J. Phys. Chem. 1996, 100, 7603-7609
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Structure of Block Copolymers Adsorbed to Perfluorocarbon Emulsions Clive Washington* Department of Pharmaceutical Sciences, UniVersity of Nottingham, UniVersity Park, Nottingham NG7 2RD, U.K.
Stephen M. King and Richard K. Heenan ISIS Facility, Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX, U.K. ReceiVed: October 11, 1995; In Final Form: January 19, 1996X
We have used small-angle neutron scattering (SANS) to study the structure of block copolymers (Poloxamer 188, Poloxamer 407, and Poloxamine 908) adsorbed at the perfluorodecalin-water interface in a submicrometer emulsion system. The data were analyzed using an established transform method to yield the polymer volume fraction profile as a function of distance from the oil-water interface. There was a good correlation between the derived adsorbed amounts of polymer and those measured experimentally, and between the adsorbed layer dimensions from SANS and earlier light scattering measurements. The profiles are consistent with a model in which the poly(propylene oxide) blocks are bound at the interface and the poly(ethylene oxide) blocks are largely present as tails. An electrolyte (sodium chloride at 0.1-0.2 M concentration) was found to significantly reduce the bound fraction of Poloxamer 188, and caused a significant increase in the extension of the adsorbed layer, without affecting the amount of polymer adsorbed.
Introduction An understanding of the structure of adsorbed layers of polymer or surfactant molecules in colloidal systems is of fundamental importance, and also relevant to a wide range of technical systems, such as paints, surface coatings, lubricants, drilling muds, cosmetics, agrochemicals, and foodstuffs.1,2 A particularly active area at present is that of biomedical colloids. There is extensive literature concerning the use of synthetic colloidal systems as carriers for the targeted delivery of a wide range of drugs in the treatment of diseases such as AIDS and cancer.3-5 Recent work from a number of authors has demonstrated that the in ViVo behavior of an injected colloidal system can be modified by the adsorption or grafting of a polymeric species at the interface.6-12 For pharmaceutical applications there is particular interest in the use of water-soluble triblock copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO-PEO) of the type marketed under the Synperonic (ICI), Poloxamer, or Pluronic (BASF) trade names. It has been shown that these copolymers can alter the surface adhesion of the proteins which mediate the body’s defense responses and which would otherwise cause injected colloidal particles to be removed from the bloodstream.10,11,13 Tetrablock copolymers of the Tetronic (BASF) or Poloxamine family, which have the starlike structure (PEOPPO)2-N-C2H4-N-(PPO-PEO)2 have also received much attention in this regard.7,14,15 Although this work is relatively recent, it is already evident that the structure of the adsorbed layer influences the fate of the colloidal particles in ViVo. For example, these copolymers which have longer PEO blocks appear to form thicker adsorbed layers on latex particles such as polystyrene, and on triglyceride emulsions, and result in the colloidal particles having longer circulatory lifetimes.16 Unfortunately this is almost certainly an incomplete picture; what is meant by the “layer thickness” can depend on the measuring technique, while a “thick” layer can just as easily arise where a few high molecular weight X
Abstract published in AdVance ACS Abstracts, April 1, 1996.
S0022-3654(95)03007-3 CCC: $12.00
copolymers are adsorbed at relatively low surface coverage as it can where many more, lower molecular weight, copolymers are adsorbed at relatively high surface coverage. Clearly any experimental technique that can resolve these questions, by providing detailed information about the structure of the adsorbed layer, is likely to be of great significance in understanding or even predicting the behavior of such colloidal particles in ViVo. A variety of spectroscopic, scattering, magnetic resonance, hydrodynamic, and, very recently, rheological techniques have all been successfully used to study the structure of adsorbed and grafted polymer layers in colloidal systems.1,17-19 The disadvantage of most of these techniques is that they generally only provide one piece of information: a layer thickness or the amount of polymer adsorbed or the fraction of polymer segments bound to the interface. In this work we have used small-angle neutron scattering (SANS) to derive volume fraction profiles for some adsorbed copolymers. A volume fraction profile, φ(z), provides a complete picture of the structure of an adsorbed polymer layer.20 This is because it describes how the average volume fraction of polymer (which is directly proportional to the average number density of polymer segments) varies with distance, z, from an interface. From this one function it is also possible to obtain two measures of the