Thermal characterization of poly(ethylene glycol) poly(d,l-lactide) block copolymer micelles based on pyrene excimer formation

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1 Journal of Controlled Release 97 (2004) Thermal characterization of poly(ethylene glycol) poly(d,l-lactide) block copolymer micelles based on pyrene excimer formation Eduardo Jule a, Yuji Yamamoto a, Muriel Thouvenin a, Yukio Nagasaki b, Kazunori Kataoka a, * a Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo, Tokyo , Japan b Department of Materials Science, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba , Japan Received 5 November 2003; accepted 20 February 2004 Available online Abstract Poly(ethylene glycol) poly(d,l-lactide) (PEG PDLLA) block copolymers were prepared by anionic ring-opening polymerization, resulting in block sizes effectively controlled by initial monomer/initiator ratios and low molecular weight distributions (<1.12). A pyrene derivative (1-pyrenyl carbonyl cyanide Py) was conjugated to the end of the hydrophobic block (PDLLA) in a quantitative manner, with coupling efficiencies >95%. The so-obtained PEG PDLLA Py copolymers displayed fluorescent properties that were associated with the pyrene monomers, when placed in good solvents for both the hydrophilic and hydrophobic blocks. When placed in selective solvents, these copolymers self-assembled into micelles in the 30-nm range, also with low particle size distributions (<0.09), within which Py could be readily entrapped in the hydrophobic PDLLA core. Py entrapment resulted in the formation of excimers, as evident from fluorescence measurements. Observation of excimer formation/dissociation further conveyed information on the physicochemical properties of the core. Thermal characterization of these systems showed that an increase in the temperature resulted in changes in the properties of excimer fluorescence, an occurrence attributed to a higher mobility of the otherwise glassy PDLLA. This, in turn, greatly affected the inter-molecular distance between pyrene molecules, a crucial factor for excimer formation. The glass transition of the PDLLA block, f38 jc, defined the onset for increasing chain mobility and whence excimer dissociation. Excimer fluorescence appeared to be time-dependent. Based on these observations, chain exchange processes were clearly evidenced through the time-dependent dissociation of excimers into unimers, a process that was influenced by changes in temperature. D 2004 Elsevier B.V. All rights reserved. Keywords: Acetal poly(ethylene glycol) poly(d,l-lactide) (PEG PDLLA) block copolymers; Polymeric micelles; Pyrene excimer formation; Chain exchange * Corresponding author. Tel.: ; fax: addresses: eduardo@bmw.mm.t.u-tokyo.ac.jp (E. Jule), kataoka@bmw.t.u-tokyo.ac.jp (K. Kataoka). 1. Introduction Block copolymers are known to self-associate into nanoscaled spherical compartments called polymeric /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.jconrel

2 408 E. Jule et al. / Journal of Controlled Release 97 (2004) micelles when placed in selective solvents [1]. The soobtained macromolecular assemblies generally have a remarkable stability brought about by a low critical association concentration (CAC) below which dissociation into unimers occurs. This property renders polymeric micelles valuable for the effective transport and delivery of active molecules to targeted sites in the body, as they should remain stable after dilution following intravenous administration, a widely studied route when it comes to systemic distribution. In the field of oncology, micellar systems with a poly(ethylene glycol) (PEG) outer corona have resulted in prolonged plasma circulation times, an essential requisite to fully exploit the enhanced permeability and retention (EPR) effect [2 5] and achieve efficacious passive accumulation in solid tumors. The hydrophobic block used in this research, poly(d,l-lactide) (PDLLA), has been studied within many drug delivery systems because of its low toxicity and remarkable biocompatibility. Systems based on the combination of