pubs.acs.org/Langmuir © 2009 American Chemical Society
Homopolymer Induced Aggregation of Poly(ethylene oxide)nb-poly(butylene oxide)m Polymersomes Thomas P. Smart,† Anthony J. Ryan,‡ Jonathan R. Howse,§ and Giuseppe Battaglia*,† †
The Kroto Research Institute, Department of Engineering Materials, University of Sheffield, Broad Lane, Sheffield S3 7HQ, United Kingdom, ‡Department of Chemistry, University of Sheffield, Brook Hill, Sheffield S3 7HF, United Kingdom, and §Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom Received August 5, 2009. Revised Manuscript Received September 11, 2009 We have studied the addition of poly(ethylene oxide) homopolymer (PEO) to a range of polymersome dispersions composed of amphiphilic di- and triblock copolymers. A number of EnBm EnBmEn and BmEnBm (E = poly(ethylene oxide) B = poly(butylene oxide)) block copolymers of varying molecular weights that spontaneously form polymersomes in water were investigated. This resulted in the aggregation of the dispersed polymersomes by two mechanisms, PEO adsorption or depletion interactions, and is shown to be dependent on PEO concentration. The aggregation kinetics and the resultant structures were analyzed by dynamic light scattering (DLS), small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM). There is a critical relationship between the polymersome corona thickness t and the PEO radius of gyration Rg, where Rg must equal t to induce aggregation. This phenomenon has been reported with small self-assembling surfactants such as sodium dodecyl sulfate, but here we show an insight into how this transposes into much larger block copolymer systems which show great promise as biomimetic delivery vectors for controlled release.
1. Introduction Amphiphilic block copolymers (hydrophilic-hydrophobic copolymers) have become an incredibly important class of selfassembling systems in recent years.1-3 These synthetic analogues of natural amphiphiles such as phospholipids self-assemble into nanostructures in water because of the opposing interactions of the respective hydrophilic and hydrophobic parts.4,5 Their ability to self-assemble has been extensively investigated to reveal a vast array of different isotropic and lyotropic mesophases.2,3,6 Amphiphilic block copolymer assemblies have found applications in several areas; in particular, the fact that these assemblies form in water makes them potential candidates for biomedical applications and devices. Isotropic nanostructures such as polymeric micelles and vesicles (polymersomes7) have already been reported *Corresponding author. E-mail:
[email protected]. Tel: +44 (0)114 222 5962. Fax: +44 (0)114 222 5945. (1) F€orster, S.; Plantenberg, T. Angew. Chem., Int. Ed 2002, 41, 688–714. (2) Smart, T.; Lomas, H.; Massignani, M.; Flores-Merino, M. V.; Perez, L. R.; Battaglia, G. Nano Today 2008, 3(3-4), 38–46. (3) Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol. Rapid Commun. 2009, 30 (4-5), 267–277. (4) Discher, D. E.; Eisenberg, A. Science 2002, 297(5583), 967–973. (5) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525–1568. (6) Paschalis, A.; Bjorn, L. Amphiphilic Block Copolymers: Self-Assembly and Applications; Elsevier Science: Amsterdam, 2000. (7) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284(5417), 1143–1146. (8) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113–131. (9) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science 2003, 300, 615–618. (10) Kim, Y.; Dalhaimer, P.; Christian, D. A.; Discher, D. E. Nanotechnology 2005, 16, S484–S491. (11) Giacomelli, C.; Men, L. L.; Borsali, R.; Armes, S. P.; Lewis, A. L. Biomacromolecules 2006, 7, 817–828. (12) Lomas, H.; Canton, I.; MacNeil, S.; Du, J.; Armes, S. P.; Ryan, A. J.; Lewis, A. L.; Battaglia, G. Adv. Mater. 2007, 19, 4238–4243. (13) Lam, J. K. W.; Ma, Y.; Armes, S. P.; Lewis, A. L.; Baldwin, T.; Stolnik, S. J. Controlled Release 2004, 100, 293–312. (14) Chim, Y. T. A.; Lam, J. K. W.; Ma, Y.; Armes, S. P.; Lewis, A. L.; Roberts, C. J.; Stolnik, S.; Tendler, S. J. B.; Davies, M. C. Langmuir 2005, 21, 3591–3598.
