Poly(propylene oxide)

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C: Physical Processes in Nanomaterials and Nanostructures

Micellar Self-Assembly and Probe Exchange Dynamics of Triblock Copolymer Poly(ethylene oxide)-Poly(propylene oxide)-Poly(ethylene oxide) PEO PPO PEO 57

45

57

Eva M Villar-Alvarez, Silvia Barbosa, J. Félix Armando Soltero, Victor Mosquera, Pablo Taboada, and Yahya Rharbi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b00153 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 10, 2018

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The Journal of Physical Chemistry

Micellar Self-assembly and Probe Exchange Dynamics of Triblock

Copolymer

Poly(ethylene

oxide)-Poly(propylene

oxide)-Poly(ethylene oxide) PEO57PPO45PEO57 E. Villar-Alvareza, Silvia Barbosaa, J. F. A. Soltero b, V. Mosqueraa, P. Taboadaa,*, and Y. Rharbic. a

Grupo de Física de Coloides y Polímeros, Departamento de Física de la Materia

Condensada; Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain. b

Laboratorio de Reología, Departamento de Ingeniería Química, CUCEI, Universidad

de Guadalajara, Blv. M. García Barragán 1451, 44430 Guadalajara, Jalisco, Mexico. c

Laboratoire de Rhéologie et Procedes, UJF/INPG/CNRS, UMR 5520, B.P.53, F-38041

Grenoble Cedex 9, France *

Author to whom correspondence should be addressed: [email protected]

ABSTRACT The

dynamics

of

micellar

solutions

of

a

triblock

poly(ethylene

oxide)−poly(propylene oxide)−poly(ethylene oxide) copolymer, PEO57PPO45PEO57 where the subscripts indicate the block length, has been analysed. This copolymer is characterized by a large monodispersity and hydrophilicity if compared to previously studied commercially-available Pluronics block copolymers. Different experimental techniques were used to characterize its micellar self-assembly, elucidating the existence of a concentration-dependent transition to a “pseudo-percolated” state as the temperature increases in which interconnected polymeric micelles are formed. The kinetics of the micellar assembly and disassembly processes and solute exchange 1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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dynamics were monitored by stopped-flow time-scan fluorescence measurements using a fluorescent probe, which points to the involvement of several underlying mechanisms other than simple particle collisions: A slow dynamic regime below 45 ºC; a transition regime between 45 ºC and 50 ºC; and a fast dynamics one above 50 ºC. The slow dynamic regime was associated with fragmentation and fusion processes in competition.; the fast one was mainly regulated by a fusion-dominant mechanism in which both concentration and temperature play a role; finally, a linear increase in the fluorescence decay rate with increasing copolymer concentrations indicated that the transition regime (in which the interconnected micelles are formed) starts to be dominated by micellar fusion whereas fragmentation becomes less likely. It was observed that the kinetic constant for the fusion process, kfusion, increased ca. two-fold as the temperature rises in a concentration-dependent manner, whilst that corresponding to the fragmentation process, kfragmentation, increases until ca. 50 ºC (close to the transition temperature, Tt) and, then, it decreases independently of the micellar concentration. Above the transition temperature, kfragmentation and kfusion are related to two apparent activation energies of similar magnitude but different sign. Finally, the kinetic constants of the micellar fragmentation and fusion processes were influenced by the PEO/PPO ratio when compared to previously structure-related commercial Pluronic copolymers analysed.

1. INTRODUCTION Water-soluble triblock copolymers of poly(ethylene oxide), PEO, and poly(propylene oxide), PPO, often denoted as PEO-PPO-PEO or EO-PO-EO (where EO denoted ethylene oxide, and PO propylene oxide) are commercially available nonionic

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macromolecular copolymers commonly known as Poloxamers® (manufactured by ICI) or Pluronics® (manufactured by BASF). These copolymers are thermosensitive, biocompatible (for most varieties) and are already approved by the Federal Drug Administration (FDA) for their use in drug delivery, cosmetics and healthcare applications1-3. In aqueous solution they can form aggregates (micelles) above a critical concentration, the so-called critical micelle concentration (CMC), or a critical temperature (the critical micelle temperature, CMT). 4 The formed micelles in aqueous solution consist of a PO core surrounded by a more hydrophilic EO corona becoming suitable nanocontainers for the solubilisation, for example, of poorly water-soluble hydrophobic compounds into the inner micellar core in order to enhance their solubilisation and control their release

1-2, 5

, hence, avoiding their unspecific absorption

and favoring their extended circulation times while decreasing clearance from the body. Also, drug incorporation inside these polymeric nanocarriers can allow drug passive accumulation in solid tumours through the enhanced permeability and retention (EPR) effect and the overcoming of biological barriers, which increases the therapeutic efficacy of the bioactive cargo compound in diseases such as cancer.6-8 The selfassembly properties of Pluronic-type block copolymers in aqueous solutions can be modified by, for example, modifying the PO/EO molar ratio or the copolymer´s molecular weight.9-10 Moreover, this class of copolymers can also undergo morphological transitions from spherical micelles to, for example, rod-like structures upon temperature and/or concentration changes depending on the copolymer composition, structure and block length, amongst other factors.11-15 A detailed picture of the copolymer self-assembly and drug-micelle exchange kinetics is crucial to elucidate the physico-chemical properties of a polymeric nanovehicle and their potential biomedical applications, especially for controlled and 3 ACS Paragon Plus Environment