layer thickness: the maximum extent, l, which will be similar to a hydrodynamic thickness, and the second moment about the mean of the distribution, σ, or standard deviation of the volume fraction profile. It is also straightforward to extract the adsorbed amount (from the integral under the volume fraction profile) and the bound fraction (from the number of segments within a short distance, say 1 nm, of the interface). We have been studying oil-in-water emulsions made from perfluorodecalin with block copolymers of the Poloxamer and Poloxamine (Pluronic and Tetronic, respectively) series present as emulsifiers. These emulsions have an intrinsic pharmaceutical application since they are used as oxygen transport media. In particular, the perfluorodecalin-Poloxamer 188 system has been widely studied as “artificial blood” for clinical use.21-28 © 1996 American Chemical Society
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TABLE 1: Average Structures and Properties of Block Copolymers56 copolymer
structure
MPEO
density MPPO (g cm-3)
Poloxamer 188 PEO76-PPO30-PEO76 6700 1800 Poloxamer 407 PEO98-PPO69-PEO98 8600 4000 Poloxamine 908 (PEO119-PEO1r)2-N- 21000 4000 C2H4-N-(PPO17PEO119)2
1.160 1.141 0.914
Since water and perfluorocarbons have similar Hamaker constants (they have almost identical refractive indices), these emulsions are easily stabilized, even by weakly adsorbing polymers. This allows stable emulsions with droplet diameters from 100 to 300 nm to be easily prepared by high-shear homogenization. The perfluorocarbon-water system has several other advantages as far as SANS experiments are concerned. Using a perfluorinated oil removes the need for an expensive perdeuterated hydrocarbon oil while at the same time the coherent neutron scattering length density of the perfluorocarbon emulsion droplets can be contrast-matched to a deuterium-rich dispersion medium. This reduces background scattering due to the large incoherent neutron scattering cross-section of hydrogen. Finally, because the density of perfluorodecalin (1.94 g cm-3) is so much higher than that of the dispersion medium (water), the emulsion droplets can be sedimented by gentle centrifugation and resuspended in pure solvent without undergoing any significant coalescence. This provides for the easy removal of unadsorbed polymer and allows for a very accurate determination of the background scattering. In this paper we report on our study of the structure of three PEO-PPO-PEO copolymers adsorbed at the perfluorocarbonwater interface: Poloxamer 188, Poloxamer 407, and Poloxamine 908. We also show preliminary results of the effect of electrolyte-induced chain dehydration on the structure of adsorbed Poloxamer 188. Experimental Section Materials. Perfluorodecalin and perfluoroperhydrophenanthrene were kindly provided by Rhone-Poulenc ISC Division (now BNFL Fluorochemicals), Bristol, U.K., and were used as received. Both materials were at least 99.5 atom % F and had isomeric purities better than 95%. Deuterium oxide, 99.9 atom % D, was purchased from CDN Isotopes, Canada. Samples of Poloxamer 188 (Pluronic F68), Poloxamer 407 (Pluronic 127), and Poloxamine 908 (Tetronic 908) were donated by BASF, Parsippany, NJ, and were purified before use to remove low molecular weight contaminants.29 The polydispersities (Mw/ Mn) of the purified copolymers were typically less than 1.3; the effective polydispersity of the adsorbed copolymer in our systems is expected to be rather lower than this for reasons that will be discussed shortly. The bulk densities of the copolymers were measured directly by liquid pycnometry in ethanol. The characteristics of the copolymers are summarized in Table 1. All other materials were reagent grade from established suppliers. Preparation of Emulsions. The emulsions were prepared by homogenizing 10% (v/v) perfluorodecalin and 2% (v/v) perfluoroperhydrophenanthrene in the appropriate polymer solution (4%, w/v) using a high-pressure homogenizer (M-110 Microfluidiser, Microfluidics Inc., Newton, MA) as described previously. Perfluoroperhydrophenanthrene is a fluorocarbon oil which is completely miscible with perfluorodecalin, and reduces the Ostwald ripening rate of the droplets.25,26,30 Due to the need to prime the homogenizer with polymer solution, the exact disperse phase fractions were uncertain to
TABLE 2: Physical Properties of Fluorocarbon Emulsions with Poloxamers 188 and 407 and Poloxamine 908 as Emulsifiers Poloxomer Poloxamer Poloxamine 188 407 908 emulsion phase fraction volume mean diametera (nm) surface mean diametera (nm) polydispersityb
0.039 270 ( 3 260 ( 4 0.05
0.043 304 ( 2 278 ( 3 0.05
0.047 320 ( 3 314 ( 4 0.08
a The volume and surface mean diameters were computed from the (measured) intensity-weighted diameters by the instrument software. b The polydispersity of a particular system is defined as the relative standard deviation of the lognormal distribution using cumulants analysis. This differs from its alternative accepted use to describe molecular weight distributions in macromolecular systems.