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as drug carriers,4,8-11 delivery vectors,12-16 and image contrast agents.17,18 Battaglia reported the formation of an interconnected vesicular gel as the concentration of a low-molecular-weight polymersome dispersion was increased.19 This structure was also found to be obtainable by addition of poly(ethylene oxide) homopolymer (PEO) to the same polymersome dispersion.20 The dispersed isotropic phases can be considered to be colloidal dispersions and as such it is possible to disrupt the forces that control their stability by altering the interparticle forces. Much work has been done investigating the effects of adding polymers to colloidal systems using both adsorbing polymers21-26 and nonadsorbing polymers.27-34 In (15) Deshpande, M. C.; Davies, M. C.; Garnett, M. C.; Williams, P. M.; Armitag, D.; Bailey, L.; Vamvakaki, M.; Armes, S. P.; Stolnik, S. J. Controlled Release 2004, 97, 143–156. (16) Kakizawa, Y.; Kataoka, K. Adv. Drug Delivery Rev. 2002, 54(2), 203–222. (17) Meng, F.; Engbers, G. H. M.; Feijen, J. J. Controlled Release 2005, 101, 187. (18) Ghoroghchian, P. P.; Frail, P. R.; Susumu, K.; Blessington, D.; Brannan, A. K.; Bates, F. S.; Chance, B.; Hammer, D. A.; Therien, M. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102(8), 2922–2927. (19) Battaglia, G.; Ryan, A. J. Macromolecules 2006, 39, 798–805. (20) Smart, T. P.; Fernyhough, C.; Ryan, A. J.; Battaglia, G. Macromol. Rapid Commun. 2008, 29(23), 1855–1860. (21) Cabane, B. J. Phys. Chem. 1977, 81(17), 1639–1645. (22) Shang, B. Z.; Wang, Z. W.; Larson, R. G. J. Phys. Chem. B 2008, 112(10), 2888–2900. (23) Balazs, A. C.; Huang, K. L.; Pan, T. Colloids Surf., A 1993, 75, 1–20. (24) Meszaros, R.; Varga, I.; Gilanyi, T. J. Phys. Chem. B 2005, 109, 13538– 13544. (25) Winnik, F. M. Langmuir 1990, 6, 522–524. (26) Blokhus, A. M.; Klokk, K. J. Colloid Interface Sci. 2000, 230(2), 448–451. (27) Lu, P. J.; Zaccarelli, E.; Ciulla, F.; Schofield, A. B.; Sciortino, F.; Weitz, D. A. Nature 2008, 453(7194), 499–503. (28) Zaccarelli, E.; Lu, P. J.; Ciulla, F.; Weitz, D. A.; Sciortino, F. J. Phys.: Condens. Matter 2008, 20, 494242. (29) Ye, X.; Narayanan, T.; Tong, P.; Huang, J. S.; Lin, M. Y.; Carvalho, B. L.; Fetters, L. J. Phys. Rev. E 1996, 54(6), 6500–6510. (30) Ye, X.; Narayanan, T.; Tong, P.; Huang, J. S. Phys. Rev. Lett. 1996, 76(24), 4640–4643. (31) Jenkins, P.; Snowden, M. Adv. Colloid Interface Sci. 1996, 68(1-3), 57–96. (32) Eisenriegler, E.; Bringer, A. J. Phys.: Condens. Matter 2005, 17, S1711. (33) Vincent, B. Colloids Surf. 1987, 24(4), 269–282. (34) Abbas, S.; Lodge, T. P. Phys. Rev. Lett. 2007, 99, 137802.
