Biodegradable Star Polymers Functionalized With ... - ACS Publications

Biomacromolecules 2009, 10, 2699–2707

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Biodegradable Star Polymers Functionalized With β-Cyclodextrin Inclusion Complexes Eki Setijadi, Lei Tao, Jingquan Liu,* Zhongfan Jia, Cyrille Boyer, and Thomas P. Davis* Centre for Advanced Macromolecular Design (CAMD), School of Chemical Sciences and Engineering, The University of New South Wales, Sydney, NSW 2052, Australia. Received June 8, 2009; Revised Manuscript Received July 26, 2009

Three-armed biodegradable star polymers made from polystyrene (polySt) and poly (polyethylene glycol) acrylate (polyPEG-A) were synthesized via a “core first” methodology using a trifunctional RAFT agent, created by attaching RAFT agents to a core via their R-groups. The resultant three-armed polymeric structures were well-defined, with polydispersity indices less than 1.2. Upon aminolysis and further reaction with dithiodipyridine (DTDP), these three-armed polymers could be tailored with sulfhydryl and pyridyldisulfide (PDS) end functionalities, available for further reaction with any free-sulfhydryl group containing precursors to form disulfide linkages. Nuclear magnetic resonance (NMR) confirmed that more than 98% of the polymer arms retained integral trithiocarbonate active sites after polymerization. Intradisulfide linkages between the core and the arms conferred biodegradability on the star architectures. Subsequently, the arm-termini were attached to cholesterol also via disulfide linkages. The cholesterol terminated arms were then used to form supramolecular structures via inclusion complex formation with β-cyclodextrin (β-CD). The star architectures were found to degrade rapidly on treatment with DL-dithiothereitol (DTT). The star polymers and supramolecular structures were characterized using gel permation chromatography (GPC), static light scattering (SLS), 2D NMR, and fluorescence spectroscopy.

Introduction Complex polymeric architectures that can be cleaved into their component parts are promising candidates for applications in drug delivery and biotherapeutics. Matyjaszewski and co-workers have described the creation of biodegradable linear polymers1 and hydrogels containing internal disulfide links using ATRP polymerization. Biodegradable hydrogels cross-linked by disulfide linkages were also successfully synthesized by Armes et al.2 Disulfide linkages have also been successfully exploited for the synthesis of functional polymers and well-defined biomolecule-polymer conjugates in recent publications.3-9 Star polymers have attracted increasing interest as they have potential applications in a number of areas, particularly in the emerging fields of biological engineering and drug and gene delivery.10-13 One advantage of star polymer architectures over hyperbranched structures is their well-controlled nature. In most cases the polydispersity indices of star polymers can be controlled to be less than 1.2, similar to those of well-defined linear polymers.14-16 Different polymerization methods can be used for the generation of multiarmed polymer architectures, for instance, ionic, coordination ring-opening and catalytic condensation polymerizations,17-19 and living radical polymerizations (LRPs), for example, atom transfer radical polymerization (ATRP),20-27 nitroxide-mediated radical polymerization (NMRP),28,29 and reversible addition-fragmentation chain transfer (RAFT) polymerization.14-16,30-49 The star architectures are usually synthesized by either “arm-first”22,27,33-36,50-53 or “core-first” methodologies.14-16,23,38,39 Most star polymers consist of homopolymer arms, however, they can also be tailored with * To whom correspondence should be addressed. Tel.: +61 2 9385 4371. Fax: +61 2 9385 6250. E-mail: [email protected] (J.L.); [email protected] (T.P.D.).