PEG and PDLLA have achieved effective entrapment of a wide variety of hydrophobic anti-cancer drugs [6] or hydrophilic anti-sense DNA [7] for gene delivery, amongst others. Studies conducted at this laboratory have shown that PEG PDLLA micelles exhibit long circulation properties and are found in a stable spherical shape in the bloodstream of mice for periods of up to 48 h [8]. Extended circulation partly arises from the low immune response provided by the steric repulsion the shell generates and from the core stability, a factor directly related to the relative hydrophobicity of the core and a low CAC. Many issues related to the dynamic behavior of PEG PDLLA micelles remain to be studied. As recently reported, these micelles display temperature-related changes in physicochemical properties such as their CAC, weight-averaged molecular weight and chain exchange rates, especially at temperatures higher than the glass transition (T g ) of the PDLLA segment, i.e. under physiological conditions (f38 jc) [9]. In this study, a pyrene moiety was conjugated to the terminal end of the PDLLA block in order to characterize the thermal behavior of micelles, and more specifically, that of their core. It appears that the formation of micelles entails the accumulation of the probe in dense microdomains, which in turn generates excited-state dimers (excimers), at high pyrene contents. Among the advantages of polymeric micelles, there is the readiness with which ligand substitution degrees can be controlled, so that detailed studies of its influence on wider, macromolecular properties can thus be undertaken. As reported elsewhere, micelles bearing lactose moieties on their surface were found to interact specifically with lectins simulating a cellular surface [10]. This specific interaction provided insight into the association of unimers into micelles, as it was possible to correlate the binding ability of unimers to their structure in solution [11]. By varying the ligand density, the observed enhanced binding capacity was shown to be related to multivalency, a feature brought about by the high association number of micelles (>100). Multivalency, in turn, resulted in low equilibrium dissociation constants from lectins, in the nanomolar range, an asset of significance in the targeting of specific cellular receptors. These studies provided insight on the influence of the ligand density on the kinetic behavior of lactose-installed micelles, useful for the design of drug delivery systems [12]. 2. Experimental 2.1. Materials and methods Tetrahydrofuran (THF), 3,3V-diethoxypropanol (DEP) and ethylene oxide (EO) were purified by conventional distillation under an argon atmosphere. D,L-lactide (DLLA) was recrystallized from ethyl acetate twice, under argon and sublimed under vacuum at 110 jc. Potassium naphthalene was used in a THF solution, the concentration of which was determined by titration. Water was purified with a Milli-Q instrument (Millipore, Bedford, MA). Gel permeation chromatography (GPC) measurements of polymers were conducted using a JASCO liquid chromatograph equipped with TSK gel columns (G4000H HR and G3000H HR ) and an internal refractive index (RI) detector (930-RI, JASCO, Japan). Dimethyl formamide (DMF) containing 10 mm LiCl was used as eluent at a flow rate of 0.8 ml/min. Molecular weight calibrations were done using a series of standard PEGs (Polymer Laboratories, UK). Samples dissolved in the eluent at a concentration of 2 mg/ml were injected into the GPC circuit