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the case of nonadsorbing polymers, the aggregation or flocculation of the colloidal particles is due to depletion interactions that give rise to attractive interparticle forces,31,35,36 whereas in the case of adsorbing polymers such as PEO, it is reported that the polymer adsorbs at the colloidal interface creating a beaded necklace type structure.21,23 In our system, adding PEO to amphiphilic block copolymer assemblies we have found that the type of aggregation that occurs, both adsorption and depletion forces, can be controlled by the concentration of the added PEO homopolymer. The effects of PEO on amphiphile assemblies has been reported in the literature before, but all with small ionic surfactants such as sodium alkylcarboxylates and sodium dodecyl sulfate (SDS).21-26 In our system, the PEO homopolymer is chemically identical to the polymersome corona. In the PEO-SDS system, the added PEO partially adsorbs at the hydrophobe-hydrophile interface and loops round the micelles creating the ‘beaded necklace’ structure and the resultant aggregates sediment creating a transient gel.21,23 This is due to PEO being amphiphilic in character; it will freely dissolve in water but can also be drawn into organic phases.37 Meszaros et al. showed that the network formation of the SDS-PEO system is highly dependent on the PEO molecular weight.24 The PEO chains must be larger than a critical molecular weight (1000 Da for SDS micelles) in order to form the beaded necklace structure. We have observed a similar effect with E16B22 polymersomes, where aggregation into a network occurred only when the PEO added was above a critical molecular weight of 4000 Da. However, below the critical molecular weight small scale fusion took place between 2 and 3 polymersomes, resulting in single larger polymersomes.20 We have expanded on this work here to show how the corona thickness of the polymersomes,38 the hydrodynamic radius of the added PEO,39 and the PEO concentration control the kinetics of the aggregation process and final structure of the aggregated gel.
2. Experimental Section 2.1. Materials. Chloroform and PEO (600, 1000, 2000, 4000, 5000, 10000, and 20000 Da) were purchased from Sigma-Aldrich and used without further purification. EnBm block copolymers were synthesized using sequential anionic living polymerization and characterized by gas permeation chromatography (GPC) and 13 C NMR spectroscopy.40 Full details of the block copolymers molecular weight and volume fraction are shown in Table 1. EnBm polymersomes were prepared by thin film rehydration. The copolymer was dissolved in a minimal amount of chloroform (∼4 mg/mL) then placed under a vacuum to evaporate the chloroform, leaving a thin film of polymer. Water was then added to the desired concentration, and the solution was stirred overnight before being passed through an extruder 15 times to ensure narrow polymersome size distributions. 2.2. Methods. Dynamic Light Scattering (DLS). A Brookhaven Instruments laser light scattering system was used fitted with a Melles Griot 633mn He-Ne 75mW laser and a BIAPD detector. Data were analyzed using Brookhaven Instruments particle sizing software using the CONTIN algorithm.41 (35) Asakura, S.; Oosawa, F. J. Chem. Phys. 1954, 22(7), 1255–1256. (36) Vliegenthart, G. A.; Van der Schoot, P. Europhys. Lett. 2003, 62(4), 600– 606. (37) Spitzer, M.; Sabadini, E.; Loh, W. J. Phys. Chem. B 2002, 106, 12448– 12452. (38) Smart, T. P.; Mykhaylyk, O. O.; Ryan, A. J.; Battaglia, G. Soft Matter 2009, in press. (39) Kawaguchi, S.; Imai, G.; Suzuki, J.; Miyahara, A.; Kitano, T.; Ito, K. Polymer 1997, 38(12), 2885–2891. (40) Booth, C.; Yu, G.-E.; Nace, V. M. Self-assembly in simple and complex systems Block copolymers of ethylene oxide and 1,2-butylene oxide. In Amphiphilic Block Copolymers: Self-Assembly and Applications; Paschalis, A., Bjorn, L., Eds.; Elsevier Science: Amsterdam, 2000. (41) Provencher, S. W.; CONTIN, A Comput. Phys. Commun. 1982, 27(3), 229– 242.