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sustained drug delivery.16 Most of the knowledge regarding the dynamics of selfassembled copolymer systems comes from comparative studies with surfactants kinetics, which analyses the return to equilibrium after the application of a small perturbation.17-22 Similarly as surfactants, in the dynamics of block copolymer two wellseparated relaxation times are frequently identified: a rapid relaxation which occurs on a time scale of microseconds, and a slower process which requires milliseconds to seconds. Anianssonn and Wall23-24 assigned the first process to the formation of metastable micelles via insertion of free copolymer chains in existing micelles, which modifies micellar sizes but not their numbers.17,18,23-32 The slower relaxation involves a deeper micellar rearrangement through the formation or destruction via micellar fusion and fragmentation of existing micelles.23,29,33 Micellar fusion/fragmentation seems to be favourable at early stages of micellization whereas unimer insertion−expulsion becomes dominant at equilibrium.34 Recent experiments on triblock PEO− PPO− PEO copolymers revealed that the rates of fusion and fission are 6 orders of magnitude lower than the rates of insertion and expulsion of individual amphiphiles under the later conditions.32 Despite similarities with surfactant kinetics, some important differences are also present as, for example, the sensitivity of copolymeric chains to solvent quality, the existence of polymeric “frozen” dynamical states trapping the formed micellar structures in metastable states without reaching the thermodynamic equilibrium, the larger thermodynamic barrier to be overcome to extract a core block from a typical micelle, or the hypersensitivity to core block length and copolymer architecture of the chain exchange rates.31,35-39 In this regard, recent molecular simulations have identified the existence of three different regimes in the exit dynamics of polymeric chains from a micelle corresponding to the reorganization of the labeled chains at very short time 4 ACS Paragon Plus Environment

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scale, an unexpected central regime characterised by a nonexponential exit of almost frozen conformations in the millisecond time scale; and a terminal regime dominated by the average conformational change of the chains.40 On the other hand, the cargo exchange solubilisation dynamics and the related modifcations in the micromolecular environment (microviscosity, local polarity, etc) of self-assembled systems has been less studied.41-42 In this regard, very few studies have analysed the effect of the dynamics in micelles formed by block copolymers during drug transport and delivery.37,43 Different works have elucidated that up to three different mechanisms can be involved in solute exchange between micelles depending on the nature of polymeric chains, solute molecules, and external parameters such as pH, temperature, etc (see Scheme 1): i) Exchange via water, in which a cargo molecules exits from a polymeric micelle, diffuses through the aqueous solution and re-enters in an empty new micelle; ii) collision-exchange-separation, involving the fusion of two micelles to form a short-lived “supermicelle”, which rapidly fragments back to two “normal” micelles with a cargo molecule each one; and iii) fragmentation-growth, which involves the breakage of a normal-sized cargo-bearing micelle into two submicelles each containing a single solute molecule, followed by the growth of these sub-micelles either by fusion with empty micelles or by interactions with free monomers to give “normal” micelles again.19, 44



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Scheme 1. Main solute exchange mechanisms of a solute molecule to a neighboring empty micelle. (a) Exit-re-entry via water mechanism; b) collision-exchange-separation mechanism; and (c) fission-growth mechanism

To put further light into this topic, in this paper we describe the kinetic behaviour of the triblock copolymer EO57PO45EO57 (where the subscripts denote the block lengths) as a function of concentration and solution temperature. This copolymer displays an optimal composition and block ratios to achieve a successful balance between micelle formation, colloidal stability, drug solubilization and sustained release capabilities as inferred from previous studies.10,45,46 Moreover, it is highly monodisperse in contrast to some previously analysed commercially-available Pluronic block copolymers, which can clearly help to avoid deleterious effects on the micellar dynamics stemming from the presence of different size/species populations. To follow the polymer assembly/disassembly chain and the solute exchange dynamics, a very hydrophobic solute (a pyrene derivative) which represents the active ingredient, was incorporated inside the copolymer micelles as an aqueous insoluble model cargo.47,48 The lack of water solubility of the fluorescent probe involved that solute exchange could not occur by an exit/re-entry mechanism and, hence, this could only occur by a