miktoarms.54-58 A combination of different LRP methods can be also utilized for the generation of more complicated polymeric architectures.50,59 A number of covalent linkages are biodegradable, for example, the disulfide linkage is cleavable in the presence of glutathione (GSH),1,2,60 the acetal linkage is acid labile,61 and the ester linkage is degradable upon hydrolysis.19,62 Disulfide linkages in proteins are formed between the thiol groups of cysteine residues, they play an important role in the folding and stability of proteins and enzymes. They can be cleaved in vivo in the presence of glutathione (GSH), the most abundant intracellular thiol (0.2-10 mM) in most mammalian and many prokaryotic cells.60 Therefore, a polymeric structure intralinked by disulfide bonding could be cleaved easily into smaller fragments in vivo and subsequently excreted. In two recent publications we reported the synthesis of biodegradable three-armed and six-armed star architectures with disulfide intralinkages on their arms using both “arm first” and “core-first” methodologies, where the three-armed RAFT agent was synthesized by attaching the RAFT functionality via its Z-group to a trifunctional core.15,63 The biodegradable star polymers, thus formed, had a trithiocarbonate group between the core and polymer arms, limiting accessability for further polymer modification reactions. In this current study, a three-armed RAFT agent was synthesized via a condesation reaction between a RAFT agent (via the R-group) and a trifunctional core. The subsequent polymerizations of polystyrene and polyPEG-A using such a RAFT agent generated three-armed polymer structures with trithiocarbonate cores at the termini of the polymer arms, creating the potential for further modifications. Aminolysis of the trithiocarbonate functionality and further reaction with dithiodipyridine (DTDP) yielded sulfhydryl groups and subsequently pyridyldisulfide (PDS) terminal groups, available for

10.1021/bm900646g CCC: $40.75  2009 American Chemical Society Published on Web 08/07/2009

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further reactions with any free sulfhydryl-tethered precursors. The cleavability of these star structures were also tested using DTT. Cyclodextrin-polymer hybrid materials have attracted a lot of interest,64-67 as cyclodextrin can be utilized as a biodegradable building block, as either a carrier (e.g., for hydrophobic drugs) or as a multifunctional unit for attachment to other groups through its multiple hydroxyl groups.68,69 Several previous studies have used the formation of cyclodextrin inclusion complexes with hydrophobic molecules (such as cholesterol) in aqueous solution to build up supramolecular structures and to form cross-linked networks. In this present work, biodegradable, cyclodextrin-polymer structures have been constructed from star polymer architectures and inclusion complexes. As these structures are noncross-linked, they can be readily characterized by conventional methods. In future work, our plan is to extend the synthetic methods to form well-defined crosslinked gels from similar building blocks as potential drug delivery vectors.70-73

Experimental Section Materials. Ethanethiol (Acros, >99%), potassium hydroxide (Analar), carbon disulfide (Analar, >99%), p-toluenesulfonyl chloride (Aldrich, 98%), dichloromethane (Ajax, >98%), 2-bromopropionic acid (Aldrich, >98%), N,N′-dicyclohexyl carbodiimide (DCC; Fluka, 99%), 4-dimethyl amino pyridine (DMAP; Aldrich, 99%), chloroform (Univar, >99.8%), acetone (Univar, >99.5%), ethyl acetate (Univar, >99.5%), 2,2′-dithiodipyridine (DTDP; Fluka, 97%), poly(ethylene glycol) acrylate (PEG-A, average molecular weight 454, n ) 8-9; Aldrich), n-hexane (Ajax, 95%), deuterium oxide (D2O, Aldrich), 2,2′-azobis(isobutyronitrile) (AIBN, 98%, SigmaAldrich), styrene (Aldrich, >99%), DL-dithiothereitol (DTT; Aldrich, 99%), diethyl ether (Univar, >99%), 1,3,5-benzenetricarbonyl trichloride (Aldrich, 98%), N,N-dimethylacetamide (DMAc; Aldrich, 99%), 2-hydroxylethyl disulfide (Aldrich, technical grade), 1-pyrene butyric acid (Aldrich, 97%), thiocholesterol (Sigma, 95%), and β-cyclodextrin (β-CD; Sigma, 98%). Synthesis of 3-[Hydroxylethyl disulfide] Ethyl, 2-Ethyltrithiocarbonate Isopropionate (HDEEI; 2). RAFT agent (1; 1 g, 3.8 mmol) was added, dropwise, to a solution of hydroxyl ethyl disulfide (1.76 g, 14 mmol), DCC (0.94 g, 4.6 mmol), and DMAP (0.20 g, 0.19 mmol) dissolved in THF (40 mL). The resulting mixture was stirred at room temperature for 12 h, followed by the removal of volatiles (under vacuum). The residue was purified via silica gel column chromatography using ethyl acetate/hexane (50/50) as the eluent. The orange-brown oil product was characterized by 1H NMR in CDCl3 solution (2.1 g, 76.6% based on the feed of RAFT [1]). 1H NMR (CDCl3, 298 K, 300 MHz) δ (ppm from TMS): 1.26-1.31 (t, 3H, S-CH2-CH3), 1.50-1.70 (m, 2H, OH), 1.83 (s, 3H, CH3-C), 2.10-2.70 (m, 4H, CH2-CH2), 2.80-2.85 (m, 4H, CH2-S, S-CH2), 3.35-3.38 (q, 2H, S-CH2-CH3), 3.80-3.85 (t, 2H, CH2-OH), 4.36-4.40 (t, 2H, CH2-O-CO). 13C NMR (75 MHz; CDCl3): 13.36 (CH3), 17.13 (CH3), 31.98 (CH2), 37.35 (CH2), 37.41 (CH2), 48.16 (CH2S), 62.65 (CH2O), 63.77 (CH2O), 164.99 (CO), 171.38 (CO), 222.12 (CdS). Synthesis of Trifunctional RAFT (3). 1,3,5-Tricarbonyl trichloride (79 mg, 0.3 mmol) was added to a stirred solution of HDEEI (0.37 g, 107 mmol) and DMAP (0.11 g, 89 mmol) dissolved in THF (20 mL). The mixture was maintained with stirring at room temperature for 12 h. The resulting mixture was filtered to remove the solid byproduct and the volatiles were removed under vacuum. The residue was purified on a silica gel column using ethyl acetate/ hexane (50/50) as the eluent to afford the expected product (0.90 g 71%). 1H NMR confirmed the successful synthesis of the three-armed RAFT agent, as shown in Figure 1. 1H NMR (CDCl3, 298 K, 300 MHz) δ (ppm from TMS): 1.25-1.30 (t, 9H, CH2-CH3), 1.52-1.55 (d, 9H, C-CH3), 2.88-2.92 (t, 6H, S-CH2), 3.00-3.04 (t, 6H, CH2-S), 3.25-3.32 (q, 6H, CH3-CH2-S),

