Macromolecular Cobalt Carbonyl Complexes ... - ACS Publications

Biomacromolecules 2009, 10, 3215–3226

3215

Macromolecular Cobalt Carbonyl Complexes Encapsulated in a Click-Cross-Linked Micelle Structure as a Nanoparticle To Deliver Cobalt Pharmaceuticals Alexander B. J. Withey, Gaojian Chen, T. L. Uyen Nguyen, and Martina H. Stenzel* Centre for Advanced Macromolecular Design (CAMD), School of Chemical Sciences and Engineering, University of New South Wales, Sydney, Australia Received June 24, 2009; Revised Manuscript Received October 13, 2009

Block copolymers poly(trimethylsilyl propargyl methacrylate)-block-poly(poly(ethylene glycol) methyl ether methacrylate) (P(TMS-PAMA)-b-P(PEGMA)) were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. Subsequent removal of the trimethylsilyl protective groups on the P(TMSPAMA)24-b-P(PEGMA)40 polymer with tetra-n-butylammonium fluoride hydrate lead to the polymer P(PAMA)24-b-P(PEGMA)40 with pendant alkyne groups, which self-assembled in aqueous solution into micelles with hydrodynamic diameters of less than 20 nm. The alkyne groups in the core took on two functions, acting as a ligand for Co2(CO)8 to generate a derivative of the antitumor agents based on (alkyne)Co2(CO)6 as well as an anchor point for the cross-linking of micelles via click chemistry. The click process was shown to be highly efficient with the two types of cross-linker employed: 1,2-bis-(2-azidoethoxy)ethane and bis(azidoethyl)disulfide, with almost all of the cross-linker reacting with the micelle at room temperature. The cross-linking density was influenced by the amount of added cross-linker leaving a well-defined amount of alkyne groups that were utilized in the formation of the cobalt complexes. The successful complexation was confirmed via UV/vis and FT-IR spectroscopy. With the formation of (alkyne)Co2(CO)6 moieties in the core, the un-cross-linked and cross-linked micelles were found to almost double in size. The resulting Coloaded un-cross-linked micelles were observed to be highly toxic to L929 fibroblast cells, while the crosslinking of the micelle was shown to reduce the toxicity.

Introduction Medicinal inorganic chemistry is receiving increased interest in the field of biomedical applications. Metals are not only part of vital processes in the body, such as enzyme driven reactions, but can also be used as therapeutic and diagnostic agents. The application of metal containing compounds can range from magnetic resonance imaging agents (e.g., Gd- or Mn-based complexes), radiopharmaceutical diagnostic and therapeutic agents (utilizing 99Tc and 90Y), along with enzyme inhibitors to therapeutic agents (e.g., Li, Pt, Au, Bi).1 In oncology, platinum complexes are widely used for the treatment of a wide range of malignant tumors. Cisplatinum is the most commonly used platinum complex, but three other PtII compounds are approved for clinical use and several other PtII and PtIV complexes are currently on trial.1 A less well-known set of metal complexes with high biological activity that represent a new promising class of anticancer drugs are cobalt complexes. CoIII complexes have been investigated as prodrugs that release toxic compounds in a hypoxic environment, the same environment typically found in tumors.2 Hexacarbonyl dicobalt complexes ((hexacarbonyl[µ-h4-(alkyne)]dicobalt (CoCo) complexes) with one alkyne ligand were first discussed as an anticancer drug in 1987, when these types of complexes were observed to have growth inhibitory properties against murine leukemia cells. A cobalt complex with a ligand related to acetylsalicylic acid was identified as a leading anticancer drug where a slight modification of the ligand resulted in a decreased antiproliferative effect.3 The precursor, Co2(CO)8, in contrast, * To whom correspondence should be addressed. Tel.: +61-2-93854344. Fax: +61-2-93856250. E-mail: [email protected].

does not show any biological activity due to limited cellular uptake. The mode of action of cobalt drugs is currently unknown. Typical pathways for anticancer agents such as destructive binding to DNA, which is the widely accepted mechanism for platinum drugs, can be excluded.4 However, an interesting correlation between cytotoxicity of various low molecular weight cobalt complexes and the ability to inhibit the activity of cyclooxygenase (COX) can be found. It is therefore suggested that cobalt drugs act as a COX-inhibiting anticancer drugs, but other mode of action are under discussion.4 The disadvantage of low molecular weight anticancer drugs is the systemic distribution leading to many reported side effects and a low efficacy.5 Drug administration is improved by introducing drug carriers, which allow not only a temporal control of the drug concentration, but also provide a vehicle for targeted drug delivery. The drug delivery to tumors is usually facilitated by the enhanced permeability and retention (EPR) effect, which leads to the preferred accumulation of drug carriers in the tumor, while the ineffective lymph drainage of tumors hampers the clearance of the drug vehicle.6 The drug carrier is, therefore, trapped in the tumor. To prevent early detection of the drug carrier by the reticuloendothelial system (RES) on its way to the tumor, the size of the carrier should be below a certain threshold (approximately 100-200 nm) and have a specific surface chemistry. Nanoparticles coated with poly(ethylene glycol) are considered the “magic bullet” in drug delivery since the high hydrophilicity of the polymer prevents the early clearance of the drug carrier leading to longer circulating particles.7 Polymeric micelles, self-assembled structures composed of block copolymers, have been proposed as efficient drug carriers,

10.1021/bm901050x CCC: $40.75  2009 American Chemical Society Published on Web 11/02/2009

3216

Biomacromolecules, Vol. 10, No. 12, 2009

Withey et al.