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Table 1. Details of the EnBm Block Copolymers Useda polymer EnBm
ΦE
Mn (Da)
corona thickness, d (nm)
0.280 2300 1.99 E16B22 0.290 3840 2.71 E28B36 0.286 6550 4.06 E47B62 0.380 6600 2.92 E31B54E31 0.361 7150 5.28 E64B60 0.270 7300 4.28 E50B70 0.356 7700 5.56 E68B65 0.355 8730 3.42 B37E77B37 0.371 12500 8.92 E115B103 a ΦE is the volume fraction of the hydrophilic E block and Mn is the number average molecular weight of the block copolymer as determined by 13C NMR. The corona thickness d of the polymersomes was calculated from previous work.38
This is a way of analyzing the autocorrelation function to derive the particle size distributions. It uses an inverse Laplace transform and is ideal for polydisperse systems such as these aggregating polymersomes. All solvents were filtered through a 0.2 μm PTFE filter and all glassware was rinsed with filtered deionised water to ensure dust free sample preparation. All measurements were run at 90°. One ml of EnBm polymersome solution (0.5% w/w) was run for 7 min to determine the initial particle size distribution before 1 mL of PEO solution of varying concentration and molecular weight was added. This was run for 1 min every minute for 10 min, or greater depending on the rate of aggregation. The particle size was plotted against time to reveal the aggregation process, where the gradient is the rate of aggregation (nm min-1) (Figure 1). The rate of aggregation from each run was then plotted against each variable, PEO concentration and PEO molecular weight, to give the graphs shown in Figures 2 and 4. Small Angle X-ray Scattering (SAXS). Samples were run at beamline 6.2 at Daresbury Laboratories SRS, Warrington, UK; using an X-ray wavelength of 1.4 A˚ and a camera length of 3.25 m, giving a scattering vector range 0.014 e q e 0.230 A˚-1. Samples were also run at BM26 at ESRF, Grenoble, France; using a camera length of 2 m and an X-ray wavelength of 1.55 A˚ giving a scattering vector range 0.020 e q e 0.234 A˚-1. For static experiments a small amount of the aggregate was placed in the center of a steel washer and sealed with Kapton tape and run for 5 min. For kinetic experiments the polymersome dispersion and PEO solution were mixed then immediately placed in a liquid cell in the X-ray beam. All data were normalized and background corrected before analysis. Scanning Electron Microscopy (SEM). A FEI Inspect F field-emission gun SEM was used. 0.25 mL of the resulting aggregate was rapidly frozen on a stub to ensure no ice crystals had time to form. The frozen sample was then freeze-dried before being sputter-coated with gold. Aggregation and Phase Separation. The aggregation process was followed by taking images of the aggregates forming using a Pixelink pl-742 camera every 30 s over a 48 h period. The particle jamming42 and phase separation of the aggregates was calculated from measuring the standard deviation of the sample intensity using these images.
3. Results and Discussion Aggregation of a range of polymersome dispersions was induced by the addition of PEO. A number of EnBm, EnBmEn, and BmEnBm polymersome forming block copolymers with varying molecular weight were used. The effect the relationship between the polymersome membrane and PEO molecular weight had on the rate of aggregation was determined by the change in particle size, monitored by DLS. Varying the molecular weight of (42) Trappe, V.; Prasad, V.; Cipelletti, L.; Segre, P. N.; Weitz, D. A. Nature 2001, 411(6839), 772–775.
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Figure 1. Plot of the particle size as a function of time as determined by DLS for several EnBm, EnBmEn, and BmEnBm block copolymer polymersomes of varying molecular weight.
Figure 2. (A) Graph of rate of aggregation as a function of the radius of gyration: corona thickness Rg/d ratio using a fixed PEO molecular weight of 10 000 Da (Δ) and 20 000 Da (O) and varying polymersome corona thickness. (B) Schematic showing Rg and d used in A.