Setijadi et al.

Figure 1. 1H NMR spectrum of trifunctional RAFT agent (3).

4.33-4.38 (t, 6H, CH2-O), 4.56-4.60 (t, 6H, O-CH2), 4.70-4.78 (q, 3H, CH-CO), 8.86 (s, 3H, CHdCH). 13C NMR (75 MHz; CDCl3): 13.36 (CH3), 17.13 (CH3), 31.98 (CH2), 37.35 (CH2), 37.41 (CH2), 48.16 (CH2S), 63.77 (CH2O), 63.97 (CH2CO), 131.48 (CHdCH), 135.34 (CH2-CHdCH), 164.99 (CO), 171.38 (CO), 222.12 (CdS). The successful synthesis of the RAFT agent (3) was also supported by the presence of a parent sodium ion at m/z 1216.86 (calcd 1216.93) in its electrospray ionization (ESI) mass spectrum. Homopolymerization of PEG-A Controlled by the Trifunctional RAFT Agent (3). A solution of PEG-A (0.76 g, 1.672 × 10-3 mol), 3- armed RAFT (20 mg, 1.67 × 10-5 mol), and AIBN (1.5 mg, 9.13 × 10-6 mol) in dioxane (5 mL) was prepared. This solution was deoxygenated with nitrogen for 30 min, followed by incubation in a water bath at 65 °C. Five samples were taken from the incubating mixture using a degassed needle at 0.5, 1, 2, 3, and 5 h polymerization times. The monomer conversion for each polymerization sample was determined by 1H NMR in CDCl3. The polymers were purified by precipitation in diethyl ether and dried. The molecular weight of the pure polymer was calculated via three independent methods, namely, DMAc GPC, SLS, and 1H NMR. Homopolymerization of Styrene Controlled by the Trifunctional RAFT Agent. A solution of styrene (0.626 g, 6.02 × 10-3 mol), 3-armed RAFT agent (21 mg, 1.76 × 10-5 mol), and AIBN (2.0 mg, 1.22 × 10-5 mol) in dioxane (4 mL) was prepared. This solution was deoxygenated with nitrogen for 30 min, followed by incubation in a water bath at 75 °C. Five samples were taken from the incubating mixture using a degassed needle at 4, 8, 12, 16, and 24 h polymerization times. The monomer conversion for each polymerization sample was determined by 1H NMR in CDCl3. The polymers were purified by precipitation in hexane three times prior to drying. The molecular weight of the pure polymer was calculated via three independent methods, namely, DMAc GPC, SLS, and 1H NMR. Modification of Poly PEG-A Stars with Three Pyridyldisulfide (PDS) Groups. Poly PEG-A (MW: 41,900 from SLS; PDI, 1.14; 80 mg, 1.91 × 10-6 mol) was dissolved in THF (10 mL) and the resulting solution was deoxygenated with nitrogen for 20 min, followed by the addition of DTDP (0.25 g, 1.15 × 10-4 mol) and hexylamine (11 mg, 1.1 × 10-4 mol). The resulting mixture was then stirred at room temperature for 12 h and concentrated to less than 0.5 mL under evaporation, followed by precipitation in diethyl ether three times to afford the expected product. Synthesis of Pyrene Disulfide. 1-Pyrene butyric acid (0.750 g, 2.62 × 10-3 mol) was dissolved in THF (20 mL), followed by the addition of hydroxyl ethyl disulfide (0.184 g, 1.19 × 10-3 mol), DCC (0.54 g, 2.62 × 10-3 mol), and DMAP (16 mg, 1.31 × 10-4 mol). After 8 h the mixture was filtered to remove any precipitate. The filtrate was