3 E. Jule et al. / Journal of Controlled Release 97 (2004) through a 100 Al loop. 1 H-NMR spectra of polymer samples was measured at 80 jc in DMSO-d 6 with a JEOL GSX-270 spectrometer at 270 MHz. Quinuclidine (Aldrich), pyrene-1 carbonyl cyanide (Py) and 1-dipyrenylpropane (DPP; Wako, Japan) were used as received Preparation and characterization of the block copolymers Ac poly(ethylene glycol) poly(d,l-lactide) (Ac PEG PDLLA) and MeO poly(ethylene glycol) poly(d,l-lactide) (MeO PEG PDLLA) block copolymers were synthesized via anionic ring-opening polymerization at room temperature under argon as described previously [11], using either 3,3V-diethoxypropanol (DEP) or 2-methoxyde (MeO), as initiators. The molecular weight of the PEG segment was determined by GPC using a fraction sampled at the end of the first stage of the copolymerization. The size of the PDLLA block was calculated by correlating the number-average molecular weight (M n ) of the PEG block with the intensity ratio of methine protons on the PDLLA block (COCH(CH 3 )O: d=5.2 ppm) and methylene protons on the PEG block (OCH 2 CH 2 : d=3.6 ppm) Labeling and characterization of the block copolymers PEG PDLLA copolymers were labeled by conjugation of Py, onto the N-end of the PLA block ( OH), as previously reported [9]. Briefly, 270 mg of copolymer ( mol) were treated with 100 mg of Py ( mol) in the presence of 80 mg of quinuclidine ( mol) as catalyst, in 100 ml of anhydrous acetonitrile. The mixture was stirred for 1 h at 60 jc, shielded from light, before quenching the reaction by addition of 5 ml of methanol, followed by further stirring for 15 min. After cooling, the mixture was dialyzed against N, N-dimethylsulfoxide (DMSO), shielded from light, using a semi-permeable membrane with a molecular weight cutoff of 1000 g/mol (MWCO 1000-Spectra/ Por, Spectrum, Rancho Dominguez, CA). Dialysis was carried out for 96 h, with the dialysate being exchanged at 4, 8, 24 and 48 h. In order to remove organic solvent residues, the mixture was finally dialyzed against water for an extra 24 h, shielded from light, using a semi-permeable membrane (MWCO 3500, Spectra/Por, Spectrum). Water was changed after 2, 5 and 8 h. Micelles were finally lyophilized under vacuum. Further purification was carried out by precipitation of a THF polymer solution in ice-cooled isopropyl alcohol (IPA), followed by vacuum filtration through a 0.5-Am PTFE membrane filter (Advantec, Japan) and freeze-drying in benzene. The polymer was analyzed by 1 HNMR and GPC coupled to a UV detector (840-UV, JASCO, Japan). Samples were measured at an excitation wavelength of 354 nm. The Py functionality, defined as the number of pyrene molecules per 100 polymer chains, was determined from the 1 H-NMR spectrum based on the peak intensity ratio of the aromatic protons (d= ppm) of the pyrene moiety to the methine proton of the PDLLA segment (COCH(CH 3 )O: d=5.2 ppm) Micelle preparation and characterization Micelles were prepared by filtering a solution of PEG PDLLA copolymers in N,N-dimethylacetamide (DMAc), 5 mg/ml, through a 0.2-Am PTFE filter (Ekicrodisk, Japan) into a preswollen semipermeable membrane (MWCO 3500), and dialysis against a 100- fold excess of distilled water for 24 h. Dialysates were exchanged at times 2, 5 and 8 h. Micelles were eventually filtered through a 0.45-Am PTFE filter (Millipore). Hydrodynamic diameters and size distributions of the so-obtained micelles were determined by dynamic light scattering (DLS). All measurements were carried out at 25 jc on a light scattering spectrometer (DLS-7000, Photal, Otsuka Electronics, Japan), the scattering being carried out with a vertically polarized incident beam at 488 nm supplied by an argon ion laser. A scattering angle of 90j was used for the DLS evaluations and the size and size distribution of particles were estimated by the cumulant method [13]. The glass transition of micelles was estimated using 1,3-dipyrenylpropane (Dojindo, Japan), for which excitation was performed at 333 nm. In this evaluation, samples were equilibrated overnight with a M solution of the probe in chloroform. All concentrations used were at least an order of magnitude greater than the critical association con-