(5%, so the final disperse phase fraction was assayed by specific gravity measurements at 25 °C. This is a sensitive method of assaying the emulsions due to the high density of the perfluorocarbon. Because of the relatively large volume of the homogenizer (100 mL), the emulsions were initially prepared by dissolving the polymer in H2O. Aliquots (∼10 mL) of the emulsions were then transferred to a dispersion medium of the appropriate scattering length density (see below) by gentle centrifugation (1000g for 20 min) followed by redispersal in a 28% (w/w) H2O/72% (w/w) D2O mixture. This procedure was repeated three times to ensure that the disperse phase H:D composition was correct, and that most of the unadsorbed polymer was removed. By analogy with the adsorption behavior of homopolymers, this procedure has a fractionating effect that preferentially adsorbs higher molecular weight homologues.1 The size distributions of the emulsion droplets were measured by dynamic light scattering (Malvern 4700 PCS using the cumulants method and CONTIN for the distribution analysis). The characteristics of the emulsions formed with each copolymer are given in Table 2. Small-Angle Neutron Scattering. SANS data were obtained on the LOQ small-angle diffractometer at the ISIS spallation neutron source (Rutherford Appleton Laboratory, Didcot, U.K.).31,32 This is a fixed-geometry “white beam” time-of-flight instrument which utilizes neutrons with wavelengths between 2 and 10 Å to provide a simultaneous Q-range of 0.006-0.22 Å-1, where Q is the modulus of the scattering vector:33,34
Q ) (4π/λ) sin(θ/2)
(1)
where λ is the neutron wavelength and θ is the scattering angle. In a SANS experiment the scattering that is actually measured is a summation of the scattering from each component present in the system. The relative contribution that each of these components makes to the overall scattering is determined by the square of the difference (or contrast) between the neutron scattering length densities of that component and the dispersion medium. The emulsions were effectively three-component systems consisting of perfluorocarbon oil, water, and polymer. Since we were only interested in the scattering from the polymer, it was necessary to make the perfluorocarbons “invisible” to neutrons. This was achieved by mixing H2O and D2O in the ratio 27.7:72.3 (w/w) and using this mixture as the dispersion medium. The match concentration was achieved to within 0.1% by weighing the solvents. Relevant scattering length densities are tabulated in Table 3. The exact point of contrast match was optimized in situ on the neutron beamline in a preliminary experiment. To reduce possible multiple scattering effects (which are difficult to correct for), each emulsion sample was diluted to a disperse phase fraction of 3-4% (v/v). A portion of this diluted
Block Copolymers Adsorbed to Perfluorocarbon Emulsions
J. Phys. Chem., Vol. 100, No. 18, 1996 7605
TABLE 3: Coherent Neutron Scattering Length Densities (×1010 cm-2) H 2O D2O perfluorodecalin poly(ethylene oxide) poly(propylene oxide)
δp
bulk density (g cm-3)
-0.559 +6.355 +4.183 +0.637 +0.343
1.000 1.105 1.940 1.127 1.004
sample was then centrifuged for 15 min at 13000g to completely sediment the dispersed phase. This provided a sample of the continuous phase for use as a background. SANS data were also collected on Poloxamer 188 systems where the dispersion media contained 0.1 and 0.2 M sodium chloride. Due to the need for high-quality data for the inversion procedure, each sample and background was measured for a total of 4 h but in 1 h runs. Visual examination of the samples, and examination of the initial and final scattering curves, showed no evidence of sample degradation, coalescence, or sedimentation during this period, so the 1 h data sets were combined prior to analysis. Each raw scattering data set was corrected for the sample transmission and background scattering and converted to scattering cross-section data (∂Σ/∂Ω versus Q) using the instrument-specific software.35 The reduced data were placed on an absolute scale using a well-characterized solid blend of hydrogenous and perdeuterated polystyrene as a calibration standard.36 When the perfluorocarbon oil phase and dispersion medium are at contrast match, the only SANS observed is that from the thin shell of polymer at the interface between the two phases. In the low-Q limit, where Qσ is small, this scattering has been shown to have the form37
(∂Σ/∂Ω)(Q) (cm-1) ) {6 × 10-22πRp-1 φpM2Q-2 exp(-Q2σ2)} + background (2) where Rp is the droplet radius (Å), φp is the volume fraction of droplets, and M is a parameter related to the adsorbed amount of polymer, Γ, through
Γ (mg m-2) ) MD/(δp - δs)
(3)
where D is the bulk density of the polymer (g cm-3), δp is the scattering length density of the polymer (cm-2), and δs is the scattering length density of the (matched) droplet cores and dispersion medium. To obtain estimates for σ and Γ, the scattering data were truncated into the region 0.009 < Q (Å-1) < 0.1 and fitted to eqs 2 and 3 using nonlinear least squares with Rp (as the surface mean diameter), φp, and D restrained to their experimental values. Values for the scattering length densities of the copolymers were obtained from appropriate weighted sums of the values for PEO and PPO. The SANS cross-section in this experiment is sensitive to the averaged scattering length density distribution normal to the fluorocarbon-water interface. Equation 2 is derived by expansion of the scattering for a thin layer on the surface of a sphere of radius Rp, assuming that Rp is large compared to the distances, t, within the surface layer, and that Qt and hence Qσ are small enough to allow Taylor series expansions. Since these latter assumptions are not always valid for the range of Qt encountered here, some numerical simulations were made using more exact equations. These demonstrated that the values of σ derived here should be correct to around 10%. Such errors would not alter the comparison of the different polymers studied below. The segment density distribution, F(z), is obtained from the scattering data by Hilbert transformation.38,39 To alleviate
Figure 1. SANS (∂Σ/∂Ω versus Q) from Poloxamer 188 adsorbed at the perfluorodecalin-water interface (the line is a fit to eq 2).
