Synthesis and Aggregation Behavior of Four-Arm Star Amphiphilic

Oct 13, 2006 - We report the first synthesis of amphiphilic four-arm star diblock copolymers ... The polymerization proceeded in an ideal “living”...
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9746

Langmuir 2006, 22, 9746-9752

Synthesis and Aggregation Behavior of Four-Arm Star Amphiphilic Block Copolymers in Water Michael R. Whittaker and Michael J. Monteiro* Australian Institute of Bioengineering and Nanotechnology, School of Molecular and Microbial Sciences, UniVersity of Queensland, Brisbane QLD 4072, Australia ReceiVed June 7, 2006. In Final Form: September 3, 2006 We report the first synthesis of amphiphilic four-arm star diblock copolymers consisting of styrene (STY) and acrylic acid (AA) made using reversible addition-fragmentation chain transfer (RAFT; Z group approach with no star-star coupling). The polymerization proceeded in an ideal “living” manner. The size of the poly(AA132-STYm)4 stars in DMF were small and close to 7 nm, suggesting no star aggregation. Slow addition of water (pH ) 6.8) to this mixture resulted in aggregates of 15 stars per micelle with core-shell morphology. Calculations showed that the polyAA blocks were slightly extended with a shell thickness of 15 nm. Treatment of these micelles with piperidine to cleave the block arms from the core resulted in little or no change on micelle size or morphology, but the polyAA shell thickness was close to 29 nm (33 nm is the maximum at full extension) suggesting a release of entropy when the arms are detached from the core molecule. In this work we showed through the use of star amphiphilic polymers that the micelle size, aggregation number, and morphology could be controlled.

Introduction It is well-known that amphiphilic block polymers can selforganize into micelles, vesicles, or rods depending upon the type of solvent and weight fraction of polymer.1,2 When these blocks are dispersed in water, micelles consisting of a core-shell morphology are usually found, in which the core consists of the hydrophobic block and shell of the hydrophilic block. Micelles of this type can act as nanocontainers to deliver and release water insoluble drugs or molecules in a controlled way,3 and they have the potential for other drug and vaccine delivery devices. The formation and subsequent stability of such micelles are sensitive to temperature, concentration, and mechanical shear (such as the forces experienced in an injection), and thus are limiting their use for in vivo applications.4 Chemically crosslinking either the shell or the core can make these micelles robust, and such systems have been researched as vehicles for the delivery of therapeutic agents,5 the delivery and display of important biomacromolecules,6-10 and agents for imaging applications.11 However, in these micelles it is often difficult to control the size and number of the hydrophilic arms, which are important design features in the controlled release and cell targeting in drug delivery applications. * To whom correspondence should be sent. E-mail: [email protected]. (1) Tuzar, Z.; Kratochvil, P. AdV. Colloid Interface Sci. 1976, 6, 201-232. (2) Zang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168-3181. (3) Teng, Y.; Morrison, M. E.; Munk, P.; Webber, S. E.; Prochazka, K. Macromolecules 1998, 31, 3578-3587. (4) Attwood, D.; Florence, A. T.; Chapman and Hall: London, New York, 1983; p 388. (5) Murthy, K. S.; Ma, Q.; Clark, C. G.; Remsen, E. E.; Wooley, K. L. Chem. Commun. (Cambridge, U.K.) 2001, 8. (6) Thurmond, B. K.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. Nucleic Acids Res. 1999, 27, 2966-2971. (7) Pan, D.; Turner, J. L.; Wooley, K. L. Macromolecules 2004, 37, 71097115. (8) Pan, D.; Turner, J. L.; Wooley, K. L. Chem. Commun. (Cambridge, U.K.) 2003, 19, 2400-2401. (9) Qi, K.; Ma, Q.; Remsen, E. E.; Clark, C. G.; Wooley, K. L. J. Am. Chem. Soc 2004, 126, 6599-6607. (10) Becker, M. L.; Remsen, E. E.; Pan, D.; Wooley, K. L. Bioconjugate Chem. 2004, 15, 699-709. (11) Turner, J. L.; Pan, D.; Plummer, R.; Chen, Z.; Whittaker, A. K.; Wooley, K. L. AdV. Funct. Mater. 2005, 15, 1248-1254.