Functionalized Biodegradable Star Polymers


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Scheme 1. Synthesis of the Trifunctional RAFT Agent and the Subsequent Polymerization of PEG-A and Styrene

Table 1. Results from the PEG-A Star Formation Using RAFT Agent (3) time/h

conversion/%a

Mn, from GPC/g · mol-1b

Mn, measured from 1H NMR/g · mol-1

Mw, measured from SLS

Mtheo, theoretical/g · mol-1c

PDIb

0.5 1 2 3 5

15.0 35.1 54.2 70.0 85.0

4800 11100 13200 18600 26800

8500 17700 22700 30500 38900

9900 19500 24400 32400 41900

7400 16600 25200 32700 39200

1.12 1.12 1.12 1.10 1.14

a The monomer conversion was calculated from 1H NMR spectra of the polymerization mixtures in CDCl3. b The experimental number-average molecular weight, Mn, and the polydispersity index, PDI, were measured by GPC using polystyrene standards and dimethyl acetamide (DMAc) (0.03% w/v LiBr, 0.05% BHT) as eluent. c Theoretical value (Mtheo) calculated using the following equation: Mtheo ) (mole ratio of PEG-A to three-armed RAFT [3]) × conversion × MWPEG-A + MW3, where MWPEG-A represents MW of PEG-A monomer and MW3 represents MW of three-armed RAFT agent.

then subjected to vacuum to remove the volatiles. The residue was then purified via silica gel chromatography using ethyl acetate/hexane (10/90) as the eluent to obtain the expected product as a white solid (1.2 g, 67%). 1H NMR (CDCl3, 298K, 300 MHz) δ (ppm from TMS): 1.56 (m, 4H, CH2), 2.06-2.14 (m, 4H, CH2), 2.34-2.39 (t, 4H, CH2CO), 2.78-2.82 (t, 4H, S-CH2), 3.25-3.30 (t, 4H, CH2-S), 4.22-4.27 (t, 4H, O-CH2), 7.81-8.16 (m, 18H, HCdCH). 13C NMR (75 MHz; CDCl3): 25.62 (CH3), 31.66 (CH3), 32.66 (CH2), 36.22 (CH2), 122.22 (C aromatic), 123.74 (C aromatic), 123.76 (C aromatic), 123.88 (C aromatic), 124.02 (C aromatic), 124.80 (C aromatic), 125.68 (C aromatic), 126.31 (C aromatic), 126.36 (C aromatic), 126.42 (C aromatic), 127.67 (C aromatic), 128.93 (C aromatic), 129.82 (C aromatic), 130.35 (C aromatic), 134.51 (C aromatic), 172.18 (CO). Cleavage of Pyrene Disulfide and Subsequent Conjugation to the PDS-Modified Three-Armed polyPEG-A Star. Pyrene disulfide (10 mg, 1.44 × 10-5 mol) was dissolved in a mixed solvent of THF and H2O (7:3; 2 mL), followed by the addition of TCEP (20 mg, 7.0 × 10-5 mol). The resulting mixture was then stirred for 2 h under nitrogen. The mixture was then evaporated to remove the THF solvent, followed by the addition of degassed CH2Cl2 (2 mL). The resulting solution was then washed with degassed water (10 mL × 2) to remove any unreacted TCEP. The remaining CH2Cl2 solution was injected directly by syringe into degassed polyPEG-A (20 mg, 4.77 × 10-7 mol, MW: 41900 from SLS; PDI, 1.14) solution in CH2Cl2 (1 mL). The resulting mixture was then stirred for 5 h and concentrated to 0.5 mL by vacuum evaporation. Precipitation of the mixture in excess ether yielded pyrene-modified polymer. Attachment of Cholesterol to the Three-Armed PolyPEG-A Star and Subsequent Complexation with β-Cyclodextrin (β-CD).

