the solution to be analyzed was added to a disposable microcuvette (Plastibrand) with a round aperture. The autocorrelation functions of the backscattered light fluctuation were analyzed using Zetasizer DTS software from Malvern to determine the size distribution by intensity and volume. The measurements were performed at 25 °C, equilibrating the system on this temperature for 120 s.

behavior. This encouraged us to investigate the influence of the guest and the guest concentration on CMS nanotransporter size using two hydrophobic fluorescent reporters. Since we also wanted to localize the position of the guest molecules within the carrier system, we chose the charge transfer (CT) operated solvatochromic dyes Coumarin 153 and Nile Red which are soluble in a wide range of solvents and show a considerable red shift in absorption and emission with increasing solvent polarity.30 Moreover, although Nile Red and Coumarin 153 as CT-operated dyes are not as prone to aggregation as chromophores with a resonant emission like cyanines, xanthenes, and BODIPY dyes, both fluorophores are expected to at least slightly differ in their aggregation tendency. Only recently, examples of dye aggregation yielding H-type dimers have been reported for Nile Red bound to DNA and solubilized in a sugar-PEG based polymer,31,32 whereas aggregates of Coumarin 153 have not yet been observed, not even in densely loaded polymer particles.33 Here, we present a systematic study of the spectroscopic properties of Nile Red and Coumarin 153 loaded CMS NPs (Figure 1) and their aggregation behavior as a function of dye

Figure 1. Schematic drawing of the structures of a core-multishell nanoparticle and the charge transfer-operated solvatochromic dyes Nile Red and Coumarin 153.

loading and CMS concentration by employing steady state absorption and fluorescence spectroscopy as well as dynamic light scattering (DLS). As model systems for the assessment of the fluorophore location within these nanocarriers and their fluorescence efficiency, solutions of methoxypoly(ethylene glycol) (mPEG)-water mixtures of varying mPEG content were used, thereby mimicking the outer layer of the CMS NPs in water.

3. RESULTS AND DISCUSSION Spectroscopic Probing of the Dye Microenvironment. To assess the microenvironment faced by Nile Red and Coumarin 153 after solubilization within the CMS nanocarriers, we compared the spectral position of the absorption and emission bands of both fluorophores obtained in different water-mPEG350 (mPEG with an average molecular weight of 350 g/mol) mixtures to the absorption and emission spectra resulting for these dyes in the CMS particles. Hereby, the mPEG350-water mixtures act as a model for the outer layer of the CMS carriers dissolved in water. In the case of Nile Red, the spectral position of the absorption maximum shifted from 544

2. EXPERIMENTAL SECTION The methoxypoly(ethylene glycol) with an average molecular weight of 350 g/mol (mPEG350) was purchased from Sigma-Aldrich. Nile Red and Coumarin 153 were obtained from Fluka and had purities higher 98%. All compounds were used without further purification. The dendritic CMS nanocarriers were synthesized and analytically characterized as previously described by us.22,26 In brief, an excess of 1,18−octadecandioic acid (C18) was reacted with mPEG350 in toluene with p-toluenesulfonic acid under Dean−Stark conditions yielding mPEG350−18-oxooctadecanoic acid. This reaction product was purified by flash column chromatography, dissolved in THF, and dicyclohexylcarbodiimide and N-hydroxysuccinimide for the activation 9453


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nm for mPEG350 containing 2.5 vol % water to 592 nm for a water content of 90 vol % (Figure 2, upper and lower left

Figure 2. Polarity-induced changes in the absorption and emission of Nile Red. Top panels: absorption (left) and emission (right) spectrum of Nile Red for different volume fractions of mPEG350 in a mPEG350-water-mixture and spectra of dye-loaded CMS. Bottom panels: spectral position of the absorption (left) and emission (right) maximum in dependence on the volume fraction of mPEG350. The triangle shows the spectral position of the dye-loaded CMS.

Figure 3. Fluorescence quantum yields of Nile Red (top) and Coumarin 153 (bottom) in different mPEG350-water mixtures and comparison with the fluorescence quantum yields of the dye-loaded CMS. Dye concentration for all samples was kept constant at 5 nM.

