J. Phys. Chem. B 2009, 113, 2773–2782
2773
Hydrosoluble Cyclodextrin/Poloxamer Polypseudorotaxanes at the Air/Water Interface, in Bulk Solution, and in the Gel State Luis Nogueiras-Nieto,† Carmen Alvarez-Lorenzo,† Isabel Sandez-Macho,‡ Angel Concheiro,† and Francisco J. Otero-Espinar*,† Departamento de Farmacia y Tecnologia Farmaceutica and Departamento de Quimica Fisica, UniVersidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain ReceiVed: NoVember 06, 2008; ReVised Manuscript ReceiVed: December 18, 2008
The interactions between poloxamer 407 (Pluronic F127, PF127) and two hydrophilic derivatives of β-cyclodextrin, i.e., hydroxypropyl-β-cyclodextrin (HPβCD; molar substitution (MS) 0.65) and methylatedβ-cyclodextrin (MβCD; MS 0.57), were characterized by means of surface tension measurements, π-A isotherms, isoperibol microcalorimetry, 1H NMR, and rheometry. An effective complexation of poloxamer with the two CDs was evidenced as a change in the surface pressure of the π-A isotherm of PF127 on a subphase of CD solution, with a positive excess being observed at the expanded region and a negative excess at the collapsed region. Such changes indicated that when the CD lies with the main axis perpendicular to the interface at low pressure no complexation occurs, but as the pressure increases and the CDs eventually change their arrangement to be with the main axis parallel to the interface, the amphiphilic copolymer can form polypseudorotaxanes. Addition of CDs to PF127 micellar solutions led to the exothermic rupture of micelles, a shift in the cmc toward higher values, changes in the chemical shifts of H3, H4, and H5 of MβCD and of the methyl groups of PF127, and an increase in the gel temperature. The interaction was stronger between poloxamer and MβCD, compared to HPβCD, with the stoichiometry of the polypseudorotaxanes being preferably ca. 1:20 in both cases. SEM images revealed formation of nanorods of stacked polypseudorotaxanes. Complexation with a high affinity constant between unimers and CDs in bulk solution was also evidenced by competitive displacement of methyl orange. Feasible structural models of the PF127:CD polypseudorotaxanes at both the air-water interface and in the bulk solution are proposed. Introduction Cyclodextrin (CD) complexation and micellar solubilization are common approaches for increasing drug solubility and stability in aqueous media.1,2 The combination of both mechanisms can lead to a synergistic effect using lower proportions of each solubilizing agent.3-6 However, the magnitude of the synergism strongly depends on the nature and the proportion of both the CD and the surfactant. An antagonist effect could even occur if the surfactant competes with the drug for complexing with the CD, where the drug solubility is lower than expected for additive contributions.7 Surfactant-CD complexation results in fewer CD cavities and fewer micelles available for hosting the drug. On the other hand, addition of CD to formulations containing self-assembling components (surfactants or amphiphilic drugs) may break the micelles and alter interface-related features, e.g., the physical stability or the foaming efficiency.8,9 Stringent efforts are being carried out to develop models able to explain and predict the dependence of the features of the formulation on the relative concentration of CD and surfactant when combined.4,10 Compared to common surfactants, amphiphilic polymers capable of self-aggregation are advantageous for pharmaceutical applications owing to the lower critical micellar concentration (cmc) and the higher stability of the polymeric micelles against dilution.2 However, * Corresponding author. Fax: 34-981547148. E-mail: francisco.otero@ usc.es. † Departamento de Farmacia y Tecnologia Farmaceutica. ‡ Departamento de Quimica Fisica.
knowledge about the complexation of amphiphilic polymers with CD is still limited. Polymers can be threaded through the macrocyclic structure of various CD molecules forming necklace-like supramolecular complexes called polypseudorotaxanes,11 which are very promising for a wide range of biomedical applications.12,13 It is generally accepted that the complex formation between permethylated CD and a guest polymer, such as polytetrahydrofuran, poly(ε-caprolactone), poly(propylene glycol), and poly(acrylic acid), proceeds in three steps: (i) the polymer is included into the CD cavity aided by ultrasonication to generate hydrophilic complexes, (ii) the threaded CD molecules are partially stacked along the polymer axis upon heating, and (iii) the intermolecular hydrophobic interactions between the CD molecules in the complexes cause the precipitation of the polypseudorotaxane.14-16 Amphiphilic poly(ethylene oxide)/poly(propylene oxide) block copolymers (PEO-PPO-PEO; i.e., poloxamer or Pluronic) can penetrate the cavity of some CDs as revealed by the appearance of turbidity and changes in the size of the macromolecule aggregates and in the gel temperature.8,17-19 Nevertheless, formation of soluble polypseudorotaxanes has been less studied5,6,8 and essential aspects regarding their stoichiometry, structure, and practical interest in the pharmaceutical field have not been fully evaluated yet. The aim of this work was to gain insight into the complexation of Pluronic F127 (PF127), which has been approved for parenteral formulations with two hydrophilic derivatives of β-cyclodextrin (hydroxypropyl-β-cyclodextrin (HPβCD) and methylated-β-cyclodextrin (MβCD)) also approved for internal