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micellar fragmentation and fusion mechanisms enabling its detailed analysis. We could observe from different characterization techniques (SLS, DLS, TEM and rheometry) that under suitable solution conditions, in particular between 47 and 51 ºC for copolymer concentrations ranging from 10 to 80 mg/mL, respectively, a certain micellar structural transition took place from simple micelle formation to the presence of bridged micelles resembling somehow a percolated structure. These processes were characterized by three different relaxation regimes as a function of temperature: a slow mode in which the solute exchange is dominated by a fragmentation mechanism at diluted copolymer concentrations; a transition regime, where fragmentation and fusion mechanisms are present and compete each other independently of copolymer concentration; and a final fast mode after the percolated structure is formed, in which the fusion mechanism seems to be predominant. This view is a consequence of the behavior observed from the solute exchange dynamics in terms of fluorescence temporal variations of the used probe under different solution conditions. Moreover, the magnitudes of the kinetic constants of each underlying mechanism were derived together with their activation energies, confirming that the described behavior also depends on the copolymer structure and, in particular, on the PO/EO ratio when compared to observations previously made with structurally-related copolymers such as Pluronic P103 (EO17PO60EO17) or P104 (EO27PO61EO27), which exhibits a more hydrophobic balance. 2. EXPERIMENTAL SECTION 2.1. Materials. The triblock copolymer EO57PO45EO57 was synthesized by oxyanionic polymerization, as previously reported.45,49,50 EO57PO45EO57 possess a Mn of 7680 g mol-1 (by NMR) and a polydispersity (Mw/Mn) of 1.07 (Mw derived from GPC data), 7 ACS Paragon Plus Environment


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with a EO content of 65%. The micellar characteristics of the copolymer can be found in Table 1.

Table 1: Micellar Properties of copolymer EO57PO45EO57 Dynamic light scattering (DLS)

Static light scattering (SLS) T (°C)

10-5 Mw (g mol-1)

Nagg

δt

rt (nm)

CMC (g dm-3)

rh (nm)

35 45

0.55 1.10

6.7 13

2.6 2.8

3.8 4.8

22 0.1

10.5

Mw = mass-average molar mass, Nagg = average association number, rt = thermodynamic radii , rh = hydrodynamic radii, δt thermodynamic expansion factor. Data taken from ref. 45.

Aqueous copolymer solutions were prepared by mixing the copolymer with double deionized water under gentle agitation at room temperature for 24 h. The fluorescent probe, a hydrophobic pyrene derivative, 1-pyrenyloctadecanone (PyC18, C34H44O), was prepared via a Friedel−Crafts acylation of pyrene with stearoyl chloride in dichloroethane in the presence of aluminum chloride (AlCl3).51 Like pyrene or ethylpyrene, this probe is characterised by a Poisson distribution in aqueous solutions of nonionic micelles.20,29,42

2.2. Dynamic Light Scattering. Dynamic light scattering (DLS) measurements were performed in a Malvern Zetasizer 5000 apparatus equipped with a 7132 multibit correlator and multiangle goniometer. The light source was a He−Ne 5mW laser with a wavelength of λ=632.8 nm. The scattering intensity was measured through a 400 µm pinhole. DLS measurements were generally carried out at an angle θ =90° to the incident beam, except for dissymmetry ratio ones, for which angles at 45 and 135º were used. For DLS, 8 ACS Paragon Plus Environment

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the correlation functions were analysed by the CONTIN method to obtain the intensity distributions of decay rates ().52 From the decay rate distributions, the apparent diffusion coefficients [Dapp = () /q2, q = (4πns/λ)sin(θ/2)] were derived, with ns as the solvent refractive index. Values of the apparent hydrodynamic radii (rh, radius of the hydro-dynamically equivalent hard sphere corresponding to Dapp) were calculated from the Stokes−Einstein equation:

=

()

(1)

where is the Boltzmann constant, T the temperature of the sample and η is the solvent viscosity. For DLS measurements, a polymeric solution of EO57PO45EO57 (10 mg/mL) was directly filtered into the cleaned scattering cell and each experiment was repeated at least five times. The temperature was measured in steps of 2ºC. and solutions were left 15 min in each to reach thermal equilibrium. 2.3. Kinetic Experiments. For kinetic experiments, a solution of 10 mg/mL of the copolymer was mixed with a small amount of PyC18 and vortexed. The incorporation of the pyrene derivative within micelles was carried out by heating the mixed solution above its cloud point (ca. 150 ºC) in a oil bath followed by cooling down to room temperature. This process was repeated at least five times. Then, the solution was filtered to remove any possible aggregates. Fluorescence measurements were carried out with a Jobin Yvon SPEX Fluorolog III spectrometer in the S/R mode. For exchange kinetics measurements, two solutions were mixed in the sample chamber of a home-built stopped-flow injector with a dead time of 2 ms. In each injection, 0.35 mL of a solution containing PyC18 was mixed with 0.35 mL of a pure copolymer solution (with concentrations ranging from 10 9 ACS Paragon Plus Environment