Scheme 1. Formation of Macromolecular Complex between Block Copolymers with Alkyne Ligands and Co2(CO)8

which addresses many of the prerequisites that an optimum drug vehicle requires. The size of a micelle is typically below 100 nm with the hydrophilic shell ensuring water-solubility and the hydrophobic core allowing the entrapment of hydrophobic drugs. Micelles with poly(ethylene glycol) on the surface, which encapsulated a toxic core made from cationic polymer, were found to be taken up by cells without revealing their toxic interior.8 While polymeric micelles are very stable compared to low molecular weight surfactant, they still benefit from cross-linking, which prevents dissociation at very low concentrations (below the critical micelle concentration). The techniques for crosslinking of micelles range from shell to core-cross-linking using a variety of approaches including the formation of covalent bonds between the block copolymers or electrostatic interactions.9-11 The chemistry involved can be very versatile and includes examples where block copolymers were connected with each other using diamines,12-15 and aldehydes,16 or click chemistry.17-20 Huisgen 1,3-dipolar cycloaddition, often termed click chemistry, was first described by Huisgen et al.21 The synthesis approach was later modified by Meldal et al.,22 as well as Fokin and Sharpless,23 by introducing Cu(I) ions. Cu(I) ions not only direct the reaction leading to the formation of a single regioisomer, but also facilitate the cycloaddition, enhancing the complete reaction at low temperatures. The click reaction is now frequently applied in polymer chemistry to build up new polymer architectures by conjoining azide and alkyne endfunctionalized polymer pairs.24-26 The click approach is, therefore, a very attractive avenue to cross-link micelles because the low temperature required does not disturb the temperature-sensitive equilibrium of the selfassembly process. While permanent cross-linking of micelles can generate very stable core-shell nanoparticles, this feature is not always desirable because the drug carrier needs to be cleared from the body once it has finished distributing the drugs. Degradable cross-linked micelles can also advance drug delivery because the covalent linkage between the polymer chains can respond rapidly to changes in the environment. Cross-linked micelles, cross-linked with acid-degradable cross-linker27 or with a crosslinker carrying disulfide bridges,28-32 can be cleaved into the

underlying block copolymers upon uptake into the cell because a more acidic and reductive environment is prevalent inside the cell. In this work, we aim to develop a polymeric drug carrier for Co2(CO)8 by binding the complex via pendant alkyne groups to a polymer backbone.33-35 To ensure high solubility, biocompatibility, long circulation time, and efficient cell uptake of the drug carrier, a micellar core-shell system based on a PEO containing block copolymer, will be designed. RAFT polymerization36-39 is utilized as a pathway to generate the underlying block copolymer (Scheme 1).40 The pendant alkyne groups can now act on two fronts, as a ligand for the cobalt drug or as a possible anchor point for further stabilization of the micelle by cross-linking via click chemistry. The two different cross-linkers employed can result in permanently cross-linked micelles or in cross-linked micelles with labile linkages between the block copolymer chains, which are sensitive to a reductive environment (Scheme 2).

Experimental Section Materials. Unless otherwise specified, all chemicals were reagent grade and were used as received: trimethylsilyl propynol (Aldrich, 99%), methacryloyl chloride (Fluka, 97%), triethyl amine (Et3N, Aldrich, 99.5%), diethyl ether (Et2O anhydrous, Ajax Finechem, 99%), sodium chloride (Aldrich, 99.8%), petroleum ether (BR 40-60 °C; Ajax Finechem, 90%), tetrahydrofuran (THF; Ajax Finechem, 99.7%), dimethylsulfoxide-d6 (DMSO; Cambridge Isotope Laboratories), chloroform-d (CDCl3; Cambridge Isotope Laboratories), chloroform (CHCl3; Aldrich, 99.8%), N,Ndimethylacetamide (DMAc; Aldrich, HPLC grade), N,N-dimethylformamide (DMF; Aldrich, 99.9%), methanol (APS, HPLC grade), acetone (BDH, GPR grade), magnesium sulfate (Ajax Finechem, 70%), toluene (Aldrich, purum), tetra-n-butylammonium fluoride hydrate (TBAF · xH2O; Aldrich, 98%), N,N-diisopropylethylamine (DIPEA; Aldrich, 99.5%), 1,2bis-(2-chloroethoxy)ethane (Aldrich, 97%), sodium azide (Aldrich, 99.5%), dicobalt octacarbonyl (cobalt carbonyl, Co2(CO)8; Merck, 97%), and acetic acid (Aldrich, 99.7%). 2,2-Azobisisobutyronitrile (AIBN; Fluka, 98%) was purified by recrystallization from methanol. Poly(ethylene glycol) methyl ether methacrylate (PEGMA; MW ) 300 g mol-1; Aldrich) was deinhibited by passing through a column of basic aluminum oxide. The RAFT agent cumyl dithiobenzoate (CDB) was prepared according to the method of Oae et al.41 with cyclohexane as the solvent.

Macromolecular Cobalt Carbonyl Complexes


3217

Scheme 2. Core Cross-Linking of Micelle Using Click Chemistry and Reaction with Co2(CO)8 Using the Remaining Alkyne Groups

The copper(I) complex Cu(PPh3)3Br was prepared by the method described earlier by Gujadhur et al.42 Synthesis Procedures. Trimethylsilyl Propargyl Methacrylate (TMS-PAMA). A solution of trimethylsilyl propyn-1-ol (7.52 g, 58.6 mmol) and triethylamine (10.7 mL, 76.0 mmol) in diethyl ether (75 mL) was cooled to -8 °C in a salted ice bath, and a solution of methacryloyl chloride (6.60 mL, 69.8 mmol) in diethyl ether (57.5 mL) was added dropwise over about 40 min. NOTE: if the addition is too fast, then vapor will be generated and the reaction temperature may rapidly rise, increasing the chance of side reactions and lowering final yield. The mixture was stirred at -8 °C for 30 min after complete

addition and then at ambient temperature overnight. The ammonium salts were mostly removed by filtration. The volatile solvents were then removed under reduced pressure (780 mbar) on a rotary evaporator at 30-40 °C. A watery yellow residue was produced (20 g) that was purified by chromatography (SiO2, petroleum ether/Et2O 20:1). Further purification by a second chromatography column was sometimes necessary (SiO2, petroleum ether/Et2O 50:1), leading to a colorless liquid (8.02 g, 69.7% yield). 1H NMR (300.17 MHz, CDCl3, 301.2 K): δ (ppm) 0.15 (s, 9H, Si(CH3)3); 1.93 (s, 3H, CH2dCCH3); 4.72 (s, 2H, OCH2); 5.58 (s, 1H, HHCdCCH3); 6.14 (s, 1H, HHC ) CCH3). 13C NMR (75.48 MHz, CDCl3, 301.2K): δ (ppm) -0.24 (3C, Si(CH3)3);