concentration of 5 mg/mL, the fluorescence quantum yield of Nile Red was 0.86, which is comparable to the fluorescence quantum yield obtained for this dye in pure mPEG350. At a carrier concentration of 1 mg/mL, however, the fluorescence quantum yield reached only a value of 0.73 as found in a 95 vol % mPEG350-water mixture. The fluorescence quantum yield of Coumarin 153 reached 0.56 for both carrier concentrations, thus matching the value found for this fluorophore in an 85 vol % mPEG350-water mixture (see Figure 3). Influence of Dye Loading Concentration. To determine the possible influence of the amount of dye incorporated into the CMS nanocarriers on the spectroscopic properties of the resulting dye-loaded CMS NPs, different amounts of Nile Red (0.1 to 25 mM) and Coumarin 153 (0.1 to 25 mM) were solubilized in CMS solutions containing 1 and 5 mg/mL CMS NPs, respectively. In the case of Nile Red and a CMS concentration of 1 mg/mL, we observed a decrease in the concentration-weighted absorption at 556 nm. At higher dye concentrations, a second hypsochromically shifted absorption maximum appeared at 505 nm (Figure 4, left panels). For the higher CMS carrier concentration, similar changes in the dye’s absorption spectrum occurred (Figure 4, right panels) although the effects were less pronounced as in the case of the lower carrier concentration. The shift of the absorption maximum of Nile Red with increasing dye concentration could be due to a change of the dye environment with the observed blue shift in absorption suggesting a reduced polarity of the dye’s microenvironment.30 Alternatively, the concentration-dependent appearance of a second absorption band blue-shifted from Nile Red’s main absorption band can point to the formation of H-type dye aggregates that are non- or barely emissive.38,39 Nile Red is not known to be very prone to aggregation according to the literature,30 as the vast majority of studies with this dye did not provide any spectroscopic evidence for the aggregation of this dye in solution, in

panels). In comparison, Nile Red has an absorption maximum of 555 nm in the CMS carriers, independent of CMS nanocarrier concentration. This indicates that the polarity of the microenvironment faced by Nile Red in the CMS nanocarriers is comparable to that of a mPEG-water mixture containing 87.5 vol % mPEG350. Therefore, we believe that Nile Red, although it is a hydrophobic guest, is localized at the surface or within the outer layer of the CMS NPs rather than in the nonpolar inner shell as postulated earlier26 or within the core of the CMS architectures. Additional fluorescence studies showed that the spectral position of the emission maximum of Nile Red in the CMS nanoparticles was the same as found in pure mPEG350 (Figure 2, upper and lower right panels). This study confirms that Nile Red is located at the outer layer of the CMS carrier structure. Similar studies with Coumarin 153 revealed the same tendency for both the absorption and the emission maximum. The main absorption band of Coumarin 153 solubilized in the CMS nanocarriers peaked at 424 nm, which corresponds to a mPEG350-water mixture with 87.5 vol % mPEG350 (see Supporting Information, Figure S1, upper and lower left panels). It also had the same emission maximum as Coumarin 153 in a 95% mPEG350-water mixture (Supporting Information, Figure S1, upper and lower right panels). Additionally performed measurements of the fluorescence quantum yields of both dyes in the CMS nanocarriers and mPEG-water solutions showed a similar trend, with the size of the fluorescence quantum yields found for the CMS-incorporated dyes laying well within the range of the polarity-dependent fluorescence quantum yields of both dyes at positions that agree well with the data from the spectrally resolved measurements. In the case of Nile Red, the fluorescence quantum yields revealed a small dependence on CMS concentration (see Figure 3). At a carrier 9454


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Figure 6. Influence of dye loading concentration on the fluorescence quantum yield of Nile Red in CMS nanotransporter solution.

shape and position of the emission band are always independent of dye concentration, whereas the fluorescence quantum yield decreased with increasing dye loading concentration for both carrier concentrations. For the smallest dye loading concentration of 0.1 mM, the fluorescence quantum yield obtained for the 1 and 5 mg/mL CMS solution amounted to 0.56 and 0.68, respectively, which are lower than the value of 0.79 obtained for Nile Red in a 97.5 vol % mPEGwater mixture yielding a matching emission spectrum. With increasing dye loading concentration, these values decreased to 0.02 (dye loading concentration of 15 mM) and slightly increased again for the highest dye loading concentration (25 mM) to 0.04 (Figure 6). This reduction in fluorescence intensity combined with the absence of concentration-dependent spectroscopic changes in emission, which would be expected for polarity-related effects, confirmed our assumption of a formation of non- or at maximum barely emissive H-type Nile Red aggregates within the CMS nanocarriers. Reabsorption effects can be neglected due to the large Stokes shift of Nile Red.40,41 This observation is in agreement with reports from Varghese et al.31 and Bhatia et al.32 as well as with previous studies from us on the concentration-dependence of the fluorescence quantum yield of Nile Red-loaded polystyrene nanoparticles.42,43 Our results also underline the need for the determination of fluorescence quantum yields in conjunction with spectrally resolved absorption and emission measurements for the proper probing of dye location. Otherwise, dye aggregation can always result in a misinterpretation of the dye microenvironment. Similar measurements performed with Coumarin 153 also revealed concentration-dependent absorption spectra, yet in this case it was interesting that a new red-shifted absorption band with increasing dye concentration was observed. The intensity of this new absorption band was more pronounced for the lower CMS nanotransporter concentration. The corresponding changes are shown in Figure 6. However, in contrast to Nile Red, the corresponding emission spectra revealed a slight blue shift with increasing dye loading that can be attributed to a new emission band located at the short wavelength side of the spectrum as highlighted in Figure 7. The fluorescence of Coumarin 153 also decreased with increasing dye concentration as previously observed for Nile Red (see Figures S3 (Supporting Information) and 8). For the lowest dye loading concentration (0.1 mM), the fluorescence quantum yields of Coumarin 153 were 0.50 and 0.52 for CMS concentrations of 1 and 5 mg/mL, respectively. These values are slightly lower than the fluorescence quantum yields of 0.60