4 410 E. Jule et al. / Journal of Controlled Release 97 (2004) centration of the block copolymers used here (ca. 8 mg/ml). Fluorescence measurements were carried out on a 770 F fluorometer (Japan Spectroscopic Tokyo, Japan) coupled to a thermostat Thermal characterization of pyrene-tagged micelles Micelles containing different amounts of pyrene in their core were prepared by mixing the pyrenelabeled block copolymer with its non-labeled counterpart, in different proportions, in DMAc. After mixing, the solutions were dialyzed by conventional dialysis against distilled water, shielded from light. The temperature dependence in the fluorescence of the PEG PDLLA Py micelles was evaluated: 1.4 mg/ml micellar solutions were mixed with a concentrated phosphate-buffered saline (PBS) including NaCl to obtain 0.35 mg/ml solutions in 50 mm PBS including 150 mm NaCl. These solutions were incubated at 15, 25, 30, 37, 45, 50, 56, 60 and 65 jc, respectively. Chain exchange experiments were carried out by mixing PEG PDLLA Py and Ac PEG PDLLA 1 mg/ml micellar solutions, in a 1:1 ratio. Each mixture was then incubated at 25, 37, 45 and 60 jc and its fluorescence measured at different time points. The fluorescence of all the samples was measured with a thermostat-coupled 770F fluorometer (JASCO, Japan) using an excitation wavelength of 354 nm. 3. Results and discussion 3.1. Preparation and characterization of block copolymers and of micelles Poly(ethylene glycol) poly(d,l-lactide) (PEG PDLLA) block copolymers were prepared by anionic ring opening polymerization as described in Section 2.2; results are summarized in Table 1. All block copolymers used in these studies had PEG and PDLLA blocks displaying a comparable molecular weight of about 5000 g/mol, resulting in hydrophilic/ hydrophobic balances close to one, for which stable Table 1 Block copolymer characterization, as established by (a) GPC measurements and (b) combined GPC measurements, and on the peak intensity ratio on 1 H NMR spectra Polymer M n M w /M n (a) PEG (a) PDLLA (b) MeO PEG PDLLA MeO PEG PDLLA Py Ac PEG PDLLA Ac PEG PDLLA Py (a) Determined by GPC. (b) Calculated based on the peak area ratio in the 1 H NMR spectrum. particles in the 30-nm range are generally obtained [14]. Micelles prepared from PEG PDLLA 55/50 (where 55 and 50 represent the number average molecular weight 10 2 g/mol for the PEG and PDLLA blocks, respectively) were found to be stable even when kept at room temperature for 6 months. Particles possessed only slight changes in their size (from 35.1 to 34.3 nm) and size distribution (0.049 to 0.135). The block copolymer polydispersity, M w /M n, usually found to be <1.05 after polymerization of PEG, was lower than 1.12 after copolymerization of PDLLA. A pyrene derivative, Py, was then conjugated to the N-hydroxyl group of the PDLLA end of the copolymer, as shown in Fig. 1. Effective labeling was confirmed by UV-coupled GPC measurements, then by 1 H NMR, for which quantification of the process was obtained, by correlating the intensity of the peak cluster in the ppm range to that of the methine peak in the PDLLA block (5.2 ppm). The conjugation reaction proceeded in a quantitative manner, as the Py functionality defined as the number of pyrene molecules per 100 polymer chains was found to be f100%. Despite a small degradation detected on the PDLLA block (Table 1), the molecular weight composition of the copolymers was not greatly altered, even after conjugation of Py. Of far more importance, the molecular weight distribution of the copolymers remained narrow, an essential requirement for the copolymer to form particles with a narrow size distribution. Micelles were obtained as 1.40F0.05 mg/ml solutions following the dialysis process described in

5 E. Jule et al. / Journal of Controlled Release 97 (2004) Fig. 1. Conjugation of pyrene-1-carbonyl cyanide to the N-end of an Ac PEG PLA block copolymer. Despite a small degradation of the PLA block, the reaction proceeds quantitatively, in the presence of quinuclidine. Section 2. The hydrodynamic radii, as well as the polydispersity index of all prepared micelles are presented in Table 2. It appears that conjugation and subsequent entrapment of the Py moiety did not induce any significant alteration in the size or size distribution of micelles Characterization of the fluorescent properties of pyrene-conjugated micelles Fig. 2 presents the fluorescence spectra of a MeO PEG PDLLA Py block copolymer in (a) DMF, (b) CHCl 3 and MeO PEG PDLLA Py block copolymer micelles