Newkome et al.12 proposed an alternative methodology in which the nanostructures consisted of a single amphiphilic macromolecule, termed a “unimolecular micelle”. Heise et al.13 suggested that star block copolymers with amphiphilic arms could form such “unimolecular micelles”. The formation of unimolecular micelles in water is difficult, and in most cases star block polymers aggregate into larger micelles. It was found that the aggregation number was dependent on the number of arms: the greater the number arms the lower the aggregation number.14 The driving force for aggregation is the reduction of surface area of the insoluble block in the nonsolvent. This forces the micelle to adopt constrained geometries, and thus aggregation is in competition with the loss of entropy due to deformation of hydrophilic and hydrophobic blocks and the number of arms attached to the core molecule. The aim of this work is to first synthesize four-arm star block copolymers with amphiphilic arms, in which the size of the polystyrene core is varied while the poly(acrylic acid) block length in the shell is kept constant. The Z group RAFT methodology was used to synthesize these amphiphilic star block copolymers. The second aim is to characterize their micellization behavior in water and determine their radius of gyration, hydrodynamic diameter, and aggregation number. In particular, we wanted to investigate the correlation between micelle size and polystyrene block length in the star as well as investigate the micellization behavior after release of the arms from the core molecule. It has been shown that a variant of the RAFT process (Z group approach, see Scheme 1)15 offers a significant advantage over other R group approaches16,17 or other living free radical (12) Newkome, G. R.; Moorefield, C. N.; Baker, G. R.; Saunders, M. J.; Grossman, S. H. Angew. Chem. 1991, 103, 1207-1209. (13) Heise, A.; Hedrick, J. L.; Frank, C. W.; Miller, R. D. J. Am. Chem. Soc. 1999, 121 (37), 8647-8648. (14) Huh, J.; Kim, K. H.; Ahn, C.-H.; Jo, W. H. J. Chem. Phys. 2004, 121, 4998-5004. (15) Mayadunne, R. T. A.; Jeffery, J.; Moad, G.; Rizzardo, E. Macromolecules 2003, 36, 1505-1513. (16) Chiefari, J.; Chong, B. Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecues 1998, 31, 5559-5562. (17) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2005, 58, 379.

10.1021/la0616449 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/13/2006

Amphiphilic Four-Arm Star Diblock Copolymers

Langmuir, Vol. 22, No. 23, 2006 9747

Scheme 1. ‘Arms First’ Approach To Make Amphiphilic Four-Arm Star Diblock Copolymers

techniques such as atom transfer radical polymerization (ATRP) and nitroxide mediated polymerization (NMP) for the synthesis of star polymers. To carry out a RAFT-mediated polymerization via the Z group method, one must use a trithiocarbonate in which the Z group is located at the core molecule (junction point). Growth of the chain arms away from the core means that the trithiocarbonate is always located at the junction point of the star polymer. Cleavage of the trithiocarbonate moieties (junction points) linking the diblock arms together is facile in the presence of piperidine. The Z group approach inherently eliminates any star-star coupling reactions, and the dead polymer consists only of linear dead species. Therefore, the purity of the targeted star polymer is much higher than when using the R group approach. Considering the advantage of the Z group approach it is surprising that there are no reports of the synthesis of amphiphilic block polymer stars (consisting of polystyrene and poly(acrylic acid)) using the RAFT technique. Experimental Section Materials. tert-Butyl acrylate (tBA, 99% pure, Aldrich) and styrene (STY, 99% pure, Aldrich) were passed through a column of basic alumina (activity I) to remove inhibitor immediately prior to use. 2,2-Azobis-(isobutyronitrile) (AIBN, 98% pure, Fluka) was recrystallized twice from methanol prior to use. HPLC grade toluene (LABSCAN 99.8%pure) and tetrahydrofuran (LAB-SCAN, 99.8% pure) were used as received. Pentaerythritoltetrakis-(3-(S-benzyltrithiocarbonyl)propionate) (1): Star-RAFT Agent.15 Triethylamine (20.2 g) in 50 mL of CHCl3 was added dropwise to a stirred solution of pentaerythritol (3-mercaptopropionate) (2.44 g, 5 mmol) and carbon disulfide (15.25 g, 0.2 mol) in CHCl3 (75 mL) at room temperature. The solution gradually turned deep yellow during the addition and was allowed to stir for 1 h. Benzyl bromide (18.8 g, 110 mmol) dissolved in 50 mL of CHCl3 was then added dropwise, and the solution was stirred for 2 h. The mixture was poured into a cold solution of 10% aqueous HCl and extracted three times with ethyl acetate to afford a thick yellow oil. The oil was purified by column chromatography using 30% ethyl acetate in n-hexane as eluent to obtain the title compound as a thick viscous yellow oil. 1H NMR: 2.9 (8H, CH2), 3.6 (8H, CH2), 4.2 (8H, CH2), 4.6 (8H, benzyl CH2), 7.3 (20H, ArH).