3218


18.3 (1C, CH3CdCH2); 53.0 (1C, OCH2); 91.9 (1C, CtCSi(CH3)3); 99.3 (1C, CtCSi(CH3)3); 126.4 (1C, CH3CdCH2); 135.8 (1C, CH3Cd CH2); 166.6 (1C, COester). Synthesis of Diazide Cross-Linker A: 1,2-Bis-(2-azidoethoxy)ethane. 1,2-Bis-(2-chloroethoxy)ethane (14.22 g, 76 mmol) and excess sodium azide (27.7 g, 426 mmol) were added to a 500 mL round-bottom flask that was filled with water (200 mL) and fitted with a condenser. The reaction was allowed to stir under reflux for 4 days. Upon cooling to room temperature, the reaction mixture was extracted with dichloromethane (5 × 100 mL). The organic layers were combined and dried overnight on MgSO4, which was then filtered off, and the filtrate was concentrated by rotary evaporation at 40 °C. The colorless liquid was then further dried at high vacuum, resulting in 14.27 g (93.8% yield) product. 1H NMR (300.17 MHz, CDCl3, 301.2 K): δ (ppm) 3.35 (t, J ) 5 Hz, 4H, (N3CH2CH2OCH2)2); 3.64 (m, 8H, (N3CH2CH2OCH2)2). 13 C NMR (75.48 MHz, CDCl3, 301.2 K): δ (ppm) 50.7 (2C, N3CH2CH2OCH2)2); 70.1 (2C, N3CH2CH2OCH2)2); 70.7 (2C, N3CH2CH2OCH2)2). Synthesis of Diazide Cross-Linker B: Bis-(azidoethyl)disulfide. Bis(hydroxyethyl)disulfide (7.71 g, 0.05 mol) and triethylamine (11.13 g, 0.11 mol) were added to a 250 mL round-bottom flask, dissolved in chloroform (50 mL), and cooled to 0 °C in an ice bath. Toluene sulfonyl chloride (26.80 g, 0.15 mol) was dissolved in chloroform (75 mL) and added dropwise to the round-bottom flask via an addition funnel. Upon complete addition of the toluene sulfonyl chloride, the reaction mixture was removed from the ice bath and reacted overnight at room temperature. The reaction mixture was filtered to remove the TEA salts. The filtrate was then washed with 0.1 M sodium carbonate solution (3 × 75 mL) and then with distilled water (2 × 100 mL). The washed reaction mixture was then dried over magnesium sulfate for 2 h, which was then filtered off, and the chloroform was removed under vacuum to give the tosylate intermediate. 1H NMR spectroscopy was used to confirm the purity. The washing steps were repeated if triethylamine was present. 1H NMR (300.17 MHz, CDCl3, 301.2 K) δ (ppm) 2.46 (s, 6H, (-S-R-Ph-CH3)2); 2.84 (t, J ) 5 Hz, 4H, (-SCH2CH2O-R)2), 4.21 (t, J ) 5 Hz, 4H, (-SCH2CH2O-R)2), 7.36 (d, J ) 5 Hz, 4H, benzylic H nearest methyl group), 7.79 (d, J ) 5 Hz, 4H, benzylic H nearest sulfur). The bis-(tosylate ethyl)disulfide was dissolved in acetone (100 mL). Excess sodium azide (19.51 g, 0.3 mol) was dissolved in water (50 mL) and added to the acetone solution. The stirred mixture was refluxed at 80 °C overnight followed by removal of the solvent under reduced pressure. The product was extracted into dichloromethane (4 × 75 mL). The combined dichloromethane fractions were then dried over magnesium sulfate overnight before being filtered. The solvent was removed under reduced pressure to yield an orange-yellow oil, 8.62 g, 0.042 mol (84.7%). 1H NMR (300.17 MHz, CDCl3, 301.2 K) δ 2.86 (t, J ) 5 Hz, 4H, (N3CH2CH2S-)2); 3.59 (t, J ) 5 Hz, 2H, (N3CH2CH2S-)2). Poly(trimethylsilyl propargyl methacrylate) (PolyTMS-PAMA) Via RAFT. Trimethylsilyl propargyl methacrylate (1.00 g, 5.1 mmol), cumyl dithiobenzoate (0.05 g, 0.184 mmol), and azobisisobutyronitrile (AIBN; 6.03 mg, 0.037 mmol) were weighed into a dry 25 mL flask along with toluene (1.4 mL). The bottle was sealed with a rubber septum. Nitrogen was purged through the solution for 30 min to displace the dissolved oxygen. The reaction mixture was then placed in an oil bath at 60 °C for 6 h, resulting in a monomer conversion of 85%. The polymerization was stopped by opening the bottle to air and then quenching in ice. The polymer solution was added dropwise into a centrifuge tube filled with 48 mL of a methanol/water mixture in a 5:1 ratio to precipitate the polymer. The solution was then centrifuged at 6000 rpm for 10 min and the solvent decanted off leaving the solid polymer which was dried on the Schlenk line for 2 h and then freeze-dried resulting in a pink-colored polymer with a theoretical molecular weight of Mn (theo) ) 4700 g mol-1 (around 24 repeating units) (Mn (GPC) ) 8000 g mol-1, PDI ) 1.12). 1H NMR