10.1021/jp809806w CCC: $40.75 2009 American Chemical Society Published on Web 02/05/2009
2774 J. Phys. Chem. B, Vol. 113, No. 9, 2009 use. The potential interactions of these three common excipients may notably modify the performance of the drug delivery systems. HPβCD and MβCD are capable of forming complexes with a wide range of drugs, and the solubility in water of these cyclodextrins and their complexes is much greater than that of βCD and its corresponding complexes. These features lead to water-soluble polypseudorotaxanes,5,6 different from the previously described water-insoluble ones,18 which opens a new scope of applications for these supramolecular structures, avoiding intricate approaches for solubilization in aqueous media. Furthermore, compared to βCD, which is poorly surface active and does not adsorb at the air-water interface, we verified that HPβCD and MβCD (as reported for other methyl derivatives20) can form monolayers and therefore can interact with amphiphilic copolymers at the interface. Thus, in the present study a wide range of complementary techniques were applied for a detailed characterization of HPβCD- and MβCD-based polypseudorotaxanes at the air-water interface, in the bulk solution, and in the temperature-induced gel state. The information obtained prompted us to propose a feasible model of interaction at the interface and of supramolecular aggregation in solution. An additional aim of the work was to explore the ability of PF127 to displace a host molecule from the CD cavity using methyl orange as a probe.21,22 Experimental Section Materials. Pluronic F-127 (PF127, poloxamer 407) and methyl orange were from Sigma-Aldrich (Madrid, Spain). Hydroxypropyl-β-cyclodextrin (HPβCD with a molar substitution of 0.65 and Mw 1399 Da, Kleptose HPB) and methylatedβ-cyclodextrin (MβCD with a molar substitution of 0.57 and Mw 1191 Da, Kleptose Crysmeb) were from Roquette Laisa (Barcelona, Spain). Ultrapure water was obtained by reverse osmosis (MilliQ, Millipore, Madrid, Spain). All other chemicals were of analytical grade. Surface Tension Measurements. PF127/CD dispersions were prepared by the addition, under stirring, of adequate amounts of PF127 to 5-10 wt % CD aqueous solutions. The samples were left to stand at room temperature for at least 24 h before measurements. Surface tension measurements were made in triplicate at 25 °C by the platinum ring method using a Lauda Tensiometer TD1 (Lauda-Ko¨nigshofen, Germany), applying the density corrections needed. The critical micellar concentration (cmc) of PF127 was estimated as the concentration beyond which the surface tension remained practically constant. π-A Isotherms. The experiments were carried out with a single barrier NIMA 611 (U.K.) surface balance with total area 550 cm2, placed on an antivibration table. The surface pressure was measured with an accuracy of (0.1 mN/m, using a Wilhelmy plate made from chromatography paper (Whatman Chr1, U.K.) as a pressure sensor. Prior to experiments, the trough was cleaned with chloroform and ethanol and rinsed with water. Water or HPβCD or MβCD solutions were used as subphase (475 mL) and the temperature was kept at 25 °C. PF127 solution in chloroform (70 µL, 0.1 mg/mL) was deposited by means of a syringe (Hamilton, USA) and allowed to stand for at least 10 min to ensure complete evaporation of the solvent. The monolayer stability was verified by monitoring the change in surface pressure while holding the area constant. The monolayers were compressed at 15 cm2 · min-1 and the surface pressure, π, was recorded as a function of the area of the monolayer. The π-A isotherms of HPβCD or MβCD when included in the subphase were previously recorded at different times to ensure
Nogueiras-Nieto et al. the attainment of equilibrium before the addition of PF127 to the air-water interface. Titration Microcalorimetry. Calorimetric experiments were performed in triplicate using a Tronac-450 isoperibol microcalorimeter fitted with Tronac FS101 software (Tronac Inc., Orem, UT). In each experiment, water or a CD solution (47.5 mL) was placed in a Dewar reaction vessel, and a relatively concentrated micellar PF127 solution (10 wt %, equivalent to 8 mM), containing CD (0.1-5 wt % equivalent to 0.72-35.7 mM HPβCD and 0.84-42 mM MβCD) or not, was loaded into a 2 mL calibrated buret. The entire assembly was then immersed in a water bath at 310.0 K. After thermal equilibration, the PF127 solution was delivered at a constant rate of 0.3332 mL/ min into the reaction vessel, in which a stirrer mixed the two solutions rapidly. The rise or decrease in the temperature of the system was monitored using a thermistor, and later reproduced using a heating coil in the reaction vessel. The apparent enthalpy was calculated from the applied current and voltage and the heating time. Calibration of the system was assured by titration of tris(hydroxymethyl)aminomethane with HCl. The integral binding heat for the PF127/CD complexation process (Qcomplex) was estimated by subtracting from the measured heat produced by addition of PF127 to the CD solution (Qp), the heat effect due to the dilution/demicellization of PF127