Synthesis of Four-Arm Poly(tBAn)m Stars. A typical synthesis of a four-arm poly(tBA) star was as follows: A solution of RAFT agent (1) (29.3 mg, 2.54 × 10-5 mol) in 3.0 mL (2.598 g, 0.020 mol) of tBA monomer freshly purified from the inhibitor was added to 12 mL of AIBN solution (9.15 × 10-4 M) in toluene. The polymerization solution was then equally measured into four glass ampules, degassed by three successive freeze-evacuate-thaw cycles under high vacuum, and flame-sealed. The glass ampules were heated at 60 °C in a temperature controlled oil bath and removed at different times. The polymerization was stopped by quenching the polymerization mixture in liquid nitrogen. The polymer was dried to constant weight under vacuum, and conversion of monomer to polymer determined gravimetrically. The molecular weight distribution of the polymer samples was determined by size exclusion chromatography (SEC). Chain Extension of Star(tBA) with Styrene. The chain extension of four-arm poly(tBA132)4 star with styrene results in the formation of a star diblock copolymer in which the poly(STY) is located in the core and the poly(tBA) in the shell. A typical polymerization was as follows: poly(tBA) (Mn ) 52 000 by light scattering-SEC; 3.00 g, 5.76 × 10-5 mol) and AIBN (0.66 mg, 4.0 × 10-6 mol) were dissolved in 20 mL of styrene monomer freshly purified from inhibitor (18.18 g, 0.175 mol). This mixture was then equally weighed into five glass ampules, degassed by three successive freeze-pumpthaw cycles, and flame sealed. The glass ampules were placed in an oil bath at 60 °C, and each ampule was removed at a designated time. The polymerizations were stopped by placing the ampule in liquid nitrogen. The polymer was dried to constant weight under vacuum, and conversion of monomer to polymer was determined gravimetrically. The molecular weight distribution of the polymer samples were determined by SEC. Cleavage of Trithiocarbonate Units Linking Arms to the Core Molecule. Piperidine (0.1 mL) was added to a solution of four-arm star polymer (20 mg) in 1 mL of THF. The solution was stirred at room temperature for 17 h and dried to constant weight in a vacuum oven, and the resulting polymer was analyzed by refractive index (RI) and UV (310 nm) SEC to detect the change in molecular weight and loss of trithiocarbonate groups in the star. Synthesis of Amphiphilic Four-Arm Poly(AA-b-STY) Star. The hydrolysis of tBA side groups on the polymer to acrylic acid

9748 Langmuir, Vol. 22, No. 23, 2006 (AA) was carried out following a literature procedure.18 TFA (5 times excess to the tert-butyl groups, 133 mg, 1.17 mmol) was added dropwise to a solution of four-arm poly(tBA132)4 (30 mg, 0.23 mmol) in 0.5 mL of DCM. The reaction mixture was stirred at room temperature for 17 h after which the solution became cloudy with observed precipitation, indicating hydrolysis of the tert-butyl groups. The sample was dried to constant weight in a high vacuum oven at room temperature. The hydrolyzed material was redissolved in 1 mL of DMF. Conservation of the RAFT trithiocarbonate groups after hydrolysis was confirmed by UV-vis spectroscopy, in which no change in the absorption peak at 310 nm was observed. This procedure was used to hydrolyze all of the four-arm star diblock copolymers. Micelle Formation in Water. Micelles consisting of four-arm amphiphilic polymers were formed by the slow addition of deionized water (0.025 mL min-1; 24 mL in total) to the polymer solution (10 mg in 1 mL of DMF). The micelles were exhaustively dialyzed against deionized water (pH ) 6.8) using dialysis tubing (Pierce Snakeskin, MWCO 3K). The hydrodynamic diameter, aggregation number, and radius of gyration were determined using dynamic light scattering (DLS) and asymmetric field flow fractionation (FFF). Cleavage of Trithiocarbonate Linking Arms to the Core Molecule after Micellization. The cleavage of the trithiocarbonate linking the arms to the core molecule after micellization was carried out as follows. Piperidine (0.1 mL) was added dropwise to 10 mL of micelles dispersed in water. The reaction was allowed to proceed for 20 h at room temperature with stirring. The reaction mixture was then dialyzed against deionized water (pH ) 6.8) water for 3 days. Removal of all piperidine was found when the pH decreased and remained constant at 6.8. Cleavage of the arms from the core molecule was confirmed from the loss of UV absorbance at 310 nm corresponding to trithiocarbonate moieties. The hydrodynamic diameter, aggregation number, and radius of gyration of the resulting linear diblock copolymers in water were determined using dynamic light scattering (DLS) and asymmetric field flow fractionation (FFF). UV-vis Spectroscopy. UV-vis spectra from 200 to 800 nm were recorded on a Perkin-Elmer Lambda 2 series spectrometer at ambient temperature using a 1 cm quartz cell. Size Exclusion Chromatography (SEC). Absolute molecular weights of the star polymers were determined by size exclusion chromatography (Shimadzu system with a Wyatt DAWN DSP multiangle laser light scattering (MALLS) detector (683 nm) and a Wyatt OPTILAB EOS interferometric refractometer). THF was used as the eluent with three Phenomenex phenogel columns (500, 104, and 106 Å) connected in series operated at 1 mL min-1 with column temperature set at 30 °C. The refractive index increments (dn/dc) for the polymers analyzed were determined using the chromatographic method after calibration of the refractometer response with a linear polystyrene standard (Mw ) 110 000, dn/dc ) 0.185). The light scattering data was analyzed using the Zimm method fitting a first-order polynomial with Astra V4.90.07 software. SEC measurements of the star and cleaved arms were also performed using a Waters Alliance 2690 separations module equipped with an autosampler, column heater, differential refractive index detector, and a photodiode array (PDA) connected in series. HPLC grade tetrahydrofuran was used as the eluent at a flow rate of 1 mL min-1. The columns consisted of three 7.8 × 300 mm Waters Styragel GPC columns connected in series, comprising two linear Ultrastyragel and one Styragel HR3 columns. Polystyrene standards ranging from 2 000 000 to 517 g mol-1 were used for calibration. Dynamic Light Scattering (DLS). Dynamic light scattering measurements were performed using a Malvern Zetasizer Nano Series running DTS software and operating a 4 mW He-Ne laser at 633 nm. Analysis was performed at an angle of 90° and a constant temperature of 25 °C. Dilute particle concentrations ensure that multiple scattering and particle-particle interactions can be considered negligible during data analysis. All samples were filtered (18) Ma, Q.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 4805-4820.