Withey et al. (300.17 MHz, DMSO, 301.2 K): δ (ppm) 0.17 (s, 9H, Si(CH3)3); 0.8, 1.0 (br m, 3H, CH2-CH3C); 1.5-2 (br, 2H, CH2-CCH3); 4.65 (br s, 2H, C(O)OCH2); 7-8 (br m, 10H, RAFT benzylic H’s). Poly(trimethylsilyl propargyl methacrylate)-block-poly(poly(ethylene glycol) methyl ether methacrylate) P(TMS-PAMA)-b-P(PEGMA) Via RAFT. Poly(ethylene glycol) methyl ether methacrylate (PEGMA300; 0.375 g, 1.25 mmol) and poly(trimethylsilyl propargyl methacrylate)24 (0.12 g, 0.025 mmol), Mn (theo) ) 4700 g mol-1 (Mn (GPC) ) 8000 g mol-1, PDI ) 1.12) were weighed into a dry 25 mL flask, AIBN (0.82 mg, 0.004 mmol) was added as a 11 mg mL-1 solution in toluene (0.6 mL) with additional toluene as the solvent added to make up to 2 mL total solvent. The solution was stirred before being separated into seven portions by first filling the FT-NIR cuvette and then equally distributing the remaining solution among six small vials. The vials and cuvette were sealed and purged with nitrogen for 30 min. The small vials were placed in an oil bath at 60 °C to react for 2, 4, 6, 14, 15, and 18 h followed by evaporation of the solvent. The progress of the polymerization was followed by online FT-NIR measurements at 5 min intervals at 60 °C by measuring the integral of the vinyl absorption at 6200 cm-1. The sample obtained after 15 h was then precipitated dropwise into a centrifuge tube filled with 45 mL of petroleum ether cooled to -8 °C. The mixture was then centrifuged at 8000 rpm for 10 min. The solvent was decanted off and the remaining solid was dried under high vacuum for at least 4 h. Degree of polymerization was calculated from 1 H NMR in DMSO by comparing the integral of the RAFT end group benzylic protons from 7-8 ppm to that of the C(O)OCH2 connected to the PEG branches at 4.2 ppm while maintaining the correct DP of the TMS-PAMA block from the signal at 4.65 ppm of the C(O)OCH2 connected to the alkyne branches. The theoretical molecular weight of the final pink polymer after 15 h reaction time was calculated to be Mn (theo) ) 16700 g mol-1 (Mn (GPC) ) 20400 g mol-1, PDI ) 1.24). 1 H NMR (300.17 MHz, DMSO, 301.2 K) δ 0.17 (s, 9H, Si(CH3)3); 0.8, 1.0 (br, 3H, CH2-CH3C); 1.5-2 (br, 2H, CH2-CCH3); 3.25 (br s, 3H, OCH3); 3.4-3.6 (br m, 16H, PEGMA OCH2CH2O); 4.0 (br s, 2H, PEGMA C(O)OCH2); 4.65 (br s, 2H, TMS-PAMA C(O)OCH2); 7-8 (br m, 10H, RAFT benzylic H’s). Deprotection of P(TMS-PAMA)24-b-P(PEGMA)40 to P(PAMA)24-bP(PEGMA)40. Trimethylsilyl protected block copolymer (0.2189 g, 0.0131 mmol) and acetic acid (0.028 g, 0.472 mmol, 1.5 equiv alkyne groups on polymer) was dissolved in THF/toluene 1:1 (14 mL) and degassed with nitrogen for 10-15 min. The degassed solution was cooled in a salted ice bath (-10 °C) before 0.2 M TBAF · xH2O (1.5 equiv alkyne groups on polymer) in THF was added slowly via a degassed syringe. The reaction solution was stirred in the salted ice bath for 30 min before being warmed to room temperature. The reaction was allowed to react for 16 h for deprotection of a block copolymer and 4 h for deprotection of a homopolymer of trimethylsilyl methacrylate. To stop the reaction, the solution was passed through a short silica column and rinsed with THF, followed by the evaporation of the solvent under reduced pressure. The polymer was then precipitated in a centrifuge tube containing 45 mL of petroleum ether and centrifuged for 10 min at 8000 rpm. After the solvent was decanted off, the solid light pink or colorless polymer was dried at high vacuum for 2 h. Complete removal of trimethylsilyl protecting groups was confirmed by 1H NMR, with the disappearance of the peak at 0.165 ppm and the appearance of the alkyne peak at 2.5 ppm with an integration equivalent to one H. Mn (theo) ) 15300 g mol-1 (Mn (GPC) ) 23500 g mol-1, PDI ) 1.13). Formation of Micelles from Deprotected Block Copolymers. Deprotected block copolymer (80 mg) was dissolved in 8 mL of N,Ndimethylformamide (DMF). Distilled water was added dropwise to the polymer solution while stirring until the solution went cloudy (ca. 10 mL) and took on a slight bluish tinge to its light pink color. The polymer micelle solution was dialyzed against water using a tubular membrane (molecular weight cutoff (MWCO) 3500 Da). After dialysis, the concentration of the aqueous solution was adjusted to 2 mg mL-1.



Swelling of Micelles Hydrophobic Core. The 2 mg mL-1 solution of polymeric micelles in water from the previous step was dialyzed against a 4:1 water/THF solution (MWCO 3500 Da) for 3 days. The volume of the swelled micelle solution was then diluted to 1 mg mL-1. Click Cross-Linking of Micelles. Cu(PPh3)3Br (3.18 mg, 0.00342 mmol) and N,N-diisopropylethylamine (DIPEA; 4.41 mg, 0.042 mmol) was preweighed into a 25 mL flask along with a small length of copper wire (ca. 2 cm) and a magnetic stirrer bar. The diazide cross-linker (0.0019 mmol, in 0.4 mL THF for 25% cross-linking density) was added along with a solution of 1 mg mL-1 swelled polymer micelles (10 mL, equivalent to 0.0156 mmol alkyne groups) to the reaction flask. The reaction mixture was stirred for 3 days at room temperature before being transferred to presoaked dialysis membrane tubing (MWCO 3500 Da) and dialyzed against a 1:5 THF/water solution for 4 days with at least four solution changes. The membranes were then transferred to 2 L of distilled water for an additional 2 days of dialysis. A sample of the cross-linked micelle solution (2 mL) was kept for particle sizing and TEM imaging while the remaining solution was freeze-dried for further investigation via FT-IR. ReductiVe Degradation of Micelles Cross-Linked with Disulfide Containing Cross-Linker. The cross-linked micelle (1 mg) was dissolved in a 6.5 M DTT solution in DMAc. The degradation of the disulfide group was monitored by taking samples every 30 min at both 25 and 70 °C. The samples were immediately analyzed by GPC. The amount of cleavage was determined by comparing the integral of the crosslinked micelle with the emerging signal of the original block copolymer. Reaction with Co2(CO)8. Model Reaction with Propargyl Alcohol. Co2(CO)8 (104 mg, 0.3 mmol) was weighed into a Schlenk flask and then dissolved in chloroform (5 mL), and propargyl alcohol (17 mg, 0.3 mmol) was added in 5 mL of chloroform. The flask was swirled to dissolve the cobalt carbonyl, resulting in a very dark orange-brown solution, and left to react for 1 h. The product was isolated by removing the solvent under a stream of air. Reaction with Block and Homopolymers. Co2(CO)8 (34.1 mg, 0.1 mmol) was weighed in a flask under a nitrogen atmosphere and sealed with a rubber septa. Polymers (P(PAMA)24, 12.5 mg; P(PAMA)24-bP(PEGMA)40, 63.7 mg; P(PAMA)24-b-P(PEGMA)40 + 25% A (Mn (theo) ) 17970 g mol-1, calculated using the cross-linker density of 24% from Table 1), 98.5 mg; P(PAMA)24-b-P(PEGMA)40 + 25% B ((Mn (theo) ) 17860 g mol-1, calculated using the cross-linker density of 19% from Table 1), 91.8 mg) were dissolved in 7 mL of chloroform and transferred to a flask that was then sealed with a rubber septa. Note: the cross-linked micelles were obtained by clicking the crosslinker A or B in aqueous solution following by dialysis, freeze-drying, and redissolving in chloroform. The solutions were purged with nitrogen Table 1. Degree of Cross-Linking from FT-IR and Hydrodynamic Diameter Dh of Cross-Linked Micellesa degree of azide groups: cross-linkingc hydrodynamic sample cross-linker alkyneb (%) (%) diameterd (nm) 1 2 3 4 5 6 7 8 9 a