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to 80 mg/mL). For emission spectra and time-scan kinetics experiments, the excitation wavelength was λex=346 nm, whilst the excitation spectra were monitored at emission wavelengths of λem=375 nm for monomers and λem=480 nm for excimers, respectively. All decay profiles were fitted to an exponential function. Experiments were carried out at a temperature interval from 37 to 70 °C. Each experiment was repeated at least eight times, and the lifetimes of the individual decays from these runs were averaged. 2.4. Transmission Electron Microscopy. Prior imaging, samples were left in a oven at the requested temperatures for 24 h to ensure the formation of the structures. To acquire the images, a drop of 5 µL of the micellar solution was applied to previously heated carbon-coated copper grids, blotted, washed, negatively stained with 2% (w/v) of phosphotungstic acid, air dried, and then examined with a JEOL JEM 1011 (Japan) transmission electron microscope at 120 keV. 2.5. Rheometry. Rheological characterization was carried out using a controlled stress AR2000 rheometer (TA Instruments, DE) with Peltier temperature control. Samples were investigated using cone−plate geometry (cone diameter 40 mm, angle 0.5°) and a solvent trap to maintain a water-saturated atmosphere around the sample cell to avoid evaporation. Experiments were carried out in oscillatory shear mode, with the strain amplitude (A) maintained at a low value (A < 0.5%) by means of the auto-stress facility of the software. This ensured that measurements were in the linear viscoelastic region. The temperature dependence of storage (G') and loss (G″) moduli was measured by temperature sweep experiments in oscillatory simple-shear mode, under A = 0.5% and f = 1 Hz. A step of 1 ºC/min was used. Measurements were performed from 30 to 70 ºC, with an accuracy of ± 0.1 ºC. Experiments were repeated three times for each concentration. 10 ACS Paragon Plus Environment

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3. RESULTS AND DISCUSSION 3.1. Characterization of EO57PO45EO57 micellar dynamics In order to monitor the formation of polymeric micelles, light scattering measurements were performed. Figure 1a shows the temperature dependence of scattered light by a 10 mg/mL aqueous solution of copolymer EO57PO45EO57 in the temperature range from 15 to 70 ºC. The intensity of scattered light was rather low at temperatures below 32 ºC denoting the presence of only monomeric polymer chains in solution, in agreement with previous works.45 The observed larger and progressively decreasing rh values at this range stemmed from the presence of some aggregates composed of non-solubilised residual polymeric chains from the synthetic process (especially noted when the samples were not filtered). Above 32 ºC, the scattered intensity started to steeply increase indicating the formation of polymeric micelles with a constant hydrodynamic radius of ca. 9-10 nm. A continuous increment of the scattered light took place up to 55 ºC and, then, slightly leveled off. This intensity increase can be a consequence of the growing number of micelles formed in solution and a certain growth of their corresponding aggregation numbers; nevertheless, the existence of water dehydration effects of the micellar polymeric core results in almost negligible effects on micellar size (1 nm in 30 ºC) or their population size distribution, which remains single and monodisperse53 (Figure 1b). In addition, the quasi plateau region observed from 54 ºC (the transition temperature, Tt) might be related to a possible rearrangement of polymeric micelles or a morphological transition upon temperature increases, as also observed for Pluronics P10354 and F68.55



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Figure 1: a) Temperature dependence of the hydrodynamic radius, rh () and the scattering light intensity at 90º () for a 10 mg/mL aqueous solution of copolymer EO57PO45EO57. b) Population size distribution of 10 mg/mL copolymer EO57PO46EO57 at 40°C. In order to put some more light into this issue, the variation of the dissymmetry factor, i.e. the ratio between the scattered intensity at 45° and 135°, with temperature was analysed. Owing to interference effects caused by reflection at different points on the particle surfaces, the scattered light would be of different intensity when measured at different scattering angles. In particular, if this ratio is nearly constant particles are usually spheres or spheroids, whilst when it changes this can be an indication of the formation of other structures such as elongated particles. Figure 2 shows that the dissymmetry ratio effectively exhibits certain temperature decrease below the CMT and around Tt, which suggests micelle formation and certain changes in micelle arrangement/morphology, respectively. For temperatures between the CMT and Tt, no angle dependence on the scattered intensity was detected in agreement with the presence of spherical micelles in solution. To further confirm the possible existence of a conformational transition above Tt temperature scans of G' and G'' at f = 1 Hz and temperatures between 37 ºC and 70 ºC at different copolymer concentrations (10 and 80 mg/mL) were performed. 12 ACS Paragon Plus Environment

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Figure 2. Temperature dependence of scattered light () and dissymmetry factor, I45°/I135°, () for a 10 mg/mL aqueous EO57PO45EO57 solution. I45° and I135° are the scattering intensities measured at 45° and 135°, respectively. The dashed lines are only to guide the eye.

From Figure 3, it can be observed that at 40 ºC (below Tt) copolymer micellar solutions behave as an unstructured fluid (i.e. sols, with G′ < 10 Pa and G″ > G′). When the temperature is raised above 51 ºC at 10 mg/mL (in agreement with dissymmetry ratio data) and 47 ºC at 80 mg/mL a more viscous fluid starts to be formed. This fluid is characterized by 10 < G´< 1000 Pa and G′ > G″, i.e. a soft gel adopting Hvidt et al. notation,56 being the transition more abrupt for the most concentrated solution. A more viscous fluid developed from a sol solution should be originated from weak attractions of spherical micelles in water at elevated temperatures, where the solvent is poorer for the micelles. The transition from sol to viscous fluid may well occur when aggregates of spherical micelles would reach a percolation threshold yielding sufficient structure to cause a characteristic rheological effect,57,58 hence, also affecting the scattered light. Finally, a hard or immobile gel (arbitrarily defined by G′ > G′′ and G′ > 1000 Pa at f = 1 13 ACS Paragon Plus Environment


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Hz) is observed for temperatures above ca. 54 and 56 ºC at 10 and 80 mg/mL, respectively, probably associated with a worsening of the solvent environment as the temperature increases, thus compressing the EO-block corona.59,60

Figure 3. Temperature scans for a 10 mg/mL aqueous EO57PO45EO57 solution () and a 80 mg/mL () at f=1 Hz. In a) the storage moduli G' is showed and in b) the loss moduli G". The solid lines are only to guide the eye.