Figure 4. Absorption spectra of different concentrations of Nile Red solubilized at two different concentrations (1 and 5 mg/mL) of the CMS nanotransporters solutions. Top panels: concentration-weighted absorption of different concentrations of Nile Red solubilized in 1 mg/ mL (left) and 5 mg/mL (right) CMS nanotransporter solution. Bottom panels: corresponding normalized absorption spectra of CMS nanotransporter solutions containing 1 mg/mL (left) and 5 mg/mL (right) CMS nanotransporter solution.

membranes, or within a carrier system. Moreover, in solution and in the case of Nile Red-loaded polystyrene particles, we did not observe any hints in the absorption spectra pointing to dye aggregation like the appearance of a new hypsochromically shifted absorption band. This interpretation is in accordance with two very recent reports of Nile Red aggregation that describe similar spectroscopic changes in absorption.31,32 To further confirm this assumption, we determined the emission spectra and fluorescence quantum yields of Nile Redloaded CMS NPs as a function of dye loading and CMS concentration. As follows from Figures 5 and 6, the spectral

Figure 5. Absorption-weighted fluorescence spectra of different loading concentrations of Nile Red in 1 mg/mL (top) and 5 mg/ mL CMS nanotransporter solution (bottom). 9455


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found for Coumarin 153 in a 95 vol % mPEG−water mixture that led to a matching emission spectrum. In the case of a CMS carrier concentration of 1 mg/mL, the dye’s fluorescence quantum yield was diminished to 0.07 for the highest dye loading concentration (25 mM) while for a solution containing 5 mg/mL CMS NPs, it only decreased to 0.35 (see Figure 8). The changes in absorption and emission in conjunction with the decrease of the fluorescence quantum yield also suggest the formation of a second species with increasing dye concentration that reveals a red-shifted absorption and a blue-shifted emission (see Figure 7) and a considerably reduced fluorescence quantum yield. This could either be dye aggregates or dye molecules in a considerably more polar environment that are accordingly less emissive. Also, hydrogen bonding interactions could principally enhance the CT character of Coumarin 153 and possibly reduce its fluorescence intensity. The former explanation does not seem to be very likely as the formation of H-aggregates is always accompanied by a blue shift in absorption and typically such species are more or less nonemissive.38,39 Moreover, to the best of our knowledge, there exists not a single report on the aggregation of Coumarin 153, and in former studies, we did not observe dye aggregates of this fluorophore even in highly concentrated dye solutions and at high loading concentrations in polystyrene particles.33 An increase of the polarity of the dye microenvironment resulted in a red shift in absorption in the case of Coumarin 153 as shown in Figure S1 (Supporting Information) depicting the spectral position of the dye’s absorption maximum in different mPEG-water mixtures. However, e.g., in water containing 0.48 vol % ethanol, the absorption maximum of Coumarin 153 is located at 427 nm,33 whereas the absorption band of the newly formed species peaks at 452 nm (Figure 7, top). Moreover, although the emission of this new species is considerably diminished as was expected for Coumarin 153 in a more polar environment e.g., the fluorescence quantum yield of 0.10 found in water containing 0.48 vol % ethanol33 (Figures 8 and S3 (Supporting Information)), its emission spectrum is slightly blue-shifted (Figure 7, bottom). However, in very polar and protic environments, like water containing 0.48 vol % ethanol, the emission maximum of Coumarin 153 is also red-shifted (emission maximum at 556 nm33), similar to the absorption

Figure 7. Spectral changes in absorption and emission of Coumarin 153 between the lowest (0.1 mM) and highest (25 mM) dye loading concentration for the 1 mg/mL CMS nanotransporters solution.