Qcomplex ) Qp - Qd - Qc
(1)
in water (Qd), and the heat effect due to the dilution of CD (Qc):23-25 The final concentration of PF127 in the Dewar vessel was well below that required for gel formation at the temperature of the experiment. Therefore, no interference due to a phase transition would be expected in the estimation of the enthalpy.25 1 H NMR and Molecular Modeling. Spectra of 1:0, 10:90, 5:95, 2:98, 1:99, and 0:1 PF127:CD (MβCD or HPβCD) molar ratio solutions in D2O were recorded in a Bruker AMX 500 (Karlsruhe, Germany) spectrometer at 500 MHz. The molar ratios refer to the whole PF127 macromolecule, not to the repeating units. The concentration of PF127 plus the concentration of CD was 19.1 mM. The signal at 4.7 ppm due to residual solvents (H2O and HDO) was used as internal reference. The molecular geometry of MβCD was constructed using the crystallographic structure of the native βCD as determined from X-ray diffraction data and deposited in the Cambridge Crystallographic Databank. Crysmeb is a mixture of methylated βCDs with a low degree of substitution (from one to seven methyl groups per molecule). A MβCD molecule type with an average of four methyl groups per native βCD unit was used to obtain the molecular model. Methyl substituents were introduced into the oxygens with the higher likelihood of substitution (C2). The structure of a PPO block with 12 monomer unities was drawn using ChemDraw Ultra 11 software (Cambridge Soft Co., Cambridge, MA) and then optimized using HyperChem 7.0 software (Hypercube Inc., Boulder, CO) implemented with an MM+ force field and a Polak-Ribiere minimizer, with a maximum energy gradient of 0.01 kcal Å-1 mol-1.26 Inclusion complexation of this portion of PF127 with MβCD was modeled by manual docking in accordance with the 1H NMR results and energy minimization using molecular mechanics MM+, which supplements the standard MM2 force field by providing additional parameters.26 Transmission Electron Microscopy (TEM). Five-microliter drops of previously filtered (0.45 µm) micellar 5 wt % (4 mM) PF127 or 10 wt % CD (71.5 mM HPβCD or 84 mM MβCD)
Hydrosoluble CD/Poloxamer Polypseudorotaxanes
J. Phys. Chem. B, Vol. 113, No. 9, 2009 2775 TABLE 1: Critical Association Concentration (cac), Critical Micellar Concentration (cmc), and Free Energy of Interaction per Mole of PF127 (∆Gi) in 5 wt % CD Solutionsa cyclodextrin
cac, mM
cmc,mM
∆Gi, kJ · mol-1
HPβCD MβCD
5 × 10-4 5 × 10-4
0.5 1
-17.11 -22.82
a
Figure 1. Surface tension of aqueous solutions of PF127 in the absence and presence of 5 wt % HPβCD or MβCD.
solutions, or mixtures of both solutions, were placed on TEM grids covered with Fomvar film. After 30 s, the excess was carefully removed with a tip of filter paper and a drop of water (5 µL) was added. After 30 s, the excess was again removed; a drop of 2 wt % phosphotungstic acid (5 µL) was added and left for 30 s before removing. The samples were then dried in a closed container with silica gel, and observed using a PHILIPS CM-12 TEM apparatus (FEI Company, The Netherlands). The diameter of the micelles/aggregates was measured with a calibrated scale. Displacement of Methyl Orange from CD cavities. Different volumes of PF127 unimer solution (0.1 mM) were added to 2.5 mL of aqueous solutions containing methyl orange (0.4 mM) and MβCD or HPβCD (4.0 mM). The volume desired was then completed with water up to 25 mL. The displacement of the dye by PF127 from the complex was quantified measuring the increase in absorbance at 460 nm, where methyl orange absorbance was maximum (HP 4250A, Agilent, Germany). Rheometry. The effect of temperature on the loss (G′′) and storage (G′) moduli of PF127 micellar solutions (10-20 wt %, equivalent to 8-16 mM) with or without MβCD (5 wt %, equivalent to 42 mM) was evaluated in triplicate in a Rheolyst AR-1000N rheometer (TA Instruments, Newcastle, U.K.) fitted with an AR2500 data analyzer, a Peltier plate, and a cone geometry (6 cm diameter, 2.1°). The assays were carried out at 0.1 rad/s from 15 to 50 °C with a heating rate of 3 °C/min. Liquid paraffin was placed around the gap to avoid evaporation of the sample during the assay. Additionally, frequency sweep experiments were carried out at 25 °C from 0.05 to 50 rad/s at 0.1 Pa. Results and Discussion Surface Tension. Surface tension plots are commonly used to elucidate surfactant/polymer interactions and could also provide information about surfactant/CD complexation.27 PF127 solutions showed two points of inflection at 0.002 wt % (1.6 × 10-3 mM) and 0.5 wt % (0.40 mM) (Figure 1). These two breaks in the curves have been attributed to changes in the size and shape of PF127 aggregates28 and to the inherent broad molecular distribution of the copolymers.29 The last inflection point was assumed to be the cmc and was coincident with the values previously reported by other authors.30 The surface tension plots of the PF127/CD solutions also showed several regions. HPβCD and MβCD are themselves surface active and caused a decrease in the surface tension of water to 66 and 61 mN/m, respectively. These values are in agreement with those previously reported, which also indicates that, different from typical surfactants, these
The cmc of PF127 in water was 0.4 mM.