in (c) water, measured using an excitation wavelength of 295 nm. In DMF and CHCl 3, pyrene moieties conjugated to the N-end of the PDLLA have virtually identical fluorescence spectra. Further, these spectra are very similar to the spectrum of free pyrene in similar solvents reported in the literature [15,16]. Table 2 Micellar size and size distribution, as established by dynamic light scattering through the cumulant method [13]. Samples were measured at a scattering angle of 90j at 25 jc Micelle batch Diameter (nm) MeO PEG PDLLA (57/54) MeO PEG PDLLA Py (57/51) Ac PEG PDLLA (55/50) Ac PEG PDLLA Py (55/46) Polydispersity index The fluorescence spectrum of the Py-tagged block copolymer micelles in (c) water is quite broad, and thus, contrasts with the sharp spectra presented in Fig. 2(a) and (b). Of more relevance, the shape of the former spectrum (c) is quite similar to that found in literature reports, corresponding to that of pyrene excited-state dimers (excimers). This observation may be explained as follows. Water is a good solvent for only the PEG block, so that MeO PEG PDLLA Py block copolymers spontaneously selfassemble into structures for which the PEG block constitutes a palisade of chains surrounding the PDLLA block shielded from the aqueous environment in a compact core [17]. It is in the compact core, a hydrophobic environment, that pyrene molecules can be entrapped, come into close contact and thus generate excimers. In contrast, MeO PEG PDLLA Py polymers dissolve as unimers in (a) DMF and (b) CHCl 3, as these solvents are good solvents of both the PEG and PDLLA blocks. Hence, the copolymer adopts a classical random coiled structure and pyrene molecules behave much as if they were simply dissolved in those solvents. Returning to excimer formation, this phenomenon originates when two pyrene molecules come in close contact and form an excited dimer. Excimers generally exhibit a wide, characteristic peak centering at a wavelength close to 500 nm [18]. In order to confirm this, micelles containing different amounts of Py (0 100%) were prepared, by simple mixing of Pymodified polymer and its unmodified counterpart, in DMAc solution, followed by conventional dialysis

6 412 E. Jule et al. / Journal of Controlled Release 97 (2004) Fig. 2. Fluorescence spectra of PEG PLA Py block copolymers in different solvents: (a) DMF, (b) CHCl 3, (c) water. against water. Predictably, the size of the so-obtained particles did not vary much from one batch to another (data not shown). Fig. 3 presents fluorescent spectra of micelles containing different Py functionalities, as recorded between 370 and 650 nm at 25 jc. The first observation is that micelles with large Py amounts (>80%) show the typical excimer peak centering at about nm. Then, as the amount of pyrene in the micellar core decreases, the intensity of this excimer peak decreases, until it virtually disappears for 30% Py. A decrease in the Py concentration results in an increase in inter-probe distances, leading to the progressive dissociation of dimers. Excimer formation can only occur when pyrene molecules are in close proximity, as will be discussed later. A decrease in the Py functionality results in the concomitant emergence of first a peak at 417 nm (Py<70%), then a second peak at 393 nm (Py<60%). The intensity of these two peaks increase considerably as the Py contents in the micellar core decreases. These two peaks correspond to Py unimers, as observed in either DMF or CHCl 3 (Fig. 2). Thus, all of these observations support pyrene excimer formation as a result of molecular condensation in the compact PDLLA core. In order to measure excimer formation, the ratio of two fluorescent intensities of the peak at 480 nm (I 480 ), characterizing the formation of excimers, and at 417 nm (I 417 ), corresponding to pyrene monomers, were recorded. Fig. 4 presents the I 480 /I 417 ratio as a function of Py functionality in the micelles. The I 480 / I 417 ratio increases with an increase in the amount of Fig. 3. Ac PEG PDLLA micelle fluorescence spectra at different Py functionalities.