TEM pictures of polymeric micellar solutions (10 and 80 mg/mL) below and above their respective transition temperatures corroborated this picture (Figure 4). Below Tt, well-defined spherical micelles are formed whereas above Tt larger micelles probably as a result of monomer insertion and/or micellar fusion,13 and strings of micelles resembling a percolating structure can be observed. In addition, the observed micellar strings at 10 mg/mL are thinner than at 80 mg/mL probably as a consequence of a decreased number of micelles in solution. Despite some caution is needed 14 ACS Paragon Plus Environment

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considering the procedure followed for TEM imaging, it is clear that a change in the micellar arrangement is observed as a result of the change in solvent quality and polymer dehydration as the temperature raises. Hence, Tt might be interpreted as a micellar growth temperature (MGT).54 The relative hydrophilicity of EO57PO45EO57 might favor the formation of this “pseudo-percolated” structure while precluding a true shape transformation from spherical to rod-like micelles as temperature raises as observed for more hydrophobic polymers as Pluronics P105, P104 or P103, which possess larger PPO/PEO ratios and are much polydisperse.61 In fact, the observed presence of a small population with larger sizes and a more hydrophobic character in these copolymers might act as a reservoir for favoring the shape transformation.

Figure 4. TEM images of 10 mg/mL at 40 ºC (a) and 80ºC (b); and 80 mg/mL at 40ºC (c) and 80 ºC (d) copolymer EO57PO45EO57 aqueous solutions. 15 ACS Paragon Plus Environment


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3.2. Characterization of the exchange process dynamics In order to study the solute exchange dynamics, it was firstly necessary to determine the optimal probe solubilization concentration to ensure a Poisson´s distribution of the probe inside micelles.13,18,19 In this regard, the excimer/monomer intensity ratio (IE/IM) is a very sensitive parameter to determine the distribution of a probe inside micelles. Here, the fluorescent hydrophobic molecule 1-pireniloctadecanone, C34H44O (or PyC18) was used and incorporated to a 10 mg/mL EO57PO45EO57 block copolymer micellar solution13,18,19,42 Poisson´s distribution of the probe cargos would be characterized by a linear increase in the excimer/monomer intensity ratio (IE/IM) with the number of probe molecules within the micelles, ,62 as =[PyC18]/([copolymer]-CMC)

(2)

where [PyC18] is the dye concentration and [copolymer] is the copolymer concentration. Namely, whenever this linearity is kept ( < 0.5) a random Poisson distribution of PyC18 molecules in the polymeric micelles should be obtained.18 Thus, the fluorescence spectra of probe-loaded copolymer micellar solutions were characterized by a strong excimer emission at ca. 480 nm denoting that the probe molecules are very close one each other, for example, micelles bearing two or more molecules in their core. To obtain the average number of fluorescent probe molecules per micelle an exchange probe kinetic experiment was performed by diluting the dyeloaded micelles with solutions of unloaded polymeric micelles at the same concentration (10 mg/mL). The mean occupancy, in this case, was = 0.02. Upon mixing of a PyC18-loaded copolymer solution with empty polymeric micelles a broad


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excimer emission at 480 nm still emerges at short incubation times (Figure 5a); however, as incubation proceeds IE exponentially decreases while IM at ca. 375 nm rises, hence, pointing to the inclusion of a single molecule in each polymeric micellar core (Figure 5b). Hence, the existence of a probe exchange mechanism between polymeric micelles is further corroborated.

Figure 5: a) Emission spectra (λex = 346 nm) of PyC18 solubilized in aqueous solutions of E57P45E57 micelles. The spectrum labeled as “prior to exchange” corresponds with the PyC18 loaded and unloaded polymeric micelles mixed solution at 0 min. The spectrum labeled as “after exchange” refers to the mixed solution in equilibrium after 40 min. b) Time-scan experiments monitoring the increase in monomer emission IM (λem= 375 nm) and the decrease in excimer emission IE (λem= 480 nm) after stopped-flow mixing of a solution of PyC18-loaded EO57PO45EO57 micelles with empty micellar EO57PO45EO57 solution of identical concentration.