CDs monotonically decrease surface tension as the concentration increases.31 Addition of 5 wt % (35.7 mM) HPβCD slightly shifted the surface tension of the PF127 solutions to greater values; the profiles of the solutions with or without HPβCD are almost parallel and show similar cmc’s. This indicates that a few PF127 molecules interacted with the HPβCD and were removed from the air-water interface toward the bulk. More remarkable changes were recorded in the presence of 5 wt % (42 mM) MβCD. A crossover of the surface tension profiles recorded in the presence and the absence of MβCD was observed at 6.3 × 10-4 wt % PF127. This copolymer concentration can be considered the minimum required for interacting with MβCD, i.e., the critical aggregation concentration, cac (Table 1). Above this concentration and up to 1.26 wt % PF127, the surface tension values were greater in the presence of MβCD. Beyond 1.26 wt % PF127, the excess of block copolymer can self-assemble and form micelles. These findings indicate a strong complexation of the block copolymer with MβCD, which causes an important shift in the cmc. The strength of the interaction was estimated as the free energy per mole of
CD-copolymer T copolymer + CDfree T micelle + CDfree ∆Gi ) RT ln(cac/cmc) (2) copolymer using an expression similar to that applied for surfactant-polymer aggregates:32,33 Table 1 illustrates the greater strength of the interactions between MβCD and PF127 compared to HPβCD. In the
cmcapp ) [PF127unimer · CDn] + cmcreal
(3)
presence of CDs, the apparent cmc of PF127 can be estimated as Differing from drug:CD complexes in which the predominant stoichiometry is 1:1, the formation of polypseudorotaxanes usually involves a great number of CD molecules per polymer chain.34 In the case of Pluronic copolymers, the most likely block to form complexes with the CD is the most hydrophobic one, i.e., the PPO. It has been previously found that the stoichiometry of polypseudorotaxanes of Pluronic L61 (POE3-PPO30-POE3) with dimethyl-βCD is 1:18.20 In the case of Pluronics L81, P85, F87, and F88, which contain 39 PO units and 6, 27, 67, and 96 EO units per molecule, respectively, 11 dimethyl-βCD molecules were threaded onto the PPO block,17 while for Pluronic F105 with 28 PO units the number of βCDs was estimated to be ca. 20.35 Considering that, owing to steric restrictions, a maximum of two CD molecules can cover three PO units and assuming a high complex affinity, the greatest number of CDs that could be threaded onto the PPO block of each PF127 molecule is 40. In our case, the CD concentration was set to be of 38.17 mM (i.e., ca. 5.0 wt %), and thus interferences caused by CD self-assembly were discarded.36,37 A minimum of 0.95 mmol/L PF127 would be required to form 1:40 complexes with
2776 J. Phys. Chem. B, Vol. 113, No. 9, 2009 all the available CD molecules. If this were the case, the cmcapp should be 0.95 mM greater than the cmc in the absence of CD. If the complexes had a 1:1 stoichiometry, the shift should be 38.17 mM. The minor changes produced by HPβCD on the surface tension profiles revealed a low tendency to interact with PF127 in aqueous medium. A more intense interaction has been previously reported in acetate, lactate, or phosphate buffers owing to the salting-out effect caused by the ions and the consequent dehydration of the block copolymer.5 Such a decrease in hydrophilicity of PF127 may facilitate the penetration in the HPβCD cavity. In the case of MβCD, the shift in cmc was 0.5 mM, which is lower than that expected if a full complexation occurs (0.95 mM). This imbalance indicates that free CDs are in equilibrium with those forming complexes with PF127, and/or that some unimers remain free or forming complexes with such a stoichiometry that does not significantly alter their surface activity. π-A Isotherms. HPβCD and MβCD are surface active by themselves,31 and we observed that they can indeed form monolayers at the air-water interface. Thus, the surface properties of the systems will depend on the number of PF127 and CD molecules at the interface and on the way they are arranged. Pluronic-CD interactions may cause the drainage of PF127 unimers toward the bulk (owing to an increase in the hydrophilicity of the macromolecule as the PPO block is threaded by CDs), a change in the CD concentration at the water surface, and the appearance of polypseudorotaxanes in the interface. Such complex behavior was evidenced by recording the π-A isotherms of CD aqueous solutions and of PF127 using water or CD solutions as subphase (Figure 2). HPβCD and MβCD are highly soluble in water and require some time to diffuse from the bulk solution to the interface and to provide a constant value of π. After recording the π-A isotherms of the CD solutions at several time intervals, we observed that 1 h was enough to attain equilibrium. A clear dependence of CD concentration on the pressure exerted at the air-water interface was observed (Figure 2A). HPβCD has a higher tendency to move toward the interface than MβCD. Smooth isotherms without plateaus were obtained for both CDs, with a progressive change in the plot slope as the compression went further. The area occupied per molecule of HPβCD and MβCD at the interface was previously estimated to be between 306 and 333 Å2, applying the Gibbs adsorption equation to surface tension data.31 These data are close to the values found for the area occupied per molecule of hydrophobic derivatives of βCD when low π values are applied to the interface.38 Taking into account the dimensions of βCD, a surface of 306-333 Å2 per molecule indicates that the CD molecules are adsorbed with the axis of the cavity perpendicular to the interface. In the collapsed state, a much lower area per molecule (120 Å2) was found, which is attributed to that the hydrophobic derivatives of βCD are positioned with the axis parallel to the interface (Figure 3A). In the cases of HPβCD and MβCD, it is very unlikely that all CDs move to the interface; more probably the concentration of CD at the interface is proportional to that in the bulk as observed when surface tension is recorded.31 The superimposition of 1 and 10 wt % HPβCD profiles suggests that, when the subphase is prepared with 1 wt % HPβCD, the interface is saturated with HPβCD. The same assessment can be made for subphases containing 10 wt % MβCD. Taking into account the total surface of the balance device (550 cm2) and the area occupied per CD unit, roughly a maximum of 1.72 × 1016
Nogueiras-Nieto et al.