7 E. Jule et al. / Journal of Controlled Release 97 (2004) as indeed, the latter will change according to the behavior of PEG PDLLA micelles and more specifically, the PDLLA core. As seen in Section 3.2, the extent to which excimer formation can occur will be directly affected by the core mobility as the interpyrene distance is to be heavily influenced by polymer chain mobility. Fig. 4. I 480 /I 417 as a function of Py functionality, expressed in percentage, at 25 jc. The ratio denotes the evolution in pyrene excimer formation in the core of PEG PDLLA block copolymer micelles. pyrene in Py-micelles. This is primarily due to a gain in the intensity of the excimer peak at f500 nm as the higher the pyrene concentration in the PDLLA core, the more excimers can be formed. There is a decrease in excimer formation for a Py content of 100% that might be explained by self-quenching. As reported previously [9], and as confirmed in this paper, PDLLA block in the micelles has a glass transition (T g ) of about 38 jc. Hence, the mobility of the glassy core will increase above this temperature. As a result, the fluorescence of micelles becomes time dependent above the T g, making interpretation of spectra more difficult. All that can be said regarding the I 480 /I 417 ratio is that it decreases at any given Py content with an increase in temperature, and that this trend becomes even more obvious at high Py contents (data not shown). These observations reflect the behavior of the PDLLA core. The higher mobility of the core as its T g is approached results in looser polymer aggregates and, as a result, lower local pyrene concentrations. Moreover, as will be demonstrated in Section 3.3.3, pyrene molecules are not randomly distributed in the core, but seem to concentrate in microdomains in which pyrene concentrations are high enough for excimer formation to occur Glass transition of PEG PDLLA micelles In order to substantiate the influence of polymer chain mobility on the properties of the core and the supra-molecular properties of micelles, the viscosity of the PDLLA block in the micellar form was evaluated using the environment-dependent fluorescence of DPP (Fig. 5). Thomas was among those who first explored the use of fluorescent probes to study surfactant micelles and amphiphilic copolymers [19]. Properties such as the viscosity and polarity within a macromolecular assembly can be evaluated using the intensity of emission, or the intensity ratio of two emission bands of probes that are sensitive to these properties [20]. DPP is composed of two pyrene molecules at each end of a short propane chain and hence its excimer formation is quite unique, in that it is mainly an intra-molecular process. That is, the ability of the pyrene molecules to rotate around the propane linker will result in monomers coming into close contact and excimer formation. The emission spectrum of DPP presents four peaks between 370 and 395 nm corresponding to probe monomer emission, and a large band centering at about nm corresponding to excimer emission. The ratio of the intensity of the excimer peak (I e ) to that of the monomer (I m ), displaying maxima at 480 and 398 nm, respectively, has been used to assess the microviscosity of polymeric micelles [21]. In the case of 3.3. Temperature-related change in the fluorescence properties of pyrene-conjugated micelles Another parameter that requires attention, then, is the temperature dependency of excimer fluorescence, Fig. 5. Structure of DPP.

8 414 E. Jule et al. / Journal of Controlled Release 97 (2004) PEG PDLLA micelles, the I e /I m ratio is This ratio is quite low and consistent with the very limited mobility of the PDLLA core that severely limits rotation around the propane chain in DPP. As a comparison, the I e /I m ratio of sodium dodecyl sulfate micelles was measured and found to be much higher (0.553), consistent with the nature of those assemblies that display liquid-like cores in which DPP can freely rotate. To evaluate the temperature dependence of the core mobility, the I e /I m ratio was measured as a function of temperature (Fig. 6). Clearly, the plot presents two domains with a break point corresponding to a temperature of f40 jc, readily attributed to the glass transition of PEG PDLLA micelles Temperature and time-related changes in the fluorescence of pyrene excimer As the mobility of the core of PEG PDLLA micelles is temperature dependent, it is not unreasonable to assume that time also influences this parameter. Fig. 7 presents fluorescence spectra recorded at different temperatures and incubation times. Time-related changes in the spectra are negligible at 25 jc, clearly demonstrating the quasi-static state of the glassy PDLLA core. At 37 jc, close to the T g, a slight decrease in the excimer fluorescence intensity at 500 nm, accompanied with an increase in the intensity at 417 nm is observed. These changes are of no consequence Fig. 6. Intensity ratio I excimer /I monomer (I 480 /I 398 ) of dipyrenylpropane entrapped in PEG PLA block copolymer micelles, as a function of 1/T. under physiological conditions, clearly demonstrating the stability that the core is to confer on encapsulated compounds. Additionally, this stability extends over periods that overlap those of micelle circulation in the bloodstream [8], confirming the stability of PEG PDLLA block copolymer micelles. In contrast, changes in excimer fluorescence are remarkably accentuated at 45 (Fig. 7c)) and 60 jc (Fig. 7d), again due to the amplified mobility of the PDLLA segment above its T g Excimer formation theory It is known that pyrene excimer formation can occur via two different processes [22]. (i) Dynamic encounter of excited-and ground-state pyrene molecules (a process known as dynamic excimer formation). (ii) Excitation of ground-state pyrene aggregates (a process known as static excimer formation). Assuming that what is observed here is the formation of dynamic excimers, excimer fluorescence should decrease with an increase of viscosity, following Eq. (1) [23,24]. ½excimerŠ=½monomerŠ ¼A=viscosity ð1þ where A is a constant. For PEG PDLLA micelles, the viscosity of the PDLLA core surrounding Py molecules decreases at temperatures above its glass transition. This is inconsistent with the dynamic model. At lower viscosities, we have proven that excimer concentration decreases because the inter-probe distance is heavily influenced by polymer chain mobility. It is logical to assume that static excimers form inter-molecularly at room temperature by and between Py molecules chemically bound to the N-end of the PDLLA block and are dissociated with an increase in block mobility. These observations suggest that the excimers observed in this study are static in nature. The dynamic model would, on the other hand, fit the behavior of DPP, for which excimer formation occurs through the intra-molecular encounter of pyrene molecules in both excited and ground states. In this particular case, an increase in the mobility

9 E. Jule et al. / Journal of Controlled Release 97 (2004) Fig. 7. Changes in the fluorescence spectra of MeO PEG PDLLA Py micelles at different temperatures (kex=354 nm). of the core facilitates pyrene molecular rotation around the propane linker enhancing the possibility of molecules in both the excited and ground state to come into close contact and thus result in excimer formation. The average distance between Py molecules in the micellar core is calculated to be 21 Å, assuming the density of the PDLLA core is 1. It is known that the distance between pyrene molecules forming excimers is 3 10 Å [16]. This suggests that Py molecules would not be randomly and homogeneously distributed, but rather concentrated in microdomains in the core. When I 417 /I 480 was plotted as a function of the incubation time, the resulting plots were almost linear at all studied temperatures (15 70 jc, data not shown). This linearity in the relationship between I 417 /I 480 and the incubation time is attributed to the initial term of the change in I 417 /I 480, representing the transition of excimers into monomers, in which the concentration of excimer can be assumed to be constant. The transition of one excimer into two monomers can be described as follows: Excimer! 2 monomer: In the initial period, the concentrations of excimer and monomer are: ½excimerŠ ¼B½monomerŠ ¼2 ½excimerŠkt; where B and k are constants. Therefore, in the initial term of the transition, I 417 /I 480 is described by the following equation: I 417 =I 480 cc½monomerš=½excimerš ¼2Ckt ¼ Dt;

10 416 E. Jule et al. / Journal of Controlled Release 97 (2004) where C and D are also constants. Hence, the slope of the I 417 /I 480 ratio against the incubation time, d(i 417 / I 480 )/dt was plotted against the incubation time, as shown in Fig. 8. d(i 417 /I 480 )/dt shows a temperature dependency corresponding to the increase in the methine proton in the 1 H-NMR spectrum in D 2 O reported in a previous paper [9], further confirming that the transition of excimers into monomers is due to the increase in the mobility of PDLLA above the T g Chain exchange reactions Py molecules appear to be distributed heterogeneously in the PDLLA core, in microdomains with short inter-py distances, thus resulting in the formation of excimers. Should any change in the structure of the core arise, the resulting change in the distance between Py molecules should result in an alteration of excimer emission. Inter-micellar chain exchange was assessed by mixing (no stirring was applied to the solution) 90% Py-labeled and unlabeled micelles in a 1:1: ratio, at a final concentration of 1 mg/ml. For each temperature studied (25, 37, 45 and 60 jc), micelles were mixed, then incubated in a water bath shielded from light, and the fluorescence measured at different time points (1 min 170 h). Interestingly, it appears that the fluorescence spectrum of micelles becomes a function of time as soon as micelles are mixed (Fig. 9). Fig. 9. Fluorescence spectra of a 1:1 mixture of micelles with a Py functionality of 90% and of reference micelles (no pyrene), at different mixture times. The mixture was incubated at 37 jc from time t =0 when the two micellar solutions were mixed. Based on plots of the I 480 /I 417 ratio against time, the apparent transfer of pyrene-labeled polymer chains between micelles occurred very rapidly after mixing (Fig. 10) but slowed at longer times as evident from the declining slope of the plot. The transfer of polymer chains was accelerated as the temperature is increased, induced by an increase in polymer chain mobility. Fig. 8. Relationship between the incubation temperature and the slope of the time-differentiated I 417 /I 480 ratio. Fig. 10. I 480 /I 417 ratio as a function of time, at different temperatures. Micelles were simply mixed (no stirring was applied to the mixture) and incubated straight away, shielded from light.