A A A A A A A B

150 95 75 71 50 48 25 0 25

99 93 69 64 57 51 24 0.0 19

9(2 11 ( 2 8(3 16 ( 3 11 ( 3 14 ( 3 17 ( 2 15 ( 3

Using cross-linker A or B and un-cross-linked micelles based on P(PAMA)24-b-P(PEGMA)40 in relation to the amount of cross-linker employed. b Molar ratio of azides of diazide cross-linker used to the available alkyne functional groups in the micelles. c Molar ratio of reacted alkynes to unreacted alkynes obtained from the integration of alkyne and carbonyl band via FT-IR spectra; % degree of cross-linking ) 100 - [((alkyne/carbonyl)integralscross-linkedmicelles)/((alkyne/carbonyl) integralsun-cross-linkedmicelles)] × 100 d Diameter measured by dynamic light scattering in water at concentration of 1 mg mL-1 at 25 °C.

3219

for 10-15 min. The polymer solutions were transferred to their respective Co2(CO)8 flask via a degassed syringe. The complexing reactions were allowed to take place at room temperature for at least 1 h. The majority of the solvent was removed under reduced pressure. The solid complexes were reddish brown. Analytical Methods. Gel Permeation Chromatography (GPC). Gel permeation chromatography was performed using a Shimadzu modular system containing a DGU-12A degasser, LC-10AT pump, SIL-10AD autoinjector, CTO-10A column oven, and a RID-10 refractive index detector. The following columns were used for analysis, a Polymer Laboratories 5.0 µm bead-size guard column (50 × 7.5 mm), four linear columns (300 × 7.8 mm; 500, 103, 104, 105 Å pore size). N,NDimethylacetamide (HPLC grade, 0.05% w/v BHT, 0.03% w/v LiBr) with a flow rate of 1 mL min-1 was used as the mobile phase. The injection volumes were 50 µL. Samples were prepared at concentrations of approximately 1 mg mL-1 and were filtered through 0.45 µm RC filters. The unit was calibrated using commercially available linear polystyrene standards (0.5-1000 kDa, Polymer Laboratories). Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR analyses were performed using a Bruker DPX-300 (resonance frequencies of 300.2 MHz for 1H nuclei and 75.48 MHz for 13C nuclei). Transmission Electron Microscopy (TEM). Analyses were performed using a JEOL1400 TEM with a beam voltage of 100 kV and a Gatan CCD for acquisition of digital images. Samples were prepared by placing a droplet of a 1 mg mL-1 polymer solution on a formamide and graphite coated copper grid and draining the excess using filter paper after 60 s. To negatively stain the samples a droplet of 2% (w/v) phosphotungstic acid solution was placed on the copper grid for 30 s before being drained with filter paper. Dynamic Light Scattering (DLS). Particle sizes were determined using a Malvern Nano-Zetasizer (laser, 4 mW; λ ) 633 nm; angle, 173 backscatter) and 1 mg mL-1 solutions in distilled water that were filtered through 0.45 µm filters before analysis. The averaged data from five runs, each consisting of five scans of 10 s each, were used for each sample. The mean diameter was obtained from the arithmetic mean using the number distributed diameter of each particle size. Fourier-Transform Infrared (FT-IR) Spectroscopy. The FT-IR measurements were performed using a Bruker IFS 66/S Fourier transform spectrometer equipped with a tungsten halogen lamp, a KBr beam splitter, and a DTGS detector. Each spectrum in the spectral region of 4000-800 cm-1 was calculated from the coadded interferograms of 32 scans with resolution of 4 cm-1. Fourier-Transform Near-Infrared (FT-NIR) Spectroscopy. FT-NIR spectroscopy was used to determine the monomer conversions by following the decrease of the vinylic stretching overtone of the monomer at 6200 cm-1. A Bruker IFS 66/S Fourier transform spectrometer equipped with a tungsten halogen lamp, a CaF2 beam splitter and a liquid nitrogen cooled InSb detector was used. The sample was placed in a FT-NIR quartz cuvette (1 cm × 2 mm) and polymerized at 60 °C. Traces were analyzed with OPUS software. Each spectrum in the spectral region of 8000-4000 cm-1 was calculated from the coadded interferograms of 64 scans with a resolution of 4 cm-1 with measurements taken every 5 min. UV/Vis Spectroscopy. Ultraviolet-visible spectroscopy was carried out using a Cary 300 Bio UV-Vis spectrophotometer (Varian). Absorption spectra were measured in chloroform from 200 to 800 nm with a resolution of 1 nm in a UV cuvette with 10 mm path length. Thermal GraVimetric Analysis (TGA). Thermal decomposition properties of polymers were recorded using a Perkin-Elmer Themogravimetric Analyzer (Pyris 1 TGA). Analyses were conducted over a temperature range of 30-500 °C with a programmed temperature increment of 30 °C per min. Cell Assay. Preparation of Samples. The block polymer P(PAMA)b-P(PEGMA) was dissolved in water, while the cobalt containing polymer or micelles P(PAMA)-b-P(PEGMA)-Co2(CO)6, P(PAMA)-b-