Figure 2. π-A isotherms recorded in the absence of PF127 when CD solutions were used as subphases (A), π-A isotherms of PF127 on water (dotted line) or on HPβCD subphase (B), excess surface pressure for PF127 isotherms recorded on HPβCD subphase (C), π-A isotherms of PF127 on water (dotted line) or on MβCD subphase (D), and excess surface pressure for PF127 isotherms recorded on MβCD subphase (E). CD concentrations at the subphase were 0 (a), 1.1 × 10-5 (b), 1.1 × 10-3 (c), 10-2 (d), and 0.1 wt % (e).
molecules of CD at the interface is expected. In the case of 1 wt % CD solutions, this amount of molecules is ca. 1/105 of the total of CD molecules present in the system.
Hydrosoluble CD/Poloxamer Polypseudorotaxanes
J. Phys. Chem. B, Vol. 113, No. 9, 2009 2777 sorption equilibrium at the interface was ensured. Since the exact number of HPβCD or MβCD molecules at the interface in the steady state is unknown, the surface pressure plots were referred to the total area of the balance. In all cases the solutions were prepared to provide an amount of HPβCD or MβCD sufficient to saturate the binding to PF127; e.g., even when the most diluted cyclodextrin solution was used as subphase, the number of HPβCD or MβCD molecules per PF127 macromolecule in the system as a whole was 95. The isotherm pattern evolved as a function of CD concentration in the subphase, from a profile similar to that recorded on water (when a diluted CD solution was used) to a markedly different isotherm when the subphase was a concentrated CD solution. Furthermore, the isotherms recorded on concentrated CD solutions did not show the typical pseudoplateau of PF127 (Figure 2B,D). The changes in surface pressure as a function of the composition of the subphase were plotted to elucidate if PF127 and the CDs interact at the interface or, on the contrary, they coexist as separate phases (Figure 2C,E). If the two components form an ideal mixture, the surface pressure at any given molecular area, A, should be equal to the pressure recorded
πideal,A ) πPF127,A + πCD,A Figure 3. Schematic view of conformational changes of CDs (A), PF127 (B), and CD/PF127 systems (C) at the air-water interface as pressure increases.
The isotherm of PF127 on water clearly indicated that the monolayer is expanded at low surface pressures and undergoes a transition to a condensed film when further compressed (Figure 2B,D). The significant lateral compressibility of the π-A isotherm of PF127 at low surface pressures is related to a relaxed conformation of the copolymer at the interface. When no pressure is exerted, the copolymer lies on the interface with a flat (“pancake”) conformation parallel to the surface plane39,40 and the area occupied is a function of the number of PO and EO units.41 As pressure increases, the hydrophobic PPO blocks initially on the interface are lifted away and the copolymers reorganize into a mushroom phase. In the 7-11 mN/m region there is a slight change in the surface pressure while the surface area decreases. This plateau42 or “pseudoplateau”43 is due to the rearrangement of the PPO coils into “loops”, within the monolayer regime, and the immersion of more EO units in the aqueous subphase (Figure 3B). The end of the pseudoplateau corresponds to an area of ∼1850 Å2/molecule, which indicates that all EO units are in the subphase and that the surface is only occupied by the PPO blocks. The area occupied per hydrated PO unit is 26.5 Å2 as previously reported.41,44 Further compression causes a rapid increase in π. PF127 molecules become closer and closer, the movement of the blocks becomes restricted due to space limitations and to an increase in lateral interaction, and the copolymer molecules reorganize into a “brush conformation”.40,44-46 In the subphase the helical PEO chains entangle with those of neighbor copolymer molecules, while at the interface the PPO blocks can form loops. If the area is further restricted, both hydrophilic and hydrophobic blocks become stretched (condensed state).41 The addition of the same amount of PF127 (i.e., 3.35 × 1014 copolymer molecules) on HPβCD or MβCD solutions of different concentrations caused the initial pressure to increase gradually with the concentration of cyclodextrin in the subphase (Figure 2B,D). This indicates the coadsorption at the interface of both PF127 and cyclodextrin. It must be noted that, before deposition of PF127, the attainment of HPβCD or MβCD
(4)
for PF127 on water plus the pressure recorded for the CD solution, as follows: Thus, the difference between the apparent surface pressure and
∆π ) πexp,A - πideal,A
(5)
the ideal surface pressure was used to quantitatively measure deviations from the ideal behavior:47 The plot of excess surface pressure for the PF127-HPβCD systems (Figure 2C) clearly revealed that in the expanded state (large molecular areas) a slight increase in the surface pressure occurred, which corresponds to an area-expansion effect or a net expelling interaction between the two components. This finding indicates that, when the HPβCD lies on the surface with the axis of the cavity perpendicular to the interface, no complexation with PF127 occurs. By contrast, at small molecular areas (condensed state), a negative excess pressure was observed; the deviation from ideal behavior was more intense as the concentration of HPβCD on the subphase increased. Nevertheless, the difference between 10-2 and 0.1 wt % was small, indicating that there are a similar number of HPβCDs at the interface available to interact with PF127. The negative excess pressure indicates a pressure reduction effect of HPβCD on the PF127 monolayer at a given molecular area because of an area-condensing effect; i.e., the area occupied by both components when they are together is smaller than the sum of the areas occupied by each component alone. This means that a net attractive interaction between PF127 and HPβCD has occurred; i.e., the HPβCD molecules oriented with the axis parallel to the interface can form polypseudorotaxanes at the interface with the PF127 molecules, as schematically depicted in Figure 3C. The results obtained when MβCD solution was the subphase followed the same pattern (Figure 2E). Klyamkin et al.20 reported an increase in the monolayer area when heptakis(2,6-di-O-methyl)-βCD was introduced into the aqueous subphase under Pluronic L-61 monolayer (constant pressure 5 mN/m). The increase was progressive with the CD concentration at the subphase until a leveling off was attained. The authors suggested that this effect was caused by the
2778 J. Phys. Chem. B, Vol. 113, No. 9, 2009
Nogueiras-Nieto et al.