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The exponential decrease of the number of micelles bearing initially two or more of probe molecules, P(t), upon incubation is characterized by a pseudo-first-order rate constant, kobs, containing contributions from the various exchange processes described above. When the fluorescence decay behavior is consistent with a Poisson-distribution model, IE and IM are proportional to the fraction of micelles that contain two molecules inside. Once determined the PyC18 distribution inside micelles, the polymeric micellar dynamics was analyzed. To do that, different solutions of PyC18-loaded micelles in equilibrium were mixed with an excess of unloaded polymeric micelles in the chamber of a stopped-flow injector at different temperatures (between 37 and 70 °C) and micelle concentrations (from 10 to 80 mg/mL) in order to determine the main probe exchange mechanisms. As the probe molecules are insoluble in water, probe exchange via water diffusion is unlikely to occur, so it is assumed that the exchange only can take place by means of micellar fusion (Scheme 1b) or fragmentation (Scheme 1c).18


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Figure 6. Time-scan experiments monitoring the decrease in excimer emission IE (λem= 480 nm) after mixing a solution of PyC18-loaded EO57PO45EO57 micelles with a solution of unloaded EO57PO45EO57 micelles of similar concentration a) at c = 10 mg/mL and different temperatures (45 ºC, 50 ºC or 60 ºC); and b) at different concentrations (10 mg/mL, 30 mg/mL or 80 mg/mL) and constant temperature (45 ºC). λex= 376 nm.

As soon as the micelles begin to collide, break and share their content the number of molecules in each micelle is expected to be either zero or one, thus decreasing the probe emission intensity until reaching the equilibrium state. This can be observed in Figure 6, in which time-scan experiments of excimer emission show that it progressively disappears as the solute probe exchanges upon addition of an excess of empty micelles. It is also observed that a dependence of IE with the copolymer concentration is detected (Figure 6b): As copolymer concentration increases micelles are closer one each other and, thus, whenever an exchange of molecules occurs, the time required for such is lower because the probability of micelle collision is greater. Moreover, a strong temperature dependence of IE is also observed (Figure 6a), specially in the range between 45 and 50 ºC, which confirms the occurrence of a slow to fast transitional dynamics at the measured concentration (10 mg/mL), which is in agreement with the formation of percolated structures noted by light scattering, rheology and TEM data. It is well-known that molecular Brownian diffusion is faster as the temperature increases63 and, hence, the probability of micellar collisions is higher; however, the observed transition does not monotonically increases with temperature denoting the existence of additional processes as the observed micellar rearrangement in the form of micellar strings (“pseudo-percolation”). 19 ACS Paragon Plus Environment


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To analyze in more detail the observed temperature- and concentrationdependency of the exchange dynamics, the relaxation rates derived from excimer emission decreases were calculated following the modellization of Infelta and Gratzel.64-66 Under our experimental conditions (low and large excess of empty micelles), the fraction of micelles P(t) bearing a pair of PyC18 molecules decreases exponentially with time, following a first-order kinetics characterized by a relaxation rate kobs which contains contributions from all three exchange mechanisms:20 IE ∝ P(t) = P(0) exp(-kobs·t)

(3)

kobs= kexit + kfragmentation + kfusion [Micelle]

(4)

where kexit is the rate constant for the exit-reentry mechanism (Scheme 1a); kfragmentation corresponds to the fragmentation rate constant related to the fracture of micelles into smaller parts (Scheme 1c); and kfusion refers to the fusion rate constant of loaded micelles with an empty one and their subsequent fission in two (Scheme 1b). The fluoresence decay profiles were fitted to a mono-exponential function. As a result, a linear relationship between kobs and the micellar concentration could be observed at each measured temperature.


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Figure 7. Plots of kobs against micellar concentration of copolymer EO57PO45EO57 in the temperature interval from 37 to 70 ºC. Three regimes can be distinguished: a "slow " dynamics regime below the dashed line, a transition regime between the dashed and dotted line, and a "fast" dynamics regime above the dotted line. Dotted and dashed lines are only to guide the eye. Inset: Plots of kobs against [micelles] of copolymer EO57PO45EO57 at 50ºC ("fast" dynamics) are shown.

In Figure 7, three different kinetic regimes for copolymer EO57PO45EO57 can be observed as a function of the solution temperature: A slow dynamical zone below 45 ºC, a fast one above 50 °C, and a transition area between 45 and 50 ºC. The slow dynamical region is characterized by an almost negligible influence of micelle concentration in the relaxation rates. Moreover, kobs exhibits very little temperature dependence within this region. A similar behavior is observed for the fast dynamic regime at low micelle concentrations (see inset in Figure 7). In contrast, at higher micelle concentrations, the temperature influence on the relaxation rates is much stronger, with kobs being largely enhanced (for example, the slope is one hundred-fold higher at 70 ºC than at 50 ºC). It 21 ACS Paragon Plus Environment