the polymersome membrane changes the corona thickness by the relation d ≈ N, where d is the corona thickness and N is the number of ethylene oxide units in the EnBm block copolymer.38 Increasing the molecular weight of the added PEO affects the radius of gyration of the PEO in solution by the equation R2g ¼ 4:08 10 -18 Mw1:16
ð1Þ
where Rg is the radius of gyration in cm and Mw is the molecular weight of the PEO in Da.39 The particle size measured by DLS was plotted over time with a linear increase in particle size observed (Figure 1). The slope of this plot gave the rate of aggregation and was used in further analysis to monitor the effects of molecular weight and concentrations of PEO and polymersomes on the rate of aggregation (Figures 2 and 4). The PEO molecular weight was fixed at 10 000 and 20 000 Da (constant PEO Rg) while varying the molecular weight of polymersome dispersions (varying corona thickness d ). In a previous experiment, the polymersome membrane molecular weight (and so corona thickness d), was fixed at 2300 Da, where a series of different molecular weight (varying Rg) PEO was added, Figure 1b from ref 20. The rates of aggregation were plotted as a function of the radius of gyration: corona thickness ratio, Rg/d (Figure 2). Langmuir 2010, 26(10), 7425–7430
As we showed in Figure 1b from ref 20, a critical PEO molecular weight is required to induce mass aggregation. Here, we use 10k and 20k Da PEO to induce aggregation and vary the corona thickness of the polymersomes. Shown in Figure 2a is the ability to control the rate of aggregation by changing the Rg/d ratio. Figure 2b shows a schematic of the polymersome membrane including the corona thickness, d, and the radius of gyration of the added PEO, Rg. The aggregation kinetics was also studied by SAXS to monitor the structure formation, where it was seen that the structural peaks were seen instantly (Figure 3A). Clearly the formation of the EnBm-PEO aggregates and the subsequent change in structure are rapid. The change in structure happens as the polymersomes aggregate on an equally fast time scale. Figure 3A shows a shift in the d-spacing of the structural peak over time which is plotted alongside the increase in scattering intensity in Figure 3B. The change in d-spacing was fitted with a power law y = a þ bx0.5 showing that the aggregation process is diffusion-dependent. Second the effect of PEO concentration on EnBm polymersomes was also investigated. Different molecular weight PEO samples were made up to a range of concentrations and added to E16B22 polymersome dispersions giving a concentration of 0.25% w/w E16B22. The particle size was measured over time for the each DOI: 10.1021/la902898w
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Figure 3. SAXS data of the aggregation of E16B22 polymersomes with 5% w/w added PEO (10000 Da). (A) Scattering peaks of the forming E16B22-PEO aggregate over the first hour after PEO addition. (B) Plot of intensity (4) and d-spacing (o) of the q* peak with time fitted to y = a þ bx0.5.
Figure 4. DLS and SAXS data for E16B22-PEO aggregates formed with PEO of varying concentration and molecular weight. (A) Rate of aggregation of 0.25% w/w E16B22 polymersomes as a function of PEO molecular weight and concentration, obtained by DLS. (B) Rate of aggregation of E16B22 polymersomes as a function of both PEO (10 000 Da) and PMPC (42000 Da) concentration obtained by DLS. (C) Structural information obtained by SAXS of E16B22-PEO aggregates. Aggregates formed by 0.25% w/w E16B22 and 10000 Da PEO at 8, 12, and 15% w/w concentration.
PEO concentration range to determine the rate of aggregation which was plotted as a function of PEO concentration, (Figure 4A). The rate of aggregation increased with increasing PEO concentration but there was also a critical PEO concentration needed to induce aggregation of E16B22 polymersomes ,which shifted with different molecular weight PEO. For lower molecular weight PEO a higher concentration was needed to induce aggregation. This shows the rate of aggregation can be carefully and accurately controlled by manipulating both the concentration and molecular weight of the PEO. For comparison a nonadsorbing hydrophilic homopolymer poly(2-methacryloxyethyl phosphorylcholine) (PMPC) was added in a range of concentrations to the E16B22 polymersome dispersions. This is shown alongside PEO data in Figure 4B. Figure 4B shows that a steady rate of aggregation, approximately 600 nm min-1 is observed for increasing PEO concentration, followed by a further increase in rate at much higher concentrations of added PEO. At these higher PEO concentrations the rate of aggregation increased rapidly becoming impossible to measure accurately using DLS. The addition of the non adsorbing PMPC did not have the same effect on the rate of aggregation. As with PEO, a critical PMPC concentration was needed to induce aggregation of the E16B22 polymersomes; however, no steady rate was observed at higher concentrations. The rate of aggregation increased rapidly with PMPC concentration, equal to that of higher PEO concentrations. This suggests two regimes of E16B22 polymersome aggregation depending on PEO concentration compared to only one seen 7428 DOI: 10.1021/la902898w