Whittaker and Monteiro through a 0.2 µm low-absorbing filter immediately prior to analysis. The number-average hydrodynamic particle size is reported (DH). Asymmetric Field-Flow Fractionation (FFF). Apparent molecular weights and the radius of gyration (Rg) of the polymer micelles were determined in Millipore water pH ) 6.8 using an FFF instrument equipped with a Wyatt DAWN EOSP multiangle laser light scattering (MALLS) detector, a Wyatt OPTILAB rEX refractive index detector and Agilent Technologies G1314A UV detector. Flow control and sample injection were controlled with an Agilent Technologies G1310A pump and G1329A autoinjector. Separation was achieved using an FFF membrane (Eclipse 2, Wyatt Technology). The light scattering data was analyzed using the Zimm method fitting a firstorder polynomial with Astra V5.1.9.1 software. Estimates of dn/dc used in the calculation of the apparent molecular weight of the micelles were determined via the Astra software using the quantitative mass recovery technique. Transmission Electron Microscopy (TEM). A drop of the micelle solution was allowed to air-dry onto a formavar precoated copper TEM support grid. To obtain a positive stain the samples were carefully exposed for 5 min to the vapor of a 10% ruthenium tetroxide solution in a sealed container prior to examination. To obtain a negative stain the samples were exposed to a drop of a 2% solution of uranyl acetate for 1 min after which excess staining solution was removed via careful blotting. The polymer nanoparticles were characterized on a JEOL-1010 instrument utilizing an accelerating voltage of 80 kv operating at ambient temperature.

Results and Discussion Design of Multifunctional RAFT Core. The multifunctional RAFT agent (1, core molecule) used in this study was specifically designed with the Z group approach to fragment in such a way as to eliminate unwanted star-star coupling and dormant linear polymers capped with a trithiocarbonate functionality (Scheme 1).15,19 This was achieved by controlling the fragmentation of the multifunctional 1 so that the core was attached to the Z stabilizing group. Radical addition to the trithiocarbonate groups of 1 will result in a benzyl radical (R group) that will react to monomer. Fragmentation of the CH2-S bond is kinetically very slow and insignificant. A comprehensive explanation of this mechanism has been described by Mayadunne et al.15 The trithiocarbonate moieties, which link the arms to the core, are readily cleaved using simple chemistries and provide a facile method to make linear diblock copolymers (Scheme 1). Synthesis of Four-Arm Amphiphilic Star Block Copolymers. The ‘living’ free radical polymerization of tBA with 1 to give the four-arm star polymer, (polytBAn)4, with predetermined number-average molecular weight (Mn) and low polydispersity (PDI) was carried out in toluene at 60 °C. Table 1 gives the Mn and PDI values as a function of conversion, x, when analyzed by SEC using polystyrene (polySTY) standards and absolute determination (using SEC with both light scattering and RI detectors). The Mn values using only a RI detector gave a linear increase in Mn with conversion and a PDI that increased from 1.19 to 1.42. At first sight these results would suggest poor control of the RAFT polymerization. However, when these polymers were analyzed using absolute means (SEC-RI/light scattering), the Mn corresponded closely with that calculated from theory20 and the PDIs were all low and below 1.1, suggesting excellent ‘living’ behavior. The difference between the two analysis methods is that the hydrodynamic volume of the polytBA stars is different to linear polySTY standards due to both the structure (19) Stenzel, M. H.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 4498-4512. (20) Monteiro, M. J. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 31893204.