Thiocholesterol (26 mg, 6.46 × 10-5 mol) was dissolved in degassed CH2Cl2 (1 mL), followed by injection of the degassed solution of PDS functionalized polyPEG-A (65 mg, 1.55 × 10-6 mol, MW: 41,900 from SLS; PDI, 1.14) solution in CH2Cl2 (1 mL) using a syringe. The resulting mixture was then stirred for 5 h and concentrated to 0.5 mL by vacuum evaporation. Precipitation of the mixture in excess ether yielded the cholesterol functionalized polymer. Cholesterol functionalized polyPEG-A (10 mg, 2.34 × 10-7 mol) was dissolved in D2O (1 mL), followed by the addition of β-CD (4 mg, 3.5 × 10-6 mol). The resulting mixture was stirred for 3 h prior to analysis by 2D NMR (NOESY 1H). Analyses. Gel permeation chromatography (GPC) was performed in N,N-dimethylacetamide (DMAc; 0.03% w/v LiBr, 0.05% BHT stabilizer) at 50 °C (flow rate: 0.85 mL · min-1) using a Shimadzu modular system comprising a DGU-12A solvent degasser, an LC-10AT pump, a CTO-10A column oven, and an RID-10A refractive index detector. The system was equipped with a Polymer Laboratories 5.0 mm bead-size guard column (50 × 7.8 mm2), followed by four 300 × 7.8 mm2 linear PL columns (105, 104, 103, and 500). Calibration was performed with narrow polydisperse polystyrene standards ranging from 500 to 106 g · mol-1. 1 H NMR spectra were obtained using a Bruker AC300F (300 MHz) spectrometer or a Bruker DPX300 (300 MHz) spectrometer. Data are reported by the chemical shifts (δ) measured in parts per million (ppm) downfield from TMS, multiplicity, and proton count. Multiplicities are reported as singlet (s), broad singlet (bs), doublet (d), triplet (t), and multiplet (m). 13C NMR spectra were obtained on Bruker AC300F (75 MHz) spectrometer. 13C chemical shifts (δ) are reported in parts per million (ppm) downfield from TMS and identifiable signals are given.

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Figure 2. Polymerization of PEG-A using the RAFT agent (3) in dioxane at 65 °C ([M]/[RAFT]/[AIBN] ) 100:1:0.55). (a) Monomer conversion at varying polymerization times. (b) Molecular weight (MW) and PDI of the polyPEG-A against monomer conversion (filled and empty diamonds represent the experimental (obtained from SLS) and theoretical MW values, respectively, while the filled triangles represent PDIs). (c) GPC traces at different polymerization times (from DMAc GPC). (d) 1H NMR spectrum of the purified polyPEG-A (Mw 9900 g/mol from SLS, PDI 1.12 in CDCl3). Table 2. Results from the Polymerization of Styrene Using RAFT Agent (3) time/h

conversion/%a

Mn, from GPC/g · mol-1b

Mn, measured from 1H NMR/g · mol-1

Mw, measured from SLS

Mtheo, theoretical/g · mol-1c

PDIb

4 8 12 16 24

11.5 38.6 58.0 76.0 87.0

3700 5800 11800 13800 18100

3800 9400 14300 21800 24500

4300 9800 15500 23400 26400

4800 13200 19300 24900 28300

1.17 1.15 1.12 1.15 1.19

a The monomer conversion was calculated from 1H NMR spectra of the polymerization mixtures in CDCl3. b The experimental number-average molecular weight, Mn, and the polydispersity index, PDI, were measured by GPC using polystyrene standards and dimethyl acetamide (DMAc; 0.03% w/v LiBr, 0.05% BHT) as eluent. c Theoretical value (Mtheo) calculated using the following equation: Mtheo ) (mole ratio of styrene to three-armed RAFT [3]) × conversion × MWSt + MW3, where MWSt represents MW of styrene and MW3 represents MW of three-armed RAFT agent.