Figure 2. SANS (∂Σ/∂Ω versus Q) from Poloxamer 407 adsorbed at the perfluorodecalin-water interface (the line is a fit to eq 2).
instabilities in the inversion procedure, it was first necessary to truncate the data to their region of acceptable statistics (Q e 0.05 Å-1 in these systems) and add a Porod law (Q-4) extrapolation to higher Q. This procedure has been previously validated although more elegant methods now exist.40 The volume fraction profile, φ(z), is then obtained from F(z) according to
φ(z) ) F(z)Γ/D
(4)
A number of systematic errors might affect the measurement of the adsorbed amount Γ from eqs 2 and 3, by perhaps a few percent. These include errors in the absolute scaling of the SANS data, the determination of Rp by light scattering, the assumption of the bulk polymer densities in Table 3, and the use of the mean composition when calculating the polymer scattering length densities. The average fraction of segments bound to the interface was obtained by integrating the area under the φ(z) curve for each polymer between the surface and 1.3 nm.41 This outer limit corresponds to the extent of one lattice layer in the ScheutjensFleer self-consistent field model of polymer adsorption.42,43 We have also estimated the effective thickness of each adsorbed polymer layer as the distance at which the integrated volume fraction equals 95% of the total. SANS is relatively insensitive to the presence of the very few, highly extended, tails. Results Figures 1-3 show the fully reduced scattering data for each emulsion system. The solid lines in each figure are fits to eq 2. The resulting values of the second moments and the adsorbed amounts of polymer are given in Table 4, while the corresponding polymer volume fraction profiles are shown in Figure 4. The general form of the scattering data from Poloxamer 188 in the presence of an electrolyte was very similar to that obtained in the absence of an electrolyte (Figure 1). For this reason we
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Figure 3. SANS (∂Σ/∂Ω versus Q) from Poloxamine 908 adsorbed at the perfluorodecalin-water interface (the line is a fit to eq 2).
TABLE 4: Adsorbed Layer Structure adsorbed copolymer
Γa (mg m-2)
Γb (mg m-2)
σa (nm)
lc (nm)
Poloxamer 188 Poloxamer 188 (0.1 M NaCl) Poloxamer 188 (0.2 M NaCl) Poloxamer 407 Poloxamine 908
1.87 1.94 1.94 3.28 1.49
1.85
2.34 2.40 2.63 3.46 3.75
4.8 8.9 8.7 16.0 15.2
2.93 1.18
a From SANS via eqs 2 and 3. b By direct measurement of unadsorbed polymer in the continuous phase. c Effective layer thickness (95% of polymer segments).
Figure 4. Volume fraction profiles, φ(z), for ([) Poloxamer 188, (9) Poloxamer 407, and (O) Poloxamine 908 adsorbed at the perfluorodecalin-water interface. The line joining the points is an aid to the eye.
do not show the scattering data, but the results of the fits to these data are also included in Table 4. The effect of an electrolyte on the volume fraction profiles for Poloxamer 188 is shown in Figure 5. The bound fractions of the copolymers, as defined above, and for comparison the fraction of PPO segments in each molecule, are shown in Table 5. Discussion The perfluorodecalin-water system is unusual in that it is unlikely that the (hydrocarbon) polymer penetrates the (fluorocarbon) oil phase to any significant extent. Indeed, PEO and PPO are insoluble in perfluorocarbon solvents.44 It is therefore likely that adsorption at this liquid-liquid interface has similarities to adsorption at the solid-liquid interface. This is an important point since it means that adsorption in these systems may be likened to, for example, adsorption at the much-studied polystyrene latex-water or silica-water interface but with one crucial difference. In these solid-liquid systems both blocks of the copolymer adsorb, and in particular, it is the PEO blocks that preferentially adsorb. As discussed above, the adsorbed layer will be composed of the higher molecular weight polymers present in the original
Figure 5. Volume fraction profiles, φ(z), for Poloxamer 188 in the presence of ([) no salt and (9) 0.1 M and (O) 0.2 M sodium chloride. The line joining the points is an aid to the eye.