for aggregation induced by PMPC; PEO adsorption at lower concentrations and depletion forces at higher concentrations. This is due to the PEO and PMPC having slightly different behaviors in water. Both PEO and PMPC are hydrophilic polymers but PEO has partial amphiphilic character and this is the key to understanding its behavior here. The PEO adsorbs at the hydrophobe-water interface of the polymersomes causing flocculation at lower concentrations before depletion forces become more dominant as the concentration is increased. This is shown by the two regimes of aggregation (Figures 4b and 5). PMPC does not have this ability; thus only one regime of aggregation is observed, induced by depletion forces (Figure 4b). The structure of several E16B22-PEO aggregates formed with different PEO concentrations were analyzed by SAXS. Figure 4C shows that the nanostructure of the aggregates does not change with increased PEO concentration; there is however, an increase in scattering intensity showing a more ordered structure is formed with increased PEO concentration. The structure of the EB-PEO aggregates is not certain but the SAXS data shows it to be a disordered lamellar type structure. Previous work20 showed the aggregates to be a network of polymersomes interconnected with tubes that fits with the lamellar type structure shown by the SAXS data here, because the polymersome and tube membranes are a highly curved lamellar membrane. The scattering peaks of the EB-PEO aggregates are near identical to those seen for E16B18 at 20% w/w concentration characterized by Battaglia et al.19 Langmuir 2010, 26(10), 7425–7430
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Figure 5. Aggregates formed by adding PEO of varying concentrations to E16B22 polymersome dispersions. (A-F) 0.25% w/w E16B22 and (G-L) 0.5% w/w E16B22. From left to right, each of the two polymersome batches were mixed with 3, 5, 8, 10, 15, and 20% w/w PEO solutions. This is the final image of a 48 h time series to determine the onset of particle jamming, shown in the plot jamming time as a function of PEO concentration.
Figure 6. SEM micrographs of E16B22-PEO aggregates. (A) Transient gel formed with 0.25% w/w E16B22 and e8% w/w 10000 Da PEO; these aggregate’s sediment. (B) Aggregate formed with 0.25% w/w E16B22 and g10% w/w 10000 Da PEO; these aggregate’s cream (the packing of these spheres is an artifact from freeze-drying).
To better understand the kinetics of aggregation in these systems a range of PEO concentrations were added to E16B22 polymersome dispersions and the vials sealed. Images were taken every 30 s for 48 h to monitor the aggregation process, to determine the onset of particle jamming. The jamming time is the point where the growing aggregates become too large to be supported in solution and precipitate out. The final image from the series of aggregates forming is shown in Figure 5. Samples A-F contained a 0.25% w/w E16B22 concentration and samples G-L were 0.5% w/w E16B22. Each sample in the series A-F and G-L used a different PEO concentration of 3, 5, 8, 10, 15, and 20% for A-F and G-L, respectively. It was observed that the jamming time increased with increasing PEO concentration (Figure 5) due to an increase in viscosity of the solution, even though the rate of aggregation increases with PEO concentration (Figure 4B). The graph in Figure 5 using data obtained from the aggregation series shows a good second- order polynomial fit of PEO concentration increasing with jamming time of the aggregates. A faster jamming time was observed for higher E16B22 Langmuir 2010, 26(10), 7425–7430
concentrations (Figure 5). The onset of particle jamming and phase separation in the solution could only accurately be measured for the lower PEO concentration samples A-C and G-I; the higher concentration samples D-F and H-J were dominated by the high viscosity of the solutions where it became less clear. Those aggregates formed with e8% w/w PEO (A-C and G-I) sedimented, whereas those with g10% w/w PEO (D-F and J-L) creamed. Samples of the different aggregates were freeze-dried for SEM analysis. Figure 6 shows the aggregates formed by addition of e8% (6A) and g10% w/w (6B) PEO, respectively. The slower rate of aggregation with lower PEO concentrations (e8% w/w) allows the formation of a large interconnected network to grow that eventually becomes too large to support itself, with a transient gel sedimenting in solution (Figure 6A). The diameter of the interconnected tubes/vesicles is in the range 100-300 nm which fits as the starting polymersomes were extruded to ∼200 nm before PEO addition. However, adding PEO concentrations g10% w/w gave such rapid aggregation that a large interconnected network did not have time to form. Instead, the polymersomes pack in to DOI: 10.1021/la902898w