11 E. Jule et al. / Journal of Controlled Release 97 (2004) Theoretically, should equilibrium be reached after complete exchange of polymer chains, the overall fluorescence of the system should be equivalent to that of standard micelles containing f45% of Py. In this evaluation, it took more than 96 h for the I 480 /I 417 ratio to attain values between that of standards at 40% and 50% pyrene at 37 jc. This comparison can only hold for temperatures below the T g of PDLLA, for which the fluorescent spectra of pyrene excimer can be assumed to be time-independent as a first approximation (see Section 3.3.1). At temperatures higher than the glass transition of PDLLA, distinguishing between the temperature-dependent change in excimer fluorescence and the also time-dependent chain exchange between micelles is difficult and would certainly require the use of far more complicated analyses and modeling. This shall not be done for this evaluation, but might be further studied in detail in the future. However, it is clear from the above evaluations is that there is an active chain exchange between micelles, and that this exchange is accelerated as the core mobility is increased above its T g. Chain exchange occurs at an overall slow rate and it takes these systems many hours to reach equilibrium. Hence, PEG PDLLA micelles are stable in physiological conditions and should not be prone to drug leakage due to chain exchange processes. This stability goes beyond temperatures above the glass transition of PDLLA, since no remarkable changes in the core of micelles is observed for periods overlapping those of blood circulation [8]. At this point, there is no explanation as to why there is polymer chain exchange at temperatures below the T g of PDLLA. Note that similar observations were previously reported [9]. An aqueous block copolymer solution above the CAC is certainly composed of micelles. But in addition to these structures, unimers and assemblies of smaller association degrees have been suggested to exist [11]. These smaller assemblies of a looser and less stable nature would explain why the CAC is not a well defined concentration, but rather a broad concentration range, as well other phenomena. Unfortunately, the detailed feature of these assemblies can not be evaluated by light scattering due to detection limits. A study on the interaction of ligandinstalled micelles and a protein bed using surface plasmon resonance [11] showed that the amount of complex formed by and between the two entities does not occur in a linear manner. Instead, this property was directly related to the amount of ligand and hence to the association degree of micelles, which suggested that assemblies of different association degrees exist in equilibrium even at concentrations above the CAC of full micelles. But relevant to this report, it is suggested that small associates undergo excimer formation in their core. Based on the observation of smaller degrees of association and greater looseness, these assemblies and more specifically, their cores would be less stable. As a result, chain migration to other small associates and/or full micelles would be facilitated and explain why chain exchange is observed even at temperatures below the T g. 4. Conclusion PEG PDLLA micelles form core-shell structures in the 30-nm size range, in which the glassy core can readily entrap hydrophobic molecules such as pyrene. Given the inherent compactness of the core and the highly hydrophobic character of pyrene, excimer formation is observed with high amounts of probe present in the core. Pyrene is not randomly distributed in the core, but rather accumulates in microdomains. In such conditions, its local concentration is so high that inter-molecular distances are short enough for excimer formation to occur. The PDLLA core exhibits a glass transition occurring at 38 jc, evident from the environment-dependent fluorescence of 1-dipyrenylpropane. The increase in chain mobility generated by this transition considerably alters the properties of excimer emission. An increase in the temperature loosens the core, resulting in increased inter-pyrene distances and hence decreased excimer emission. Despite their remarkable stability, PEG PDLLA micelles are not immutable systems and are subject to chain exchange processes as also evidenced by fluorescent techniques. And even though a sensible quantification of these processes has not been obtained, it can be concluded that chain exchange processes are strongly influenced by the core mobility. A series of fundamental studies on the dynamic character of polymeric micelles has provided insight into not only

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