3220


P(PEGMA)-Co2(CO)6 + 25% cross-linker A, and P(PAMA)-bP(PEGMA)-Co2(CO)6 + 25% cross-linker B were dissolved in approx 5 mL of DMF (with a brief immersion in a sonicator) and then added to presoaked dialysis membrane and dialyzed against water for 3 days. The solution was then diluted to concentrations of Co-alkyne complex of 5.5 × 10-6 M, 1.1 × 10-5 M, 5.5 × 10-5 M. These samples were sterilized by γ radiation. Cell Treatment. The cell line for use in this experiment was the mouse fibroblast Earle’s L cells-NCTC Clone 929 (L929). Cells were cultured in Eagle’s Minimal Essential Medium (EMEM) + 10% fetal calf serum (FBS) + antibiotics (5000 U · mL-1 penicillin and 5000 µg · mL-1 streptomycin). Cells were seeded to plates at a concentration of 50000 cells mL-1 media and each plate contained 2 mL media. Each sample was prepared in triplicate. The dishes were placed into a 5% CO2 incubator maintained at 37 °C ( 2 °C. After 24 h, the media was then poured out and replaced with 2.0 mL of fresh media. A total of 200 µL of polymer samples were added to each plate to achieve a final concentration of Co complex of 5.0 × 10-7 M, 1.0 × 10-6 M, 5.0 × 10-6 M. The samples were then returned to the incubator for 3 days. After 3 days, cells were washed with Osmosol, detached by a 2 mL of the 1:1 mixture of tripsine and osmosol and analyzed by a Beckman Coulter Vi-CELL.

Results and Discussion The synthesis of the monomer trimethylsilyl propargyl methacrylate (TMS-PAMA) was extensively described in earlier publications when it was used in the polymerization via ATRP43,44 and RAFT,45 respectively. In this work we employed the RAFT process using a similar procedure reported in an earlier publication.45 The synthesis of low molecular weight polymers based on TMS-PAMA were targeted by engaging high RAFT agent concentration (ratio of monomer to RAFT ) 28:1). To avoid too much loss of monomer, the polymerization was carried out to approximately 90% conversion leading to polymers with 24 repeating units. Higher conversions were found to result in the loss of RAFT functionality as evidenced by the subsequent block copolymerization with PEGMA, which resulted in broad molecular weight distributions indicative of dead polymer.40 P(TMS-PAMA)24 with a theoretical molecular weight of Mn (theo) ) 4700 g mol-1 (Mn (GPC) ) 8000 g mol-1, PDI ) 1.12) was used as a macroRAFT agent in the polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) to generate amphiphilic block copolymers (Scheme 2). A linear pseudo first-order kinetic plot was obtained in the first 300 min of the polymerization, equivalent to a monomer conversion of around 40%, which was followed by a subtle retardation, suggesting the loss of radicals (see ESI). At the same time, the molecular weight distributions were observed to broaden (Figure 1), indicating the occurrence of termination reactions. However, up to a monomer conversion of 50%, the polymerization was well-controlled leading to polymers with narrow molecular weight distribution (PDI < 1.2; Figure 1). P(TMS-PAMA)24-b-P(PEGMA)40 (Mn (theo) ) 16700 g mol-1, Mn (GPC) ) 20400 g mol-1, PDI ) 1.24) was employed for further investigations. All calculations are from now on based on the theoretical molecular weight because the calibration of the GPC system, which is based on polystyrene, does not give reliable information. In the next step, the TMS protective group, which was necessary to prevent uncontrolled cross-linking during the RAFT process,45 had to be removed. Tetra-nbutylammonium fluoride hydrate (TBAF · xH2O) buffered with acetic acid was utilized in a mixture of THF and toluene leading to the complete formation of the unprotected alkyne group (see ESI). A mixture of solvents was necessary to ensure the full

Withey et al.

Figure 1. Molecular weight Mn and PDI vs monomer conversion x of the polymerization of poly(ethylene glycol) methyl ether methacrylate in the presence of poly(trimethylsilyl propargyl methacrylate)24 macroRAFT agent in toluene at 60 °C. Samples were taken after a reaction time of 2, 4, 6, 14, 15, and 18 h and the conversions were determined using NMR. [RAFT] ) 0.0125 mol L-1, [PEGMA] ) 0.625 mol L-1, [AIBN] ) 2 × 10-3 mol L-1.

solubility of TBAF · xH2O and the polymer at the same time. Only then was full conversion achieved. Using THF or toluene alone resulted in incomplete removal of the TMS protective group. As depicted in Scheme 1, the RAFT end group is partly removed from the polymer, which results in decoloration of the polymer (see ESI). Evidence for the replacement of the RAFT end group by hydrogen has confirmed earlier using mass spectroscopy analysis.45 Closer inspection of the NMR (see ESI) confirmed indeed the disappearance of the signal around 7 ppm, which belongs to the RAFT agent, while all other functional groups including alkyne are still present. The cleavage of the RAFT end group can vary widely from batch to batch and ranges from 100% removal to sometimes less than 10%. The resulting polymers range, therefore, from pink to colorless in appearance. The exact amount in each case can be determined by UV/vis spectroscopy using the absorbance at 515 nm (ε ) 100 L mol-1 cm-1; see ESI). So far, it is unknown why the RAFT end group is frequently removed, while a repeat experiment using similar concentrations does not affect the RAFT end group to a significant extent. The resulting polymer had a narrow molecular weight distribution, indicating that no side reactions such as cross-linking reactions occurred (Mn (theo) ) 15300 g mol-1, Mn (GPC) ) 23500 g mol-1, PDI ) 1.13). The increase in molecular weight, as measured by GPC after deprotection, is the result of the altered hydrodynamic diameter. Because GPC results do not suggest any signs of cross-linking or other side reactions and NMR results confirm the complete removal of the protective group, a change of hydrodynamic diameter might have occurred leading to higher measured molecular weights when compared with polystyrene standards. The polymer was dissolved in DMF followed by the slow addition of water under vigorous stirring to generate micelles. The solution was then dialyzed against water to remove the organic solvent, resulting in a bluish-purple aqueous solution. Dynamic light scattering analysis at 25 °C revealed a hydrodynamic diameter of 17 ( 2 nm, which is complemented by the size obtained via TEM (see ESI). The micelles were cross-linked via click chemistry using two different types of cross-linker (Scheme 3). The purpose here was the design of two different types of cross-linked micelles: a cross-linked micelle of high stability and a cross-linked micelle