TABLE 5: Derived Values of the Bound Fraction and the PPO Segment Fraction adsorbed copolymer
bound fraction
fraction of PPO segments
Poloxamer 188 Poloxamer 188 (0.1 M NaCl) Poloxamer 188 (0.2 M NaCl) Poloxamer 407 Poloxamine 908
0.32 0.22 0.20 0.14 0.15
0.16 0.16 0.16 0.26 0.13
polydisperse sample. As a consequence the volume fraction profile and adsorbed layer thicknesses are likely to be the maximum attainable in these systems. Volume Fraction Profiles. All of the volume fraction profiles shown are smoothly-decaying functions which bear a resemblance to profiles obtained from systems where homopolymers physically adsorb at the solid-liquid interface.20 The profile for Poloxamine 908 appears to have a character slightly different from those of the Poloxamers, but this is not altogether unexpected. Since the neutron contrasts (δp - δs)2 for PEO and PPO are similar in our experiments (1:1.2, respectively), the similarities between these profiles and those reported for PEO homopolymers,45 together with the absence of any maxima away from the surface, suggests that neither block particularly dominates the structure of the adsorbed layer, though this may be less true of Poloxamine 908. The conclusion is that both blocks must be adsorbed to some extent, although the more hydrophobic PPO segments are likely to be preferentially adsorbed in this system. In other words, in our system segregation of the blocks into an anchor and a buoy is not clear cut. This argument does, however, assume that there is no micellar adsorption at the surface (a possibility given the differential solubility of the blocks and the fact that the polymer concentrations used in this work are above published cmc values46,47). Again, the absence of distinct maxima does tend to mitigate against this, as does the fact that any micelles would be reversible (both blocks have glass-transition temperatures well below room temperature), but the most revealing evidence comes from measures of the layer thickness, and these will be discussed later. The volume fraction profiles in Figure 4 are of shape similar to and cover length scales similar to those reported by Mallagh46 for the adsorption of PEO32-PPO56-PEO32 and PEO140PPO56-PEO140 onto polystyrene latex from water. These authors also measured the profiles of the same copolymers at the toluene-water interface. Unlike the present system, their results showed significant penetration of the oil phase by the polymer. More recently the volume fraction profile for Poloxamer 407 at the hexane-water interface has been measured by neutron reflectometry.52 The PPO segments were found to
Block Copolymers Adsorbed to Perfluorocarbon Emulsions penetrate the hexane phase, but the less soluble PEO segments remained extended in the water phase, with an Rg 4-5 times greater than that of the free EO block. The differences in the starting height of each profile can be attributed to differences in the fraction of polymer segments bound to the interface, while the area under each curve is related to the mass of polymer adsorbed. Bound Fractions. The bound fractions shown in Table 5 correlate better with the fraction of PPO segment in the copolymers than they do with the fraction of PEO segments. This is consistent with the PPO segments predominantly forming the trains and loops and the PEO segments predominantly forming the tails. There are noticeable differences however. The bound fraction of Poloxamer 188 (32%) is much higher than its fraction of PPO segments (16%). This suggests that, in addition to the expected adsorption of the PPO segments, a proportion of the PEO segments in this polymer are also to be found adsorbed, or very close, to the interface. By analogy with the known behavior of homopolymers this would in turn imply a relatively low adsorbed amount with many more segments in trains than in loops. The situation with Poloxamer 407 appears to be the complete opposite. In this instance the bound fraction (14%) is lower than the PPO segment fraction (26%). Again, by analogy with homopolymers, this implies a relatively high adsorbed amount with more segments in loops than in trains, and fewer adsorbed PPO segments. The bound fraction of Poloxamine 908 is only marginally higher than its PPO segment fraction. This is probably related to the very asymmetric nature of each of its four “arms”. Since the PPO blocks are very short in this copolymer, there is much less scope for the formation of loops. This, together with steric considerations imparted by the much longer PEO blocks, probably limits the amount of this polymer that can be adsorbed. Thickness of the Adsorbed Layers. The volume fraction profiles in Figure 4 show that in the case of Poloxamer 188 the adsorbed layer extends almost 5 nm from the surface while that of Poloxamer 407 extends just over 3 times as far. Both of these values are less than the maximum theoretical extensions of the copolymers, 14.4 nm for Poloxamer 18848 and approximately 18-22 nm for Poloxamer 407, but much larger than the calculated radii of gyration, Rg, of the individual chains in solution, 3.4 and 4.1 nm, respectively. These adsorbed layer dimensions are consistent with those obtained by previous researchers studying adsorption onto polystyrene latex from water. For example, Killmann et al.49 obtained hydrodynamic thicknesses by dynamic light scattering of 5 nm for Poloxamer 188 and predicted 8 nm for Poloxamer 407 on 56 nm diameter polystyrene latices. Similarly, Muller16 reported values of 5.8 and 9.8 nm, respectively, on 60 nm diameter polystyrene latices. It is interesting that there is a much greater discrepancy between our data and these literature values for Poloxamer 407 than there is for Poloxamer 188. This will be returned to later. The second moment of the segment density distribution, σ, may be likened to the distance of the center of mass of the adsorbed layer from the interface. This means that it reflects how far the loops could (were it not for the weighting effects of the segments in trains) extend from the surface. (Strictly speaking this measure is given by the root-mean-square thickness.50 Consequently σ should be significantly smaller than any hydrodynamic thickness due to the higher density of segments near the interface. The full extent of the volume fraction profiles is determined by the small number of segments in tails that extend much further from the surface. It is these tails that influence hydrodynamic measurements.