Figure 5. 1H NMR chemical shifts of MβCD (A) and PF127 (B) observed for PF127/CD systems. The molar ratios refer to the whole PF127 macromolecule.
Figure 4. Calorimetric titration curves recorded during addition of small volumes of 10 wt % PF127 aqueous solution to a Dewar containing water or 5 wt % CD solution (A), of 10 wt % PF127 and 5 wt % CD solution to a Dewar containing water or the corresponding cyclodextrin solution (5 wt %) (B), and of 10 wt % PF127 solution to a Dewar containing water or 0.1 wt % CD solution (C). The open symbols represent Qcomplex, i.e., the difference between the heat evolved in the presence of HPβCD (up triangles) or MβCD (down triangles) and its absence. The PF127 millimole values used as the x-axis scale refer to the whole macromolecule.
complexation between the copolymer and methyl-CD at the interface. However, according to the model of interaction that we propose, the reported initial increase in pressure is caused by coadsorption; the complex is formed not at low pressures but at a pressure high enough to cause the CDs to flip up. Microcalorimetric Titration. The thermodynamics of the interaction between PF127 and HPβCD or MβCD was investigated using isoperibol microcalorimetry at 37 °C. Recent studies have shown that calorimetry titrations may provide detailed information on the complexation process of cyclodextrins with different host molecules.5,48 This technique is also useful to analyze changes in the micellization processes of surfactants and amphiphilic copolymers in the presence of additives.33,49,50 The changes in temperature measured after the
addition of a micellar PF127 solution (from a microburette) to a CD solution (in a Dewar vessel) can be analyzed taking into account the following contributions: (i) demicellization and dilution of PF127; (ii) dilution of CD solution; (iii) interaction of PF127 with CD to form polypseudorotaxanes, accompanied by changes in the conformation and the solvation state of both components; and (iv) interactions among PF127/CD aggregates. The dilution enthalpies of HPβCD and MβCD were negligible. Figure 4A shows the enthalpy changes that occurred when 2 mL of a 10 wt % PF127 micellar solution was added to 47.5 mL of water or 5 wt % CD solution (the final number of MβCD and HPβCD molecules per PF127 macromolecule was 107 and 125, respectively). When injected into water, the micelles broke up and the unimers separated until the concentration in the Dewar was again above the cmc. The demicellization process was strongly exothermic (enthalpy change negative) owing to hydrogen-bonding formation between the triblock copolymer unimers and the water molecules after breakage of water-water and surfactant-surfactant hydrogen bonds.25 Differing from most ionic surfactants, the micellization of polyoxyalkylene surfactants, although enthalpically unfavorable, occurs spontaneously due to entropy-driven hydrophobic interactions, in which core (polyoxypropylene)-shell (polyoxyethylene) structures are formed.30 The apparent enthalpy change associated with the demicellization of PF127 when the Dewar contained 5 wt % CD solution was more exothermic. The heat evolved in the Dewar containing MβCD solution was remarkably greater than that recorded with an HPβCD solution. These findings suggest that the complexation with the CD enhances the hydrophilic character of the copolymer and promotes its interactions with water, especially in the case of the MβCD. In agreement with surface tension measurements and π-A isotherms, MβCD can effectively host PF127 through an enthalpy-driven process that might be related to van der Waals interactions between the PPO blocks and the inner surface of the CD cavity, but also through hydrogen bonds between PEO blocks and the -OH groups at
Hydrosoluble CD/Poloxamer Polypseudorotaxanes
J. Phys. Chem. B, Vol. 113, No. 9, 2009 2779
Figure 6. Molecular model representing a fragment of 12 PO units forming inclusion complexes with MβCD. Lateral (A) and frontal (B) views.