3221

Scheme 3. Structure of Co2(CO)6(alkyne) complex

in which the disulfide can be cleaved to form the thiol in a reductive environment such as in the presence of glutathione, which is found in abundance inside the cell. Reason for employing two different cross-linkers is to compare the differences of the Co-loaded cross-linked micelles, with permanent or degradable cross-linker, in its biological activity. The advantage of the click reaction is that it can be carried out under ambient conditions, which prevents disturbance to the dynamic equilibrium of the micelles. While the cross-linker A (Scheme 2) has been already reported in literature,17 a synthesis procedure for cross-linker B needed to be established. Bis-(hydroxyethyl)disulfide was reacted with toluene sulfonyl chloride prior to the nucleophilic substitution with sodium azide. An alternative pathway using methane sulfonyl chloride was found to be incomplete. The click reaction to cross-link micelle has been carried out earlier showing that good results can be achieved when crosslinking the core,17 while shell cross-linking seemed to be hampered by the hydrophobic nature of the cross-linking agent.18 The core was successfully cross-linked by either utilizing a polymer with pendant alkyne groups in combination with an azide cross-linker17,18 or by exploring the opposite pathway with an azide containing polymer and a propargyl ether.19 To allow better access to the two types of hydrophobic crosslinkers employed in this work (Scheme 3), the core of the micelle was slightly swollen by the addition of 20% THF to the aqueous solution. The micelles were then stirred with various amounts of diazide cross-linker (Table 1) for 3 days in the presence of Cu-catalyst and copper wire, followed by a thorough purification using dialysis against a water/THF mixture and then water. The consumption of azide cross-linker after 3 days was monitored using FT-IR. The successful click reaction can be evidenced by the disappearance of the strong signal at ∼2100 cm-1 (CtC stretch) and alkyne signal at ∼3300 cm-1 (HsCtC stretch), which are replaced by triazole bands at ∼800, 1650, and 2800 cm-1. A range of cross-linker concentrations were employed. A ratio of 100% in Table 1 is equivalent to a 1:1 molar ratio between azide functional groups (from cross-linker A) and alkyne groups (from polymer), which is equivalent to a molar ratio between propargyl methacrylate repeating units and cross-linker of 2:1. Ratios below 100% were mainly targeted in this study because a significant amount of pendant alkyne groups are required for complexation with Co2(CO)8. Table 1 summarizes the experiments carried out; the results listed were obtained from the FT-IR spectra displayed in Figure 2. The signal corresponding to the alkyne functionality decreases with increasing cross-linker concentration. The area of the signal was compared to the carbonyl stretch vibration as internal standard (Figure 2) to calculate the amount of reacted alkyne groups. The results obtained would indicate that, within an estimated error of 20%, considering the reproducibility of the integration of the signal areas, complete cross-linking has been achieved and one cross-linker connects indeed to two polymer chains

with each other. However, this scenario is not likely considering that a certain amount of azide group of a cross-linker, once connected to a polymer chain, is trapped in its position and cannot reach unreacted alkyne functionalities. While the FTIR results indicate the reaction did occur, the calculated results should, therefore, be treated with caution. This result also needs to be seen in context that the suggested azide and triazole stretch, and bend vibrations mentioned above are absent, suggesting that FT-IR seems not sensitive enough to detect vibrations of low intensity. This is especially obvious when an excess of crosslinker (150%) was used where it is expected that some azide pendant group would appear as the result of cross-linker surplus. When a smaller amount of cross-linker has been used to target cross-linking densities well below 100%, it is more likely that the diazide cross-linker has indeed clicked with two alkyne groups. Each azide group is now surrounded by an excess of alkyne groups and the scenario of having a cross-linking density, which is close to the targeted density (determined by the chosen alkyne-azide ratio) is highly likely. It is also interesting to note that the cross-linking process is independent from the type of cross-linker as long as the cross-linker has a similar base structure. The degradable cross-linker B results in a similar outcome (Table 1). The occurring cross-linking can also be observed in the decline of the hydrodynamic diameter of the micelle in water. With increasing amount of cross-linker, the micelle is more and more compact, thus, the hydrodynamic diameter in water decreases (Table 1 and ESI). The resulting cross-linked micelles have a superior stability in many solvents maintaining their size at various concentrations. This is especially evident in solvents such as DMAc, a good solvent for both blocks, where the

Figure 2. FT-IR spectra of core cross-linked micelles of P(PAMA)24b-P(PEGMA)40 using varying amounts of cross-linker.

3222


Withey et al.

Figure 4. TEM image (negatively stained with 2% PTA at 250K magnification) of a 1 mg mL-1 solution of cross-linked P(PAMA)24b-P(PEGMA)40 in water using 75% cross-linker.

Figure 3. Top: GPC curve of block copolymer and cross-linked micelles using various amounts of cross-linker; Bottom: GPC curve of the block copolymer used for cross-linking with the residue after cross-linking (magnified for better comparison). Normalized height indicates that all curves have a uniform area.

structure of the micelle is disrupted. Cross-linked micelles in contrast retain their core-shell structure, even at concentrations as low as typical concentrations used in the GPC system (1 mg mL-1). The high stability of the resulting structure also needs to be seen in the context that high pressures, high temperature (here 50 °C), and a high shear rate are applied in a typical GPC system confirming that a very stable core-shell system has been created. In Figure 3, the molecular weight distribution of the micelle is displayed together with the molecular weight distribution of the block copolymer used. The resulting molecular weight of the micelle is around 400000 g mol-1, which is probably immensely undervalued because linear polystyrene standards were used. A small amount of low molecular weight polymer remained after the cross-linking procedure. Closer inspection revealed that the peak was not congruent with the block copolymer signal. Instead, the molecular weight is closer to the molecular weight of the P(PEGMA)40 block alone. Attention needs to be drawn to the mechanism of RAFT polymerization. For detailed discussion the reader is referred to more specialized articles, but it should be pointed out that the RAFT process leads to a range of side products including homopolymer and various block copolymers.36-38,40,46 P(PEGMA) homopolymer, which is generated during the block copolymer synthesis, is not capable of taking part in the crosslinking process. This water-soluble P(PEGMA) byproduct can easily be removed using a dialysis membrane with a molecular weight cutoff of 50000 g mol-1. The resulting product does not show a low molecular weight product during GPC analysis.