J. Phys. Chem., Vol. 100, No. 18, 1996 7607 As can be seen from the segment density data in Table 4, the bulk of each copolymer lies within 2.3-3.5 nm of the surface. The upper of these limits is for Poloxamer 407, and so the σ data correlates well with the bound fraction data discussed above. It is also interesting to note that for each copolymer σ lies within Rg. This implies that the adsorbed chains adopt conformations which are on the whole flatter than random coils. Since the Rg values for these copolymers are very similar to those for PEO homopolymers of comparable molecular weights,41,53 it is unlikely that any micellar adsorption takes place or that any organized surface phases are formed. The volume fraction profile for Poloxamine 908 extends approximately 15 nm from the interface. This is consistent with Muller,16 who reports a hydrodynamic thickness of 13.4 nm, and similar to the extent of the Poloxamer 407 profile. Our data also show that Poloxamine 908 has a second moment similar to that of Poloxamer 407. The total number of adsorbing segments in Poloxamine 908 is very similar to the number in Poloxamer 407, and the bound fraction data suggest that, in the former case, these segments must lie quite flat at the interface. Since Poloxamine 908 has longer PEO blocks than Poloxamer 407, the tails in the latter must be subject to a greater degree of stretching. The most likely cause of such stretching could, quite simply, be that more Poloxamer 407 adsorbs at the interface. Again, this would be consistent with the bound fraction data. Adsorbed Amounts. Two sets of adsorbed amount data are shown in Table 4. One set is derived from the SANS data while the other was obtained by analytical assay of unadsorbed polymer. The agreement between the two techniques is very encouraging and indicates that the sedimentation and redispersal procedures, used to transfer each emulsion to a contrast-matched medium and to remove free polymer, did not cause significant desorption of the physically adsorbed polymer from the droplet interfaces. This was potentially a cause for concern, since the strength of adsorption in this system is unknown. As mentioned above, a number of errors are inherent in the estimation of the adsorbed amounts. These include errors in SANS scaling, emulsion droplet size, and polymer scattering properties. In addition it should be noted that the emulsions were not monodisperse; they had lognormal size distributions with a polydispersity (relative standard deviation of the lognormal distribution) of 0.05. Consequently it was necessary to use the appropriate mean diameter, which in this case is the surface mean diameter. Due to the relatively low polydispersity of the emulsions, the number, surface, and volume mean diameters all fell within 30 nm of each other. In light of these sources of potential error, we would estimate that the precision inherent in the measurement of the adsorbed amounts is 2025%, although a detailed error analysis has not been undertaken. Since these errors are in many cases systematic rather than random, the relatiVe values of the adsorbed amounts should be much more accurate. The adsorbed amounts are much higher than is normally seen in systems where both homopolymers and copolymers physically adsorb as independent chains at the solid-liquid interface. In such cases adsorbed amounts of ca. 0.6-1 mg m-2 are typical. In the polystyrene latex-water system, for example, Kayes and Rawlins48 report a pseudoplateau adsorption of 1.01 mg m-2 for Poloxamer 188. It is interesting to compare this value, and the data in Table 4, with the data of Cohen Stuart et al., 51 who report a remarkably linear relationship between the adsorbed amount and log(molecular weight) for PEO adsorbing on polystyrene latex from water. Using their data, we can interpolate adsorbed amounts of 0.3, 0.4, and 0.6 mg m-2 for
7608 J. Phys. Chem., Vol. 100, No. 18, 1996 PEO homopolymers with the same total molecular weight as Poloxamers 188 and 407 and Poloxamine 908, respectively. The latices of Kayes and Rawlins and of Cohen Stuart et al. were of comparable size to the PFC emulsion droplets, 312 and 240 nm diameters, respectively, and both were prepared by surfactant-free dispersion polymerization methods so they should have similar surface charge densities. It is not altogether unsurprising that we should observe adsorbed amounts on our emulsion droplets which are so much higher than those measured on latex particles since, to form a stable emulsion, the emulsifier must fill the interface efficiently. However, it does also show that more Poloxamer molecules, and therefore fewer PEO segments, are being adsorbed at the fluorocarbon-water interface, in more extended conformations, than at the latex-water interface. This supports the idea that it is the PPO blocks which preferentially adsorb at the