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can be observed that the fast dynamical mode takes place at Tt, that is, at the temperature at which micelles starts to grow and form pseudopercolated structures. Thus, the diffusion of the fluorescent probe molecules may be faster through the “channels” formed by the “bridged” micelles present in such structures. In this regime, kobs increases as the copolymer concentration becomes higher. This can be a direct consequence of the development of more and larger “bridged” micelles, facilitating probe exchange along the interconnected structures, in agreement with rheological and microscopy experiments (see Figures 3 and 4). On the other hand, it is worth mentioning that the relaxation rates calculated from the fits of the individual decays varied from 4·10-4 to 6·10-3 s-1, much shorter than those found for other Pluronic-type copolymers studied.25,66 Several parameters can influence the exchange rate, in particular, the PO/EO ratio, the interfacial tension between the hydrophobic block and the solvent,67 and the glassy nature of the micellar core.68,69 In particular, we consider that the relatively low PO/EO ratio of the present copolymer is responsible for the observed fast relaxation rates if compared, for example, to those of structurally-related Pluronic block copolymers P84 (EO19PO43EO19) bearing a similar PO length. For the latter copolymer, the shorter hydrophilic blocks involves a much packed inner hydrophobic core to avoid interaction with water, thus, increasing the friction of the chains and reducing their mobility. Similar conclusions can be also inferred when comparing the relaxation times previously obtained for Pluronic F88 (EO103PO39EO103).70 Solute exchange can, in principle, occur by the three mechanisms indicated in Scheme 1. As mentioned previously, PyC18 solubility in water is so low that kexit can be considered negligible. Then, considering that the exchange of an aqueous insoluble


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probe as PyC18 makes its transition from one micelle to another via water diffusion very unlikely, equation (4) remains as follows: kobs = kfragmentation + kfusion [Micelles]

(5)

Therefore, the exchange mechanism described here by kobs consists of two processes: a second-order process with linear dependence of kobs on [Micelles], and a first-order process with a rate constant independent of the empty micellar concentration.20,29,41 To determine these rate constants, it is previously required to establish the micellar concentration in solution, which can be obtained as71 [Micelle] = (C-CMC(T) )/ Nagg (T).

(6)

with Nagg(T) denoting the temperature dependence of the micellar aggregation number obtained through fluorescence quenching measurements72,73 (see Table 2). Here, it is necessary to bear in mind that commercial Pluronic copolymers have larger Nagg as should correspond to their stoichiometric formula due to their intrinsic polydispersity in contrast to monodisperse lab-synthesized PEO57PPO45PEO57 copolymer. In addition, it is necessary to stress that larger aggregation numbers can enhance the fraction of micelles sufficiently to undergo fusion, i.e. the aggregation number can influence kfusion.41 Table 2. Temperature dependence of aggregation number of EO57PO45EO57 micelles. Temperature Aggregation number (ºC) (Nagg) 35 6.7 37 8.1 40 10 45 13 47 14 50 16 55 19 60 23 23 ACS Paragon Plus Environment


65 70

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26 29

kfragmentation and kfusion rate constants were calculated by fitting kobs shown in Figure 7 at different temperatures following eq. 4. Values for kfragmentation and kfusion are shown in Table 4. It can be noted that kfusion increased by ca. two orders of magnitude as the temperature rises in a concentration-dependent manner. On the other hand, kfragmentation increases until ca. 50 ºC (close to Tt) and, then, decreases independently of the micellar concentration. Table 3. Fragmentation and fusion rate constants at different temperatures calculated by the linear fitting of kobs against micellar concentration of copolymer EO57PO45EO57. Temperature (ºC) 37 45 47 50 55 60 65 70

10-3 kfragmentation (s -1) 4.4 ± 0.1 5.3 ± 0.1 6.3 ± 0.6 11.7± 0.2 11.0± 0.3 8.5 ± 0.4 5.0 ± 0.1 2.0 ± 0.1

kfusion (M-1 s-1) 2.2 ± 0.1 3.3 ± 0.1 7.4 ± 0.2 15.1 ± 0.5 24.5 ± 1.2 47.3 ± 1.9 135 ± 8.7 226 ± 11

Figure 8 shows the Arrhenius plots of both rate constants and their behavior as a function of temperature (see Figure 8 insets). As seen in Figure 8a and Table 3, there exists a change in the behavior of kfragmentation with temperature. This increases until 50 ºC and, then, it linearly decreases at larger temperatures. This inflection point matches with the temperature interval at which the dissymmetry factor starts to decrease (Figures 1 and 2). Hence, this abrupt change can be related to the observed transition from pure spherical micelles to “bridged” ones (“pseudo-percolated”. It is obvious that negative


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acrivation energies involve the ansebce of an energetic barrier to preclude structures). Conversely, an exponential relationship can be derived as observed from the linear Arrhenius plot, log (kfusion)∝(1/T) in Figure 8b. No changes in the slope are found at 50 ºC in contrast to kfragmentation. Thus, in this particular case the fusion process is not abruptly influenced by the observed structural rearrangement, being linearly enhanced as temperature rises. In summary, kfragmentation is not a concentration-dependent constant, opposite to kfusion, which is influenced by both temperature and micelle concentration. In the slow dynamic regime kfragmentation ∼ kfusion, at large polymer concentrations, which implies that both mechanisms are in competition; However, for smaller micelle concentrations, kfusion < kfragmentation, and the fragmentation process seems to be predominant in a diluted polymeric solution below Tt where only spherical micelles are present. In the transition regime, both processes are in competition, independently of the micellar concentration. Conversely, when the fast dynamical regime is reached, the solute exchange mechanism is characterized by micellar fusion provided that kfusion is the main contribution to kobs, in particular, at elevated micelle concentrations and temperatures. From Figure 8 apparent activation energies (Ea) can be calculated using the Arrhenius equation. These energies, together with the pre-exponential Arrhenius factor or frequency factor (A) (a measure of the probability of favorable collisions), are shown in Table 4. It can be observed that negative values of Ea of similar magnitude are achieved for both process below Tt. Negative activation energies involves the absence of an energetic barrier able to impede a determined kinetic process to occur; hence under these conditions both mechanisms, fragmentation and fusion, are favorable to take place and are in competition. Nonetheless, the fragmentation process exhibits an abrupt change at Tt (Figure 8a). In the fast regime (T > Tt ºC), kfragmentation and kfusion are 25 ACS Paragon Plus Environment