Figure 8. Dependence of the fluorescence quantum yield on Coumarin 153 loading concentration for solutions with 1 and 5 mg/ mL CMS nanotransporters.

Table 1. DLS data of Unloaded and Nile Red Loaded CMS Nanoparticlesa CMS conc. [mg/mL]

guest conc. [mM]

1 5

− −

1

0.1

5 1 5

0.1 0.5 0.5

1 5 1 5 1 5

1 1 10 10 25 25

rhint [nm] 15.8 8.2 82,4 7.9 102.6 27.2 110.9 121.1 5.2 102.2 139.1 99.0 102.4 93.6 103.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.1 8.9 1.3 1.7 1.6 2.7 2.9 0.7 0.2 1.4 1.0 1.5 2.0 1.0

intensity [%] 100 79.7 20.3 86.6 13.4 100 100 92.1 7.9 100 100 100 100 100 100

± ± ± ±

2.3 2.3 1.5 1.4

± 0.4 ± 0.4

rhvol [nm] 7.8 5.1 68.2 6.3 80.6 7.8 108.2 4.2 49.6 119.4 147.5 95.3 98.4 86.0 97.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.1 9.5 1.1 5.8 0.5 2.4 0.6 1.7 8.0 8.3 0.7 1.4 2.0 0.9

volume [%]

Z-average [nm]

PDI

100 99.9 0.1 98.8 1.2 100 100 99.3 0.7 100 100 100 100 100 100

11.9 ± 0.3 8.2 ± 0.1

0.20 ± 0.01 0.34 ± 0.01

45.5 ± 3.2

0.51 ± 0.06

15.5 ± 0.1 85.8 ± 0.7 64.7 ± 0.9

0.37 ± 0.04 0.23 ± 0.01 0.51 ± 0.04

79.8 94.3 86.0 84.8 79.0 76.1

± ± ± ± ± ±

0.9 0.8 0.7 1.0 0.4 0.7

0.22 0.33 0.14 0.18 0.16 0.24

± ± ± ± ± ±

0.02 0.04 0.25 0.01 0.01 0.02

a

The hydrodynamic radius (rh) is given based on the intensity distribution (rhint) and the volume distribution (rhvol). The values represent the maximum of the distribution and the mean standard deviation. 9456


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Figure 9. Scheme of dye-induced CMS nanoparticle aggregation upon loading with Nile Red. Prior to dye addition, the CMS nanoparticles form smaller aggregates (depending on the CAC). Upon addition of hydrophobic Nile Red, the CMS nanoparticles start to form bigger aggregates. Depending on the concentration of the dye added, Nile Red is incorporated into the CMS nanocarrier aggregates either as single emissive dye molecules (low dye concentrations; upper panel) or as dye aggregates in the case of high dye concentrations (lower panel).

nevertheless point to an influence of the guest on the aggregation behavior of the CMS NPs and the size and structure of the resulting aggregates. Here, more systematic studies with guests of different hydrophilicity and aggregation behavior are needed to derive structure−property relationships for such guest-loaded CMS nanocarriers. Such a dye-induced aggregation has been reported, e.g., for surfactants in the presence of azo-dyes.44 Moreover, dyes can lower the critical micelle concentration (CMC) of the surfactants as was reported for Nile Red.45 These effects were mainly attributed to hydrophobic effects.46 So far, to the best of our knowledge, this has not yet been reported for dendritic carrier systems. However, CMS nanocarriers also have an amphiphilic character and a critical aggregation concentration (CAC). The CAC is the concentration at which the CMS nanocarriers start to form aggregates comparable to the formation of micelles above the CMC (see Figure 9). Typically, the CAC for such CMS-type particles lies in the order of 6−7 μM.22 In case of our dendritic nanocarriers, the CAC can be also lowered due to hydrophobic effects.