fluorocarbon-water interface but the PEO blocks which predominantly adsorb at the latex-water interface. The self-consistent nature of our data is nicely displayed by the fact that, as predicted above, Poloxamer 188 does indeed have a lower adsorbed amount than Poloxamer 407. In addition, the amount of Poloxamine 908 adsorbed is lower than that of either Poloxamer. The adsorbed amounts correlate well with the fraction of PPO in the copolymers as would be expected if the PPO is the anchor block. In the case of the two Poloxamers, the adsorbed amounts approximately scale with their total molecular weights. The scaling against the molecular weights of either the PEO or PPO blocks is less satisfactory. The adsorbed amount of Poloxamine 908 does not scale in a similar manner; indeed, it is significantly lower. This reinforces our view that the Poloxamers and Poloxamines form adsorbed layers with rather different structures. The surface areas occupied by Poloxamers 188 and 407, as calculated from the adsorbed amounts, are 7.5-7.6 and 6.47.1 nm2 molecule-1, respectively. These values are quite similar, which, in turn, means that in the case of Poloxamer 407 the additional bulk of this copolymer must be moved away from the interface into the solvent in order to maintain the small molecular footprint. This helps to explain the larger second moment and, more importantly, the much greater extent of the volume fraction profile for this copolymer. Poloxamine 908 occupies a much larger surface area (27.9-35.2 nm2 molecule-1) than either of the two Poloxamers. This no doubt reflects the greater degree of steric hindrance present in the Poloxamines where four PPO blocks, each terminated with a PEO block, are joined at the center of the molecule. Essentially a Poloxamine is four PEO-PPO diblock copolymers joined through the PPO blocks. In solution the PEO blocks in the Poloxamines will be well-solvated and randomly arranged around the hydrophobic PPO blocks, but in an adsorbed layer, they must be oriented away from the surface and compressed into a smaller solid angle about the center. An estimate of the degree to which the copolymers are perturbed from their free solution conformation can be obtained by comparing the surface areas they occupy when adsorbed with πRg2, the cross-sectional area each polymer coil might reasonably occupy in solution. The calculation of Rg for a copolymer such as Poloxamine 908 is more difficult than it is for linear block copolymers like the Poloxamers, so the approach we have adopted assumes that Poloxamine 908 can be approximated by a linear block copolymer with the composition PEO119-PPO34PEO119. The result is that the calculated Rg for such a copolymer is very similar to that for Poloxamer 407, about 4.1 nm. Hence, we estimate cross-sectional areas of approximately 36 nm2 molecule-1 for Poloxamer 188 and 53 nm2 molecule-1 for
Washington et al. Poloxamer 407 and Poloxamine 908. These values are significantly larger than the adsorbed molecular areas for all three polymers. Consequently the adsorbed polymers must be quite closely packed at the interface with a significant degree of conformational restriction, though this is more true of the Poloxamers than it is of Poloxamine 908. Effects of an Electrolyte. The effect of adding dilute sodium chloride to the Poloxamer 188 emulsion system is complex. Although there is a negligible change in the adsorbed amount, an increase in the electrolyte concentration results in a reduction in the bound fraction. For 0.2 M NaCl the bound fraction is almost reduced to the PPO fraction of the copolymer. This reinforces our view that in the absence of electrolyte a proportion of the PEO segments are adsorbed at the fluorocarbon-water interface. We suggest that adding an electrolyte weakens the interaction that these segments have with the interface and releases them. However, this behavior is accompanied by a small, but nonetheless significant, increase in the second moment of the layer and an almost doubling of its overall extent. Both sets of effects are clearly visible in the volume fraction profiles in Figure 5. It is important to stress here that the emulsion did not show any signs of flocculating at these electrolyte concentrations. Since the adsorbed amount was unchanged, this increase in the layer dimensions was not due to the adsorption of more copolymer molecules from solution; it must be the result of conformational rearrangements within the layer itself. Researchers studying the short-chain, nonionic, ethylene oxide monoalkyl ether (the so-called CnEm) surfactants have reported that electrolytes dehydrate the ethylene oxide portion of the chain.55 In extreme cases nonsolvent conditions for the EO segments can be induced, resulting in phase separation. On this basis one might expect that the addition of an electrolyte would bring about a collapse