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1-2 μm spheres (Figure 6B) that cream. This phenomenon corresponds very well with the DLS data measuring rate of aggregation as a function of PEO concentration (Figure 4B). The plot in Figure 4B shows a steady rate of aggregation with lower PEO concentrations with a rapid increase at 8% w/w PEO where the rate became too fast to measure accurately by DLS. The data shows two different mechanisms of polymersome aggregation dependent on PEO concentration. For lower PEO concentrations e8% w/w, the PEO partially adsorbs at the hydrophobewater interface, looping around and creating a beaded necklace structure as seen in work on PEO and SDS micelles;21-26 this allows the formation of the large interconnected transient gel (Figure 6A). At higher PEO concentrations g10% w/w, the PEO causes mass aggregation of the colloidal polymersomes by depletion forces; in this case, the polymersomes aggregate rapidly in to the large spheres (Figure 6B) as there is not time for a large interconnected gel to form. PEO has a density of F = 1.12 kg dm-3 so is denser than water. The transient gel formed with PEO forming part of the structure thus sediments, while PEO is not present in the aggregates formed by depletion forces and so creaming occurs instead. Similarly, all the E16B22-PMPC aggregates cream. PMPC has a density F = 1.20 kg dm-3 thus the fact that the rate of aggregation of E16B22 by nonadsorbing PMPC is similar to that by higher concentration PEO, and both aggregates cream proves the aggregation mechanism is depletion forces. The SAXS data shown in Figures 3A and 4C shows the same scattering peaks for the aggregates at 5, 8, 12, and 15% w/w added PEO. This shows there is no change in the disordered lamellae nanoscale structure of the aggregates, but as Figures 5 and 6 clearly illustrate, there is a difference in the larger scale structure which leads to the sedimenting or creaming of the aggregates. The rate of E16B22 aggregation by addition of PMPC was similar to that of high concentration PEO samples. As PMPC is a non adsorbing polymer (unlike PEO) the only mechanism for aggregation is by depletion forces, and indeed the rates of aggregation indicate this is the case. The E16B22-PMPC aggregates cream in solution at all concentrations, the same as high concentration E16B22-PEO aggregates, rather than forming a large interconnected transient
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gel. The PMPC data confirms the proposed mechanisms for E16B22 aggregation by PEO.
4. Conclusion In conclusion, we report the formation of EnBm-PEO aggregates and their structures that strengthens our understanding of our previous work on polymersome fusion and aggregation induced by PEO addition.20 Here we have shown the rate of polymersome aggregation can be carefully controlled by altering the ratio between the polymersome corona thickness d, and the added PEO radius of gyration Rg. The rate of aggregation increased as d was decreased relative to the PEO Rg. SAXS data of the aggregation kinetics showed an increase in the scattering intensity and a shift in the d-spacing consistent with a system that is diffusion dependent. The concentration of the PEO added lead to control over the rate of aggregation as well as the structure of the aggregate formed. Changing the added PEO concentration gave control over whether the EnBm-PEO aggregate sediments or creams. SAXS data showed there was no change in the nanostructure of the aggregates whether they sedimented or creamed, however, SEM micrographs did reveal a change in the microstructure. A large interconnected network was formed with low PEO concentrations (PEO adsorption) sediments, whereas the 1-2 μm spheres formed with higher PEO concentrations (depletion forces) cream. The porosity of these aggregates and their non toxic nature being composed of PEO makes them an ideal candidate for a 3D scaffold for tissue engineering applications. Also, their in situ formation and indeed the ease of their formation could lead to the entrapment of cells for their transport and delivery. Further work is currently being undertaken to test their ability to entrap cells. Acknowledgment. We thank the EPSRC Doctoral Training Account for funding T.S. studentship, the Worshipful Company of Armourers and Brasiers for a bursary Mr. Adam Blanazs for the synthesis of the PMPC homopolymer and Dr. Shaomin Mai and Dr. Christine Fernyhough for the synthesis of the EnBm block copolymers used in this study. G.B. thanks Biocompatibles Ltd for sponsoring part of his lectureship.
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