Important protons and carbon signals from NMR spectra are highlighted in bold and italic character. Electrospray ionization mass spectroscopy (ESI-MS) was carried out on a Finnigan LCQ Deca mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an atmospheric pressure ionization source operating in the nebulizer-assisted electrospray mode. The instrument was calibrated in the m/z range 195-1822 Da using a standard containing caffeine, Met-Arg-Phe-Ala acetate salt (MRFA), and a mixture of fluorinated phosphazenes (Ultramark 1621; all from Aldrich). The molecular weights of the three-armed polystyrene and polyPEG-A were also analyzed at 25 °C using static light scattering (SLS) on a Malvern static light scattering analyzer (Laser type: HeNe gas laser; beam wavelength: 633 nm) using dn/dc of 0.185 for polystyrene and 0.134 for polyPEG-A in THF.

Fluorescence spectra of pyrene functionalized polyPEG-A were obtained on a Perkin-Elmer LS50B scanning instrument employing a slit width at 5 nm. The excitation wavelength was 347 nm (Data shown in Figure 4c). The emission wavelength was located at 379 nm for the pyrene fluorophore.

Results and Discussion Synthesis of Trifunctional RAFT Agent. The synthesis of the trifunctional RAFT agent and the subsequent polymerization of PEG-A and styrene are summarized in Scheme 1. A trithiocarbonate RAFT agent (1) was first reacted with excess hydroxyethyl disulfide to yield 3-[hydroxylethyl disulfide] ethyl, 2-ethyltrithiocarbonate isopropionate (HDEEI; 2), followed by a condensation reaction with 1,3,5-benzenetricarbonyl trichloride



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Figure 3. Polymerization of styrene using RAFT agent (3) in dioxane at 75 °C ([M]/[RAFT]/[AIBN] ) 342:1:0.69). (a) Monomer conversion vs polymerization time. (b) Molecular weight and PDI of the purified polySt versus monomer conversion (filled and empty diamonds represent the experimental (obtained from SLS) and theoretical MW values, respectively, while filled triangles represent PDI). (c) Normalized GPC traces of purified polySt at different conversions. (d) 1H NMR spectrum of purified polySt (Mw 26400 g/mol calculated from SLS, PDI 1.19) in CDCl3.

in the presence of 4-dimethylamino pyridine (DMAP) to afford a trifunctional RAFT agent (3). The trifunctional RAFT agent (3) was characterized by 1H NMR, where the signal at 8.80 ppm originates from the three unsubstituted protons in the phenyl core (Figure 1). The integration ratio of the signal at 8.80 ppm to the remaining signals from the RAFT agent confirmed complete RAFT functionalization. The successful synthesis of the trifunctional RAFT agent was also supported by the presence of a parent sodium ion at m/z 1216.86 (calcd 1216.93) via electrospray ionization mass spectrometry (ESI-MS). Synthesis of Three-Armed Poly PEG-A Stars Using the Trifunctional RAFT Agent (3). The results of the synthesis of three-armed star polymer of PEG-A using the trifunctional RAFT agent are summarized in Table 1 and Figure 2. It is evident from the data in Figure 2a that the monomer conversion increased concomitantly with polymerization time and the radical concentration remained constant with conversion as indicated by the pseudofirst order plot. The data summarized in Figure 2b shows that both the experimental (measured from SLS) and the theoretical molecular weights were found to be proportional to the monomer conversion. The theoretical MW values were slightly smaller than the experimental ones and the polydispersity indices (PDI) of the purified homo-polyPEG-A were less than 1.14, indicating a well-controlled polymerization, consistent with the known traits of living radical polymerization. The GPC traces of polyPEG-A obtained at different conversions