Amphiphilic Four-Arm Star Diblock Copolymers

Langmuir, Vol. 22, No. 23, 2006 9749

Table 1. Experimental Results from the Polymerization of tert-Butyl Acrylate (1.35 M) in Toluene at 60 °C entry poly(tBA19)4 poly(tBA65)4 poly(tBA102)4 poly(tBA132)4 poly(tBA19) poly(tBA65) poly(tBA102) poly(tBA132)

[RAFT(1 )] (mmol/L)

[AIBN] (mmol/L)

time (min)

x

Mn (theory)

Mna

PDIa

Mnb

PDIb

diameterc (nm)

1.70 1.70 1.70 1.70

0.73 0.73 0.73 0.73

71 243 300 480

0.088 0.330 0.522 0.676

10663 (22512d) 34297 (40080d) 53515 (59048d) 68542 (69684d) 2500e 8533e 13213e 16970e

39039 45951 51945 -

1.03 1.02 1.10 -

7434 26576 31567 39237 5340 9732 14474 17133

1.26 1.19 1.42 1.41 1.23 1.22 1.13 1.16

3.5 5.8 6.3 7.1 -

a

Determined from double detection SEC (RI and light scattering). b Determined from RI detection SEC. c Determined from DLS analysis in THF. [M]ox d MWmonomer Calculated from SEC analysis of the cleaved arms. e Calculated from Mn ) ([RAFT]o - [RAFT]x) + af([I]o - [I]x)

Figure 1. UV-vis spectrum (using a photodiode array detector) taken at SEC peak maximum of the MWD for (A) poly(tBA132)4 star (Mn ) 51 945, PDI ) 1.10, x ) 0.68) before (solid line) and after cleavage of arms (dashed line), and (B) poly(tBA132-block-Sty186)4 star (Mn ) 143 524, PDI ) 1.05, x ) 0.26) before (solid line) and after cleavage of arms (dashed line).

(usually lowers the hydrodynamic volume) and chemical composition and possibly due to band broadening.21 The arms can be cleaved from the core of the star through the trithiocarbonate functionalities using piperidine (a strong nucleophile). Table 1 shows the Mn and PDI analyzed using only an RI detector. The Mns were all close to theory suggesting that most of the stars were converted to the corresponding linear arms. Verification and quantitative determination of cleavage is shown in Figure 1A when analyzed by SEC over a range of UV wavelengths (i.e. from 200 to 400 nm). Prior to piperidine treatment, the absorption curve (curve a, solid line) showed a distinct peak centered at approximately 310 nm corresponding to the trithiocarbonate groups on the core, and after treatment with piperidine this peak disappeared, confirming that the arms were no longer attached to the star. The synthesis of four-arm star block copolymers, poly(tBAb-polySTY)4, in which the first block attached to the core is (21) Netopilil, M. J. Biochem. Biophys. Methods 2003, 56, 79-93.

polySTY and the outer block polytBA, was prepared by chain extension of the (polytBA132)4 (Table 1, experiment 3) with STY and AIBN in bulk at 60 °C. The Mn and PDI values as a function of conversion for the growth of four-arm star block copolymers are given in Table 2. The SEC analysis of the four-arm stars using a polySTY calibration curve gave Mns that were lower than theory and PDIs starting at 1.38 at low conversion and ranging to 1.19 at 26% conversion. Analysis using absolute determination of the molecular weight distribution (MWD) showed that the Mns were slightly greater than theory but the PDIs decreased from 1.17 to 1.05 with conversion. These results suggest that we have produced star polymers with excellent control. Cleavage of the diblock arms from the core using piperidine gave Mns that were all close to theory and PDIs that were slightly higher than the star PDIs. The reason for this is due to random coupling, where the PDI of a star is lower than the PDI of the individual arms.22 Figure 1B shows the SEC traces over a range of UV wavelengths (i.e. from 200 to 400 nm) and also supports that most if not all of the trithiocarbonate linkages have been cleaved. Synthesis of Poly(AA132-STYm)4 Copolymers and Their Micellization Properties in Water. It has been shown that polymer containing tBA units can easily and quantitatively be converted to acrylic acid (AA) units using TFA.18 The four-arm stars of poly(tBA132-STYm)4 were converted to their corresponding nonionized poly(AA132-STYm)4 in DMF using TFA at room temperature (17 h). 1H NMR showed no trace of tBA units (loss of tert-butyl groups at 1.6 ppm), and the UV-vis spectrum (for four-arm star of polytBA) under identical concentrations showed a strong band at 310 nm before and after hydrolysis with TFA (see Figure 2). The latter showed that only the tBA units and not the trithiocarbonate groups have been hydrolyzed with TFA, suggesting little or no arm cleavage. To make the corresponding linear poly(AA132-STYm) species, poly(tBA132-STYm)4 star polymers in DMF were first cleaved with piperidine and then the tBA units were hydrolyzed to AA with TFA. This provided a direct comparison between the micellization properties of stars and their linear amphiphilic diblock copolymers. Table 3 shows the hydrodynamic diameter (DH, as measure by DLS) for the linear and star diblock copolymers in DMF, a good solvent for both polyAA and polySTY. As the number of STY units increased, the values of DH also increased for both linear and star polymers. The DH for stars was greater than that found for their linear diblocks. Under these experimental conditions it can be assumed that these structures are unimolecular and do not aggregate. Deionized water (pH ) 6.8) was slowly added to the solution of polymer in DMF, and the mixture was (22) Tanaka, T.; Donkai, N.; Omoto, M.; Inagaki, H. J. Macromol. Sci., Part B: Phys 1980, 17, 211-228.