the CD surface. Binding saturation was not observed using 5 wt % CD solution, probably because of the large excess of CD units compared to PF127 molecules (135 to 1). The integral heat of complexation, Qcomplex, was estimated as the difference between the heat evolved in the presence of CD and the heat associated with the demicellization/dilution of PF127. In the case of HPβCD, a small decrease in Qcomplex (exothermic event) was observed up to 0.2 wt % PF127, followed by a plateau region. In the case of MβCD, an almost constant decrease in Qcomplex was recorded; the integral heat of binding was significantly larger for MβCD than for HPβCD (the data were very reproducible, coefficient of variation < 2%). To obtain complementary information about the affinity of PF127 for the CDs evaluated and the resistance of the complexes against dilution, a solution containing 10 wt % PF127 and 5 wt % CD was loaded into the buret and emptied into water or into a 5 wt % CD solution (Figure 4B). The dilution enthalpies in water of the PF127/CD systems were quite similar to that obtained for PF127 alone, although slightly more exothermic in the case of the PF127/HPβCD system. The calorimetric profiles of the PF127/HPβCD system (when diluted into HPβCD) or of the PF127/MβCD system (when diluted into MβCD) were superimposable to the ones obtained by adding PF127 into the corresponding CD solution (Figure 4A). The differences in Qcomplex when the binary system was added into the CD solution or into water were once again greater for MβCD. This behavior can be related to the fact that 5 wt % CD can only partially fulfill the complexation/interaction ability
of the PF127 molecules contained in a 10 wt % solution; in fact, the CD:PF127 molar ratio is only 4.8 (i.e., 4.8 molecules of CD per PF127 macromolecule). Therefore, when the binary system is added to water, it undergoes a demicellization process that resembles the one observed for PF127 in the absence of the CD. By contrast, when the binary system is poured into 5 wt % CD solution, in addition to the demicellization process an exothermic binding/complexation process of CD with PF127 (or with nonsaturated polypseudorotaxanes) takes place. This confirms that 4.8 CD molecules per PF127 molecule is far from being sufficient for saturating the formation of the polypseudorotaxanes. An additional experiment was carried out placing in the Dewar a diluted CD solution (0.1 wt %) in order to titrate the CDs with PF127. That is, differing from former experiments in which there was an enormous excess of CDs compared to PF127 and complexation did not reach saturation, the present experiment may provide adequate conditions to saturate the complexation ability of the CDs with PF127 (Figure 4C). The calorimetric profiles obtained when 10 wt % PF127 was poured into the diluted CDs were quite similar to those recorded placing just water in the Dewar. Nevertheless, some differences can be noticed. The energy released in the presence of MβCD was again greater than in the water only, confirming the exothermic character of the interaction, which may indicate that polypseudorotaxane formation is both enthalpy and entropy driven. By contrast, the lower energy released when HPβCD was placed in the Dewar suggests an entropy-driven process. The Qcomplex
2780 J. Phys. Chem. B, Vol. 113, No. 9, 2009
Nogueiras-Nieto et al.
Figure 8. Changes induced by PF127 in the absorbance of methyl orange:MβCD (solid squares) or methyl orange:HPβCD (open triangles) solutions. The molar ratios refer to the whole PF127 macromolecule.
Figure 7. TEM images of 5 wt % PF127 solution (A) and of mixtures of 5 wt % PF127 and 10 wt % CD solutions resulting in 1:23 PF127: HPβCD (B) or PF127:MβCD (D) molar ratio.
profile patterns consisted of initial peaks at HPβCD:PF127 17:1 and at MβCD:PF127 22:1 molar ratios, followed by a region of lower slope up to 5:1 molar ratio (for both CDs), and a final portion of greater slope corresponding to the micellization of the excess of PF127. The initial peak may correspond to the preferential binding stoichiometry of the polypseudorotaxanes, which become progressively poor in CDs as the addition of PF127 proceeds. To confirm these findings, complementary techniques were used as described below. 1 H NMR and Molecular Modeling. PF127 significantly changed the chemical shifts of the H3, H4, and H5 protons of
the internal surface of MβCD (Figure 5A). These shifts indicate that PF127 molecules were included in the MβCD cavity. The modifications in chemical shifts of the PF127 protons observed in the presence of the MβCD support such a hypothesis (Figure 5B). In the presence of MβCD, a significant change in chemical shift of -CH3 of PPO blocks was observed, but no changes in the signals of -CH2-CH2- of EO and -CH2-CH- of PO were detected. This means that since the -CH3 of PO is oriented to the outside of the PPO chain, it can effectively interact with the cyclodextrin cavity. The three-dimensional structure generated by molecular modeling of a polypseudorotaxane consisting of five MβCD molecules and a PPO block of 12 units (with the sCH3 groups inside the cavity of MβCD) is shown in Figure 6. The dependence of the chemical shifts on the MβCD/PF127 ratio suggests that each PF127 unimer simultaneously interacts with 20-30 CD molecules, which is in agreement with the microcalorimetric titration data. The 1H NMR spectrum registered for HPβCD-PF127 was not resolved enough to elucidate the nature of interactions between the molecules. The signal of the -CH3 groups of the PPO block appeared at the same ppm as the -CH3 groups of HPβCD. TEM Images. A 5 wt % PF127 solution is formed by monodisperse spherical micelles51 and falls in the isotropic solution region L1.52 Although the accuracy of size measurements using TEM is limited, micellar size in 5 wt % PF127 solution can be estimated to be around 12 nm. This value is in agreement with the 14 nm micellar size found by Mortensen and Talmon51 and the 6 nm core diameter reported by Lam et al.53 using cryo-TEM. Assuming that the micellar core is entirely composed of PO units, the aggregation number for the 5 wt % PF127 solution has been reported to be 18.53 Micelles were seldom observed in 50:50 mixtures of 5 wt % PF127 and 10 wt % HPβCD or MβCD solution despite the fact that 2.5 wt % (2.0 mM) PF127 is well above the cmc (Figure 7B,D). Elongated rodlike structures relatively bright against the background and surrounded by black lines were observed in both mixtures. The mean thickness of the whitish part of the nanorod is around 13-14 nm, and that of the black borderline is 5-6 nm, which renders a total thickness of about 25-28 nm. However, the length of the nanorod is variable. Taking into account these dimensions and recent studies on supramolecular nanoplatelets of water-insoluble polypseudorotaxanes of βCD and Pluronic L61, L64, and F68,54 the rod may be formed by thousands of stacked polypseudorotaxanes (Figure 7C). The
Hydrosoluble CD/Poloxamer Polypseudorotaxanes
Figure 9. Storage (G′, solid symbols) and loss (G′′, open symbols) of 10 wt % (A, B), 15 wt % (C, D), and 20 wt % (E, F) PF127 aqueous systems containing or not 5 wt % MβCD as a function of angular frequency at 25 °C (A, C, E) or as a function of temperature at 0.5 rad/s (B, D, F).