are shown in Figure 2c. It is evident that the MW of the polyPEG-A star increases with increasing conversion as shown by decreased retention times. Polymers were obtained by the repeated precipitation of the reaction mixture in diethyl ether. A purified polyPEG-A (Mw 9900 g/mol from SLS; PDI 1.12) was analyzed by 1H NMR using CDCl3 as the deuterated solvent (Figure 2d). The signals at 8.80, 4.76, 4.57, 3.01, and 2.87 ppm and other peaks labeled in Figure 2d are consistent with the presence of the core. It is well-known that the hydrodynamic volume of multiarmed polymers is smaller than equivalent linear polymers. When comparing the MW using different analysis methods, we found that the MWs of the three-armed polystyrenes obtained from SLS are approximately 1.66 times of those obtained from GPC analysis. However, the MWs obtained from both 1H NMR spectra and SLS are very close. The signals labeled a, b, c, d, e, and h correspond to the core proving its integrity postpolymerization. The shift of signal f from 4.75 ppm (Figure 1a) to 2.62 ppm strongly supports the expected transfer mechanism. Synthesis of Three-Armed Star Polystyrene Using Three-Armed RAFT Agent (3). As summarized by the data shown in Table 2 and Figure 3, the RAFT agent (3) was also used in the synthesis of three-armed star polymers with hydrophobic polystyrene arms. The RAFT controlled polymerization of three-armed styrene was much slower than that of PEG-A (Figure 3a). However, the PDIs of the polySt star were

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Scheme 2. Modification of Three-Armed PolyPEG-A with PDS Functionality and the Cleavage Reaction in the Presence of DTT

less than 1.19, indicating a well controlled living polymerization. The normalized GPC traces of polystyrene obtained at different conversions are shown in Figure 3c. A purified polySt (MW 25400 g/mol; PDI 1.19) was analyzed by 1H NMR in CDCl3 (Figure 3d). The peaks labeled a, b, c, d, e, and h corresponded to the core, proving its integrity after polymerization. The shift of peak f from 4.75 ppm (Figure 1a) to 2.62 ppm strongly supports the expected transfer mechanism. Functionalization of Three-Armed Polymers with Thiol-Reactive Pyridyldisulfide (PDS) End Groups. The three-armed star polymers possessed RAFT functionality at the end of each arm. Therefore, these star polymers can be aminolyzed in the presence of primary amine to yield armterminal thiol groups.74-76 The trithiocarbonate groups at the arm-termini were subjected to aminolysis in a one-pot reaction in the presence of DTDP. The free thiol terminal groups generated by the aminolysis reaction were instantaneously captured by the pyridyldisulfide (PDS) groups, as shown in Scheme 2. The reactivity of these terminal PDS groups was then investigated by reaction with pyrene modified with thiol functionality (Scheme 3). The 1H NMR spectrum of polyPEG-A stars modified with PDS groups (after aminolysis) is shown in Figure 4a. The signal

Scheme 3. Modification of Pyrene with a Sulfhydryl Terminal Group

a at 8.78 ppm, as labeled in Figure 4a, correspond to the three unsubstituted aromatic protons in the aromatic core and signals b, c, d, and e correspond to the protons in the PDS groups. The ratio of the integration of peak a to that of peak b is 0.98, indicating that 98% of the trithiocarbonate groups on the polymer arms have been successfully converted into PDS groups. The subsequent reaction with sulfhydyl-terminated pyrene further confirmed the successful aminolysis and subsequent PDS attachment. As shown in Figure 4b the PDS groups



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Figure 5. GPC traces of polyPEG-A stars (MW: 41900 from SLS) before (filled circle) and after cleavage (empty circle) by DTT in DMAc solution for 3 h.

Figure 4. 1H NMR spectra of polyPEG-A stars with PDS groups (a), with pyrene fluorophore (b), and the fluorescence emission of pyrenemodified polyPEG-A (c; the inset of c represents the excitation spectrum of pyrene modified with polyPEG-A).

disappeared after the conjugation reaction with pyrene and the characteristic signals corresponding to the aromatic protons of pyrene appeared. The integration ratio of signal a (corresponding to protons from the phenyl core) to the group peaks labeled as b was 1:8.8 (1:9 is the fully functionalized ratio), consistent with the complete functionalization of the stars with pyrene groups (this also provides convincing indirect evidence for the success of the aminolysis reaction and subsequent PDS modification). Fluorescence analysis also supported the successful attachment of pyrene to the star polymers (Figure 4c). In previous work we have reported the quantification of PDS functional groups on the surface of micelles via fluorescence