9750 Langmuir, Vol. 22, No. 23, 2006

Whittaker and Monteiro

Table 2. Experimental Results from the Chain Extension of Poly(tBA132)4 with Styrene at 60 °C in the Bulk entry poly(tBA132-STY31)4 poly(tBA132-STY39)4 poly(tBA132-STY57)4 poly(tBA132-STY122)4 poly(tBA132-STY186)4 poly(tBA132-STY31) poly(tBA132-STY39) poly(tBA132-STY57) poly(tBA132-STY122) poly(tBA132-STY186)

[RAFT] (mmol/L)

[AIBN] (mmol/L)

time (min)

x

Mn (theory)

Mna

PDIa

Mnb

PDIb

diameterc (nm)

2.88 2.88 2.88 2.88 2.88

0.2 0.2 0.2 0.2 0.2

480 960 1440 2880 4320

0.041 0.052 0.077 0.168 0.256

64777 (81548d) 68168 (89072d) 75808 (92948d) 102918 (121056d) 129353 (144960d) 20222e 22100e 23072e 30099e 36075e

78966 80348 91361 127810 143524 -

1.17 1.12 1.05 1.05 1.05 -

52698 48404 57523 90194 119464 22853 22875 25590 33053 39968

1.38 1.48 1.42 1.34 1.19 1.32 1.30 1.29 1.23 1.20

9.7 9.1 7.8 10.4 12.1 6.1 7.4 5.8 7.3 7.3

a

Determined from double detection SEC (RI and light scattering). b Determined from RI detection SEC. c Determined from DLS analysis in THF. [M]ox d MW + Mn(first - block). Calculated from SEC analysis of the cleaved arms. e Calculated from Mn ) ([RAFT]o - [RAFT]x) + af([I]o - [I]x)

that these results are not artifacts of precipitation. The unique and important feature of FFF is that it allowed the polydispersity of the MWD for the micelles to be determined. It can be seen that all the PDIs were low and below 1.1, suggesting that the MWD of the micelles are very narrow, resulting in a narrow micelle size distribution. The radius of the core Rc can be calculated for amphiphilic block micelles from geometric (space filling) considerations giving the following relationship based on the derivations of Antonietti et al.:24

Figure 2. UV-vis adsorption spectrum of four-arm star, poly(tBA132)4 before (solid line) and after (dotted line) hydrolysis with TFA to give the four-arm star poly(AA132)4. The concentrations of both samples were identical.

exhaustively dialyzed to remove residual DMF solvent and salts formed after hydrolysis with TFA. Upon addition of water, the AA units became ionized, and the solvent mixture gradually became a poor solvent for polySTY. When the critical ratio of water to DMF was reached, the star and linear polymers aggregated into micelles with a core-shell morphology. This transition was observed as the solution went from clear to opaque (bluish tinge). TEM micrographs using negative (Figure 3A) and positive staining (Figure 3B) showed the morphology of these aggregates under our conditions to be spherical (micellular) consisting of a polySTY core and polyAA shell. It can also be seen that the micelles were of similar size (i.e. a narrow micelle size distribution), which was confirmed by field flow fractionation (FFF). The size of the micelles formed from linear diblock copolymers in water is given in Table 3. The radius of gyration (Rg) and weight-average molecular weight (Mw) of the micelles were determined by FFF, and from the Mw values the aggregation number, Z, was calculated. The linear diblock copolymers showed an unusual trend: the DH values decreased with increased NSTY, which was the same trend found from the Rg values except when NSTY equals 122. It should be noted that for diblocks when NSTY is equal to or greater than 122, small amounts of coagulation and precipitation during the micellization process were observed. The decrease in DH and Rg can be accounted for by the decrease in the aggregation number, Z, as NSTY is increased. An increase in NSTY from 31 to 57 gave a decrease in the value of Z from 303 to 160, suggesting that as the polySTY block increased the number of linear diblock aggregates decreased. This is surprising as it has been shown that as NSTY increased the number of aggregates also increased.23 The reason for this trend remains unclear, and the correlation functions for the lower Nsty suggests