width of the central part of the nanorod depends on the number of CDs strung on the PPO block. Since the depth of the βCD cavity is 0.7 nm, 18.5-20 molecules are required to cover a length of 13-14 nm. PF127:CD 1:20 is in close agreement with the molar ratio found as preferential stoichiometry for the polypseudorotaxanes using microcalorimetric titration and 1H NMR. Competitive Displacement of Methyl Orange. The affinity of the drug:CD 1:1 or 1:2 complexes can be evaluated using competitive dye absorption methods based on the changes of dye absorbance upon CD complexation/decomplexation.21,55 The extinction coefficient of methyl orange dye decreases upon CD complexation. Therefore, if a solute competes for the CD cavity and the dye is released to the medium, the absorbance increases proportionally to the free dye concentration. This approach was used to gain insight into the strength of the PF127:CD interactions. A methyl orange:CD 1:10 molar ratio solution was prepared to ensure a complete complexation of the dye as required for competitive studies;22 the stability constants of methyl orange with randomly methylated-βCD and with HPβCD have been previously reported to be 2200 and 1060 M-1, respectively.22 When PF127 was added to the dye/CD solutions, a progressive increase in the absorbance occurred until a plateau was reached at PF127:CD 1:20 molar ratio (Figure 8). The increase in absorbance was more remarkable in the MβCD system, which indicates a greater affinity of PF127 for this CD. These results revealed that PF127 can indeed displace host molecules from CDs and that the statistically more probable
J. Phys. Chem. B, Vol. 113, No. 9, 2009 2781 stoichiometry of the polypseudorotaxanes is around 1:20. Furthermore, the capability of PF127 to displace a dye with such a high affinity constant from the CD cavity clearly indicates that the copolymer strongly interacts with HPβCD and MβCD. Rheology. The sol-gel transition that PF127 solutions undergo when the temperature rises has an enormous practical interest for preparing in situ gel systems or temperatureresponsive hydrogels.56 Since the triggering temperature depends on the strength of the hydrophobic interactions and on the amount of block copolymer available to form a threedimensional network of micelles, polypseudorotaxane formation can significantly alter the gel temperature and the structure of the network.5 We have previously observed that 5 wt % HPβCD increased by 4-6 °C the gel temperature of 15-20 wt % PF127 solutions.5 In the case of MβCD such changes were more remarkable (Figure 9); the increase in gel temperature was 15 °C (from 20 to 35 °C) in the case of 15 wt % PF127 system (Figure 9D). Consequently, the viscoelasticity at 25 °C was remarkably different (Figure 9C). Furthermore, in the case of 20 wt % PF127 gels at room temperature, addition of 5 wt % MβCD caused a 1 order of magnitude decrease in G′ and G′′ (Figure 9E). This means that even a PF127:CD 1:2.6 molar ratio is enough to cause significant structural changes in the copolymer network. HPβCD and MβCD prevent hydrophobic interactions among the PF127 chains, confirming that polypseudorotaxane formation involves the threading of the PO units. The CDs at the molar ratio evaluated did not completely prevent gel formation. The PF127:CD 1:20 molar ratio was not evaluated because it would require an extremely high CD concentration (e.g., 30 wt % CD in a 15 wt % PF127 system). Assuming that the polypseudorotaxanes are not involved in the gelling process and that the free PF127 chains can undergo the gel transition in a way similar to that in the absence of cyclodextrins, the shift in gel temperature indicates the concentration of PF127 that is not interacting with the CD. For example, in the case of 15 wt % PF127 system, addition of 5 wt % MβCD shifted the gel temperature to a value that corresponds to 12 wt % PF127 solution. Thus, 3 wt % PF127 may be involved in the polypseudorotaxane formation. This roughly corresponds to the MβCD:PF127 20:1 molar ratio, which is in agreement with the values obtained by microcalorimetric titration, 1H NMR, and competitive displacement of methyl orange assays. These findings highlight the observation that CDs able to form water-soluble polypseudorotaxanes can be useful for modulating the temperature responsiveness of poloxamer systems regarding both the gel point and the consistency of the network. Conclusions Both MβCD and HPβCD can lead to water-soluble polypseudorotaxanes with PF127, in which 3.5 PO units are covered by one CD. Since formation of polypseudorotaxanes involves the shielding of the hydrophobic interactions among PO units, properties of the systems at both the bulk solution and the air-water interface become affected. Particularly relevant are the changes in pressure of the monolayer exerted at the interface due to changes in the number of species and their relative conformation, the capability of PF127 to displace host molecules from the CD cavities, and the changes in the sol-gel transition. The interaction models developed in the present work help to explain such phenomena and to acquire knowledge on polypseudorotaxanes and their aggregation to form nanorods in aqueous medium. Polypseudorotaxane-induced changes should be taken into account when developing drug delivery systems
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