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related to two apparent activation energies of similar magnitude but different sign, i.e. Ea corresponding to the fragmentation process becomes positive, and thus the most probable process here is then the fusion of micelles since no an energy barrier must be overcome. In this regard, micelles should start to bridge to form somehow a string-ofbeads/percolated-like structure above Tt, which can be considered as a pseudo-fusion process (Figure 4b,d). This kind of structure is more probable as the temperature increases, in agreement with the enhancement of kfusion and the decrease of kfragmentation values with temperature. This behavior is in strike contrast, for example, with that observed for Pluronics P103 and P10 which undergo a morphological sphere-to-rod transition at their corresponding Tt; for the latter copolymers, kfragmentation diminishes with the reciprocal temperature until reaching Tt, (Ea > 0) and, then, increases. Besides, kfusion also has a clear inflection point at which its slope changes to positive becoming fusion an unfavourable process. Thus, for P103 and P104 the fragmentation becomes dominant close to equilibrium when rod-like micelles are formed as a result of instabilities along the rod surface.13-14 An alternative view of the observed kfragmentation, kfusion, and Ea values can be considered on the basis of the copolymer HLB value and the interfacial tension between the PO block and the solvent, At T< Tt (formation of spherical micelles), the energetic cost for a copolymer with a PO/EO ratio > 1 (considered as hydrophobic-prone such as P103 or P104, for example) to form a micelle is more favorable than a more hydrophilic one, such as EO57PO45EO57. Consequently, the energy barrier to fragment a micelle is lower for the latter copolymer and the fragmentation mechanism can be more probable as a consequence of better polymer-solvent interactions.


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Figure 8: Arrhenius plot of a) first-order rate constant, kfragmentation, b) of second-order rate constant, kfusion, versus inversed temperature. Insets: Rate constants as a function of temperature. The dashed line is only to guide the eye.



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Table 4: Apparent activation energies (Ea) and natural logarithm of the pre-exponential Arrhenius factor for fragmentation an fusion of the micelles, calculated from experimental data in Figure 8. Ea (KJ/ml) ln(A) (ln[s-1]) 125 ± 27 -50,2 ± 9,7 Micelle Fragmentation ( T >Tt ºC) 34,2 ± 3,2 Micelle Fragmentation ( T < TtºC) -103,4 ± 8,6 -127,0 ± 6,4 50,0 ± 2,4 Micelle Fusion Conversely, for copolymers P103 or P104 with a greater PPO core (PO/EO >1), hydrophobic forces dominate over the hydrophilic PEO-water interactions, maintaining a more compact structure, (Ea(fragmentation) > 0). Regarding kfusion, it has been shown that a fusion process is primarily a consequence of coronal interactions and, thus, favored for longer PEO units.24,28 Taking this into account, since EO57PO45EO57 has longer EO segments than P103 (EO17PO60EO17) and P104 (EO27PO61EO27), micellar fusion should be more favored in the former copolymer. On the other hand, at T > Tt strong water dehydration takes place as well as a progressive enhancement of PO hydrophobicity. Thus, micelle formation is strongly favored and hence, the energy barrier for fusion also decreases. Thus, fusion is started to be largely favorable independently of the length of EO segments (Ea(fusion) < 0). For the present EO57PO45EO57 copolymer this fact is reflected through the formation of “pseudopercolated” structures composed of bridged spherical micelles, and the probe molecules would diffuse through the “interconnected pores” of the interconnected micelles; hence, this phenomenon should be considered as a pseudo-fusion process Conversely, kfragmentation at temperatures above Tt does not influence the exchange kinetics becoming less favorable.


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The observed behavior is rather different when compared to that observed for other more hydrophobic Pluronic copolymers such as P103 and P104, which form selfassemble into rod-like micelles above Tt, as mentioned previously. It was reported that kfragmentation largely contributes to the exchange kinetics for these two copolymers, as opposed to EO57PO45EO57. As mentioned previously, it was argued that surface instabilities that may appear in the long axis of the micelle, contributing to decrease the energy barrier of fragmentation (Ea Tt kfragmentation and kfusion were related to two apparent activation energies of similar magnitude but different sign, i.e. Ea corresponding to the fragmentation process became positive, and the only favorable process here was then the fusion of micelles, in agreement with the formation of the bridged structure.

ACKNOWLEDGMENTS 30 ACS Paragon Plus Environment

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This work was supported by AIE (funding through Project MAT2016-80266-R and ERDF funds). E.V.A. is

also grateful to the Spanish Ministerio de Economia y

Competitividad for their FPU fellowship.

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