4π 3 R ) ZNAVo 3 c

(1)

where Z is the number of block aggregates in the micelle, NA Avogadro’s number, Vo is the molar volume (a value23 of 0.16 was used in this work). If Z is known, then the interchain distance, b, between neighboring chains at the core-shell interface can be calculated as

4πRc ) Zb2

(2)

The value of b2 (proportional to the surface area) gives an indication of the packing density of the hydrophilic arms attached to coresthe more compact the hydrophilic chains, the smaller the value of b. The distance between polyAA chains, b, calculated from eq 2 is based on the assumption that the polySTY chains are in an ideal conformation and that polyAA does not influence this conformation. In principle this is a reasonable assumption. It can be seen that the values of b in Table 3 increased with NSTY from 1.46 to 1.99 nm. This was slightly higher than that found by Burguiere et al. of 1.1 to 1.74 (depending on the block lengths and structure). The extension of the polyAA blocks in water can also be estimated from the difference between RH and Rc (where δc ) RH - Rc), and it was found that regardless of the block length of polySTY, the polyAA blocks were close to full extension (δc,max ) 33 nm). This is in agreement with other work25 where at very low salt concentrations the polyAA block through charge repulsion was fully extended. In the case of the micellization of the amphiphilic four-arm star diblock copolymers in water, it was found that as NSTY on the arms increased from 31 to 122, DH also increased from 39 to 81 nm (see Scheme 2). The Rg showed a different trend, and (23) Burguiere, C.; Chassenieux, C.; Charleux, B. Polymer 2003, 44, 509518. (24) Forster, S.; Zisenis, M.; Wenz, E.; Antonietti, M. J. Chem. Phys. 1996, 104, 9956-9970. (25) Forster, S.; Schmidt, M.; Antonietti, M. Polymer 1990, 31, 781-792.

Amphiphilic Four-Arm Star Diblock Copolymers

Langmuir, Vol. 22, No. 23, 2006 9751

Table 3. Characterization of Linear and Four-Arm Star Amphiphilic Diblock Copolymer Micelles in DMF and Water DM F

water

entry

DH (nm)

DH

Rg

poly(AA132-STY31) poly(AA132-STY39) poly(AA132-STY57) poly(AA132-STY122)

4.8 5.0 5.3 6.0

70.4 67.2 63.1 62.0

26.7 16.2 16.0 23.5

poly(AA132-STY31)4 poly(AA132-STY39)4 poly(AA132-STY57)4 poly(AA132-STY122)4

6.6 7.0 7.3 8.6

39.1 38.1 50.2 82

19.9 16.2 14.4 26.3

poly(AA132-STY31) poly(AA132-STY39) poly(AA132-STY57) poly(AA132-STY122) a

42 46.9 48.4 62.5

Za

PDI

Rc

linear diblock copolymers 303 (76) 1.07 7.19 225 (56) 1.01 7.02 160 (40) 1.02 7.12 star diblock copolymers 15 1.11 17 1.07 8 1.39

4.19 4.71 4.16

b

b′

1.46 1.66 1.99

3.83 4.05 5.21

arm cleavage of star diblock copolymers in water 21 50 (12) 1.04 3.99 1.96 12.4 54 (13) 1.02 4.42 2.09 11.2 37 (9) 1.09 4.33 2.56 15.3

1.92 2.03 2.61

δ c ) RH - Rc (nm)

δc,max (nm)

28.0 26.8 24.4

33 33 33

15.4 14.3 20.9

33 33 33

28.6 28.6 29.2

33 33 33

Z values in parentheses are the four-arm star equivalent.

Figure 3. UV-vis spectra in Millipore water pH ) 6.8 at 25 °C of (a) micellized poly(tBA132-block-Sty39)4 stars, (b) piperidine treated poly(tBA132-block-Sty39)4 stars micelles, and (c) poly(tBA132-blockSty39)4 stars micellized cleaved arms. Scheme 2. Micellization Behavior of Amphiphilic Four-Arm Star Diblock Copolymers in Water Before and After Cleavage of the Core Molecule