spectroscopy.9 In that case, fluorophore quantification was relatively facile. However, when the fluorophores are attached to different polymers or to polymers bearing functional groups such as amine, their fluorescence intensity will be significantly quenched.77 Thus, in the present work this limits the usefulness of fluorescence studies. Fortunately, the end-groups were quantified by 1H NMR spectral analysis of the polymer after PDS modification and pyrene attachment. Cleavage of PolyPEG-A Stars into Single-Armed Linear Polymers. A three-armed star polymer of polyPEG-A was degraded in the presence of DTT in DMAc solution. The cleavage was complete within 3 h in 0.1 M DTT. Upon cleavage the MW of the cleaved mixture was significantly decreased as evidenced by GPC analyses (Figure 5). The MW of the cleaved mixture was found to be 14000 from DMAc GPC, approximately one-third of the MW of its star precursor measured by SLS (MW: 41900) and 60% of that measured by GPC (MW: 23600).78 The PDI of the cleaved mixture was 1.23, slightly more than its three-armed precursor, but still in accord with successful living polymerization. Attachment of Cholesterol to the PolyPEG-A Stars and Subsequent Complexation with β-Cyclodextrin (CD). The PDS functionalized polyPEG-A stars were also reacted with thiocholesterol (Scheme 4), under anaerobic conditions to afford three cholesterol functionalized arm-termini. The cholesterolstar polymer conjugate was then used to form an inclusion complex with a host molecule, β-CD. It is known from previous work that one cholesterol molecule might complex with one or two β-CDs.79,80 The successful attachment of cholesterol to the polymer arms was confirmed by 1H NMR in CDCl3. As shown in Figure 6a (upper), the signal b at 5.27 ppm confirmed the presence of cholesterol (one double bond proton) and the signal a at 8.8 ppm corresponds to the three unsubstituted protons of the aromatic polymer core. The integration ratio of peak a to b was calculated to be 0.97, indicating the quantitative conversion of PDS functionality to cholesterol groups. However, when the 1 H NMR was carried out in D2O the signals originating from the protons of the cholesterol (double bond) were hardly visible, indicating that the hydrophobic cholesterol units self-assemble in the aqueous environment to form micelle-like structures80 (Figure 6a, lower spectrum). The formation of cholesterol/βCD complexes was confirmed by 2D NMR (NOESY 1H) analyses on their mixed solution in D2O. As shown in Figure

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Setijadi et al.

Scheme 4. Modification of PolyPEG-A Stars with Cholesterol and the Subsequent Formation of an Inclusion Complex with β-Cyclodextrin (β-CD)

6b, cross-peaks from cholesterol protons (δ 0.9-1.3 ppm) and protons at δ 3.9 ppm were observed (cross-peaks highlighted by rectangles). Both glucosidic β-CD and PEG protons resonate at 3.9 ppm. However, because the hydrophobic cholesterol and hydrophilic polyPEG-A are phase-separated in an aqueous medium, it can be concluded that these cross-peaks result from interactions between cholesterol and β-CD protons in the inclusion complex.80 A control experiment was also carried out on the mixture of polyPEG-A stars without cholesterol end groups, and no such cross-peaks were observed (result not shown).

Conclusions We have successfully synthesized a trifunctional RAFT agent and used it to synthesize three-armed polymers of polySt and polyPEG-A using a “core first” methodology. The radical polymerization of three-armed star polymers was found to be well-controlled by the RAFT mechanism. The trithiocarbonate groups at the termini of the star-arms were subjected to aminolysis generating PDS end-groups. Both thiol-functional pyrene and cholesterol were then attached to the arm-termini at high yields. Finally, the cholesterol terminal polymers were utilized to form supramolecular structures vis complexation with cyclodextrin forming inclusion complexes. Thus, in this work we have successfully produced biodegradable polymer hybrid nanostructures that can easily be adapted to form cross-linked functional gels (the subject of continuing research). Acknowledgment. T.P.D. thanks Australian Research Council for Federation Fellowship Award. J.L. acknowledges the UNSW Vice Chancellor’s Postdoctoral Research Fellowship. We thank Dr. Donald Thomas at the NMR facility unit for help with obtaining 2D NOESY spectra.

References and Notes

Figure 6. (a) 1H NMR spectra of polyPEG-A stars with cholesterol groups (upper spectrum obtained in CDCl3 and lower spectrum in D2O), and (b) 2D NOSEY (cross-peaks are highlighted by the rectangle).

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