maximum. In any case, in the CMS systems studied here, Coumarin 153 obviously forms a different type of spectroscopic species than Nile Red at a high dye loading concentration. This is astonishing since both dyes reveal a comparable hydrophobicity according to the rather similar log D values of 3.8 and 3.6 of Nile Red and Coumarin 153.43 The identification of the exact nature of this new coumarin species requires further spectroscopic studies which were beyond the scope of this investigation. CMS Nanotransporter Size and Aggregation. DLS measurements of CMS NPs loaded with different concentrations of Nile Red and Coumarin 153 were performed in order to investigate a possible influence of the guest molecule on the size and aggregation state of the CMS nanocarriers. At a concentration of 1 mg/mL, the unloaded CMS carriers showed a hydrodynamic radius of approximately 16 nm. This points to small aggregates of a few CMS particles. At a higher CMS concentration of 5 mg/mL, the DLS data shows two peaks at approximately 8 and 82 nm. The former peak is ascribed to single particles while the latter provides evidence for the selforganization of the CMS particles to bigger aggregates. With increasing loading concentration of Nile Red, only a single peak around 100 nm resulted that remained constant even for increasing dye loading concentrations. However, although the size of the CMS species was not affected by the concentration of Nile Red, interestingly, the polydispersity of the size distribution of the Nile Red loaded CMS NPs decreased (Table 1). This suggests that there exists an optimal, most likely thermodynamically favored, hydrodynamic radius for the Nile Red-loaded CMS aggregates of approximately 100 nm. A similar enhancement of CMS aggregation can also be observed in the case of Coumarin 153 (see Table S1, Supporting Information). Overall, we observed slightly bigger dye-CMS aggregates with sizes of around 120 nm for the highest concentrations of Coumarin 153 used. The polydispersity of these aggregates was only slightly lowered with increasing dye concentration and never reached the values found for the Nile Red loaded CMS NPs. Although the differences in aggregation size and size distribution between Nile Red and Coumarin 153 loaded CMS NPs are not very pronounced, the observed effects

4. CONCLUSION As demonstrated by our systematic spectroscopic studies of CMS NP loading with Nile Red and Coumarin 153, the spectroscopic properties and the size of the dye-loaded CMS are controlled by the hydrophilicity and aggregation behavior of the incorporated fluorophores, the sensitivity of the dye’s absorption and emission features to the polarity of its immediate environment, and by hydrophobic effects controlling the CMS nanocarrier aggregation. With the help of absorption and fluorescence measurements, we could show that the internalization of these hydrophobic and solvatochromic dyes occurs within the outer layer of the CMS nanocarriers. Uptake of Nile Red by CMS nanotransporters favors dye aggregation as indicated by blue-shifted absorption bands and a concentrationdependent loss in fluorescence. This presents one of the very few examples for the formation of H-type aggregates of Nile Red, a dye commonly reported not to form aggregates. These results also underline the need for the determination of fluorescence quantum yields in conjunction with spectrally 9457


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resolved absorption and emission measurements for the proper probing of dye location. In the case of Coumarin 153 loaded CMS NPs, that revealed a considerable red shift in absorption, a blue shift in emission, and a diminution in fluorescence quantum yield with increasing dye loading concentration, further spectroscopic studies are required to identify the exact nature of this newly formed species. The loading of the CMS particles with both dyes lead to CMS aggregates with an optimum hydrodynamic radius of approximately 100 nm for Nile Red and approximately 120 nm in the case of Coumarin 153. The localization and the behavior of guest molecules within supramolecular architectures designed for drug delivery and diagnostic applications as well as the size of the supramolecular carrier system itself is of high importance since these factors can have a direct influence on the uptake of the delivery system and the release behavior as well as on the activity of the active agent. Here, further systematic studies with guests of different hydrophilicity are required to derive structure−property relationships for the rational design of guest-loaded CMS. Moreover, our results also highlight the need to optimize the dye loading concentration for the construction of efficiently fluorescent dendrimer-derived optical probes.

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ASSOCIATED CONTENT

S Supporting Information *

Absorption spectra of Coumarin 153 in different mPEG350− water mixtures and for different loading concentrations, absorption-weighted fluorescence spectra of different Coumarin 153 loading concentrations. and dynamic light scattering data of unloaded and Coumarin 153 loaded CMS nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.”

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +49 3081041134. Fax: +49 3081041157. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the Helmholtz Portfolio Topic on Multimodal Imaging, the Focus Area Nanoscale of the Freie Universität Berlin, and the Federal Ministry of Economics and Technology. Financial support from the Federal Ministry of Economics and Technology (grant number BMWI VI A2−13/09; M.G.) is gratefully acknowledged.

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ABBREVIATIONS CMS, core-multishell; NP, nanoparticle; DLS, dynamic light scattering; MRI, magnetic resonance imaging; PET, positron emission tomography; hPG, hyperbranched polyglycerol; PEG, poly(ethylene glycol); PEI, poly(ethylene) imine; EPR, enhanced permeation and retention; mPEG350, methoxypoly(ethylene glycol) with an average molecular weight of 350 g/ mol; CMC, critical micelle concentration; CAC, critical aggregation concentration

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REFERENCES

(1) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347−360. 9458


Macromolecules

Article

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Aggregation Phenomena of Host and Guest upon the Loading of

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