it initially decreased from 19.9 (at NSTY ) 31) to 14.4 nm (at NSTY ) 57) and then increased to 26.3 (at NSTY ) 122). This trend was similar to that found for the linear diblocks. The aggregation number was 15 at NSTY equal to 31, increased to 17 (at NSTY ) 39), and decreased to 8 (at NSTY ) 57). In general, the aggregation based on number of arms was much lower than for linear diblocks, suggesting that tethering arms to a core influences the aggregation behavior. The lower aggregation number for the stars suggests that this conformation provided the lowest equilibrium surface energy for the polySTY core. The PDI of the micelles was low, ranging from 1.11 to 1.39, but higher than the linear diblock. For the stars, the b′ value (where b′2 ) b2/4, for a four-arm star)23 can be directly compared with the b values of the linear diblocks. The values of b′ were greater

than the b values for the diblocks and increase from 1.91 to 2.61 nm with NSTY such that the polyAA arms on the star were further apart than in the corresponding linear diblock systems. This result suggests that tethering the arms to a core molecule could change the packing of the star polymers in the micelle. Support for this postulate comes from the values of δc for the polyAA in the shell of the micelle, in which δc is much lower than for the linear diblock, ranging from 15 to 20 nm. It seems there is some entropic penalty for having the arms tethered to a core molecule and thus keeping the polyAA arms closer to the core. The next set of experiments was carried out to study the influence of cleaving the arms from the core and to determine the effect of conformational changes on the size and aggregation number of the micelles. The micelles (aggregates of the stars) were treated with piperidine in water to cleave the arms from the core molecule. Figure 4 shows the UV-vis spectra of the micelles in water. Curve a represents the micelles prior to treatment with piperidine and shows an absorption maximum at 310 nm, corresponding to the -S-(CdS)-S linkages of the diblock arms to the core molecule. After treatment with piperidine this peak was no longer observed (curve b), and the UV-vis spectrum was similar to that of the linear diblocks micellized in water. This result suggests that all the arms have been cleaved from the core molecule, and interestingly, piperidine can diffuse through the glassy polySTY core to the core molecule. The summary of micellization behavior of the star micelles after treatment with piperidine is given in Table 3. The diameter, DH, as measured by DLS showed that the micelle size did not change significantly from its parent fourarm star aggregated micelle, in which DH is 42 nm at NSTY ) 31 and increased to 62.5 nm at NSTY ) 122. Similar values of Rg for the four-arm stars were also found. However, the most compelling evidence to show that there was no linear diblock exchange between micelles was from the values of Z. It can be seen from the Z value in parentheses (which is equivalent to that of the number of four-arm star aggregates) there was little or no change in the aggregation number after treatment with piperidine (Scheme 2). The PDI of the micelles was also very low, ranging between 1.04 and 1.09. The conformation of the polyAA blocks in these micelles can be inferred from the b and δc values. Spacing between polyAA chains in the core-shell interface, b, increased with NSTY from 1.96 to 2.55 nm, which was close to the values found for the four-arm stars micelles. However, the δc values were greater than the four-arm star micelles and similar to the

9752 Langmuir, Vol. 22, No. 23, 2006

Whittaker and Monteiro

chains, resulting in reordering of both the polySTY and polyAA blocks allowing the hydrophilic block to be fully extended (see Scheme 2). This reordering, however, does not change the size or aggregation number (number of block arms) of the micelles.

Conclusion

Figure 4. TEM images of micellized poly(tBA132-block-Sty57)4 fourarm stars (A) negatively stained with uranyl acetate (scale bar: 200 nm) and (B) positively stained with ruthenium oxide vapor micelles (scale bar: 50 nm).

linear diblock. The data suggest that when the arms of the fourarm star are cleaved there is a gain in entropy of the polySTY

In summary, the synthesis of four-arm star block copolymers with amphiphilic arms, in which the length of the polystyrene core was varied while the poly(acrylic acid) block length in the shell was kept constant. The Z group RAFT methodology was used to synthesize these amphiphilic star block copolymers. It was found from the absolute molecular weight determination that the stars were of a very narrow MWD and followed ideal ‘living’ behavior. Hydrolysis of the tBA units on the outer block with TFA gave AA units in quantitative yields. More importantly, the core molecule linking the diblock arms were not cleaved. The four-arm stars of poly(AA132-STYm)4 were then dissolved in DMF and analyzed using DLS. The size of the unimolecular micelles was small and close to 7 nm, and increased slightly with the amount of STY units. Upon slow addition of deionized water (pH ) 6.8), approximately 15 four-arm star polymers aggregated to form a core-shell micelle with an estimated 15 nm extension of the polyAA blocks in the shell. After these micelles were treated with piperidine, to cleave the arms from the core to form linear diblocks, the size of the micelle did not change and the polyAA chains were close to full extension. These results showed that we were able to control the micelle size and morphology using star polymers. There is an entropic penalty in having the arms locked to a core molecule (see Scheme 2), which is released when the arms are cleaved from the core. LA0616449