of the Poloxamer 188 adsorbed layer and a reduction in its dimensions. Clearly this is not the case. Cosgrove et al.52 studied the effect of sodium sulfate on the adsorption of PEO homopolymers onto polystyrene latex and also observed thicker adsorbed layers at higher electrolyte concentrations. However, unlike the present system, this was accompanied by an increase in the adsorbed amount. Further studies of the effects of electrolyte are in progress and will be reported in a subsequent paper. Conclusions The data are consistent with a model in which the PPO blocks of the copolymers are predominantly adsorbed at the fluorocarbon-water interface and the PEO blocks are extended into the aqueous phase. Adsorbed amounts are generally higher than is usual in systems where homopolymers and copolymers adsorb at the solid-liquid interface. There is evidence that, in the absence of an electrolyte, some of the PEO segments reside at, or very close to, the interface although this behavior is dependent on the surface coverage and hence on the composition of the copolymer. The Poloxamine copolymer adsorbs more weakly than the Poloxamers. This is presumably due to the steric crowding around its hydrophobic core which results in a much larger surface area per molecule. All three copolymers display volume fraction profiles that are consistent with them physically adsorbing from solution as single chains to form densely-packed adsorbed layers. The PEO tails are slightly stretched. Many researchers in the biomedical community have suggested that highly-extended, brushlike, adsorbed polymer layers are paramount in the use of polymer-coated colloids as drug delivery systems. Attempts to develop such systems have, in general, concentrated on using polymers with ever increasing
Block Copolymers Adsorbed to Perfluorocarbon Emulsions PEO chain lengths. Indeed, the experimentally observed decrease in biological activity (related to a reduction in protein adsorption) with increasing chain length and adsorbed layer thickness appears to support this hypothesis, though at present a detailed mechanism is lacking.3,7,16 For example, Illum et al.7 found that polystyrene latices with adsorbed Poloxamine 908 were not taken up rapidly by the liver (in contrast to untreated latices) while similar latices with an adsorbed layer of Poloxamer 407 were largely taken up by the bone marrow.8 These results evidently represent a complex interplay of both structural chemistry at the interface and physiological and immunological factors. The data we have presented in this paper show that in actual fact the PEO chains are far from maximally extended. Increasing the PEO chain length does not therefore automatically improve the brushlike structure and suggests that this approach to achieving a biocompatible interface will ultimately have its limits. In order to significantly extend the adsorbed layer structure over that achievable with PEO surfactants, it may be necessary to select a polymer with a completely different structure. Acknowledgment. The authors would like to thank the Engineering and Physical Sciences Research Council for support, and the Central Laboratory of the Research Councils’ ISIS facility for the provision of neutron scattering facilities. We would also like to thank Dr. T. Cosgove, of the University of Bristol, for helpful discussions. References and Notes (1) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapman & Hall: London, 1993. (2) Everett, D. H. Basic Principles of Colloid Science; Royal Society of Chemistry: London, 1988. (3) Tomlinson, E., Davis, S. S., Eds. Site-Specific Drug DeliVery; John Wiley: Chichester, 1986. (4) Davis, S. S.; Washington, C.; West, P.; Illum, L.; Liversidge, G.; Sternson, L.; Kirsch, R. Ann. N.Y. Acad. Sci. 1987, 507, 75. (5) Illum, L.; Davis, S. S.; Wilson, C. G.; Thomas, N.; Frier, M.; Hardy, J. G. Int. J. Pharm. 1982, 12, 135. (6) Illum, L.; Davis, S. S. J. Pharm. Sci. 1983, 72, 1086. (7) Illum, L.; Davis, S. S.; Muller, R. H.; Mak, E.; West, P. Life. Sci. 1987, 40, 367. (8) Illum, L.; Davis, S. S. Life Sci. 1987, 40, 1553. (9) Bazile, D.; Prudhomme, C.; Bassoulet, M. T.; Marlard, M.; Spenlehauer, G., J. Pharm. Sci. 1995, 84, 493. (10) Allen, T. M. Trends Pharmacol. Sci. 1994, 15, 215. (11) Needham, D.; McIntosh, T. J.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1108, 40. (12) Mayhew, E. G.; Lasic, D.; Babbar, S.; Martin, F. J. Int. J. Cancer 1992, 51, 302. (13) Norman, M. E.; Williams, P.; Illum, L. Biomaterials 1993, 14, 193. (14) Stolnik, S.; Davies, M. C.; Illum, L.; Davis, S. S.; Boustta, M.; Vert, M. J. Controlled Release 1994, 30, 57. (15) Moghimi, S. M.; Muir, I. S.; Illum, L.; Davis, S. S. Biochim. Biophys. Acta 1993, 1179, 57. (16) Muller, R. H. Colloidal Carriers for Drug DeliVery; CRC Press: Boca Raton, FL, 1991.
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