The small size of the micelle and cross-linked micelle was confirmed using TEM (Figure 4). Micelle sizes of around 10-15 nm complemented the DLS data listed in Table 1. It should be noted here that cross-linking will probably not only take place between different polymer chains (interchain cross-linking). Intrachain cross-linking is certainly expected. However, the analytical techniques employed cannot distinguish between both forms of cross-linking. What is known is that sufficient intermolecular cross-linking occurred to capture all the block copolymers into the cross-linked micellar structure (Figure 3). Only the micelles obtained from a theoretical cross-linker concentration of 25% A or B (Table 1) were further investigated. The micelle was adequately cross-linked in this case but also had around 75% of alkyne groups available for further reaction with Co2(CO)8. The reaction between low molecular weight alkynes and Co2(CO)8 has been known for a long time.47,48 The deep orange colored dicobalt octacarbonyl reacts readily with alkynes forming orange, red, or purple compounds, depending on the substitution on the alkynes. Co2(CO)8 looses its bridging CO ligand, replacing it with alkyne, which is arranged in a perpendicular position to the Co-Co bond (Scheme 3). The proceeding reaction can be monitored by the loss of CO, which is visible by the development of gas during the reaction.49 FTIR analysis was used to confirm the complete reaction with the elimination of the CO band of the bridging ligand at 1859 cm-1 and the disappearance of the band of the alkyne group. The terminal CO ligands at 2090, 2050, and 2025 cm-1 in contrast remained.47 Prior to the investigation of the Co complexation of the polymers, the reaction of propargyl alcohol only, which is the reactive pendant group of the polymer, was investigated. The complex Co2(CO)6(propargyl alcohol) was described earlier47-49 as a orange-red solid. Following on from propargyl alcohol, a range of polymers were reacted with Co2(CO)8 using then the same procedure including PAMA24, P(PAMA)24-b-P(PEGMA)40, and P(PAMA)24-b-P(PEGMA)40 cross-linked with permanent cross-linker A or degradable cross-linker B. With the addition of stoichiometric amounts of Co2(CO)8 to alkyne groups the chloroform solutions turned



3223

Figure 5. FT-IR spectra of the different alkyne compounds before and after reaction with Co2(CO)8 to form (alkyne)Co2(CO)6, evidenced by the disappearance of the alkyne band at around 3250 cm-1 and the appearance of the three terminal CO bands, while the bridging CO band is absent. The block copolymer used is not cross-linked.

Figure 6. UV/vis spectra of Co complexes in chloroform. The absorption coefficient ε has been calculated based on the concentration of alkyne groups in solution.

in all cases dark orange-red. Care needs to be taken to prevent the oxidation of the reactive orange Co2(CO)8, which oxidizes to purple cobalt(II) oxide/carbonate on prolonged exposure to air, which is insoluble in the solvent used. In addition, the decomposition of orange Co2(CO)8 to insoluble black tetracobalt dodecacarbonyl (Co4(CO)12) has been observed, especially when heating the reaction mixture. The final complex Co2(CO)6(propargyl alcohol) is in contrast reasonably stable under ambient conditions. However, these complexes are reactive, and the contact with certain solvents such as DMSO should be avoided since DMSO can act as a ligand.50 The reaction was carried out for one hour to ensure completeness. No heating was applied since elevated temperature can catalyze a Pauson-Khand reaction, which may take place when monomer impurities are present.51 Earlier studies confirm that the reaction between alkynes and Co2(CO)8 usually has a half-life (t1/2) of around 300 s, depending on the type of alkynes and other factors.49 Therefore, a reaction time of 1 h is more than sufficient. The Co complexes of all investigated compounds were stable in chloroform solution for an extended period of time. However, once the solvent has been removed, the solubility of the resulting macromolecular complexes was rather low and the orange-red solids could only be redissolved in THF, chloroform, and DMAc at very low concentrations (see ESI). It should be noted here that the limited solubility was only observed when the Co complexes were completely dried and stored over an extended period. However, when chloroform was removed under reduced pressure only for a limited time, the Co complexes could be redissolved into chloroform, DMAc, DMF, THF, or DMSO at high concentrations. The solids were investigated using FT-IR. Co2(CO)6(propargyl alcohol) showed indeed the behavior described in literature with the disappearance of the bridging ligand at 1859 cm-1 and the alkyne group at around 3250 cm-1. A similar observation was made for the macromolecular complexes (Figure 5), confirming that all reactive groups involved (alkynes and Co2(CO)8) have reacted to completeness. The typical pattern with three bands of the terminal CO ligands is present in all investigated cases, indicating that the polymeric surrounding does not have any significant influence on the structure of the Co complex. In addition, a small band at 1550 cm-1 appears, which can be assigned to the formation of alkene as a result of the formation of a complex between alkyne and cobalt. The alkyne group therefore takes on double bond character52 (Figure 5).

Further analysis to corroborate the comprehensiveness of the reaction between alkyne and Co2(CO)8 was carried out using UV/vis spectroscopy. While propargyl alcohol and all the polymers are colorless or only slightly pink (Note: the purple RAFT end group has mostly been eliminated during the TMS deprotection step), the resulting Co-containing polymers are orange-red, but as a solid dark orange-brown. A characteristic transition at a wavelength of 349 nm appeared in the UV/vis spectra in chloroform. The molar absorptivity (or extinction coefficient) of Co2(CO)6(propargyl alcohol) was calculated to be around 4000 L mol-1 cm-1. A similar result was obtained using different polymers, where the calculation was based on the concentration of available alkyne groups. Molar absorptivity values of 4000 L mol-1 cm-1 are equivalent to Co2(CO)6(propargyl alcohol), suggesting that all Co2(CO)8 has reacted with all available alkyne ligands (Figure 6). So far, the Co-complexed polymers were investigated in solutions such as chloroform. Chloroform presents a reasonable solvent for both blocks, the P(PEGMA) block and the P(PAMA)Co2(CO)6; therefore, the formation of micelles is absent in uncross-linked polymers, and only cross-linked micelles maintain their core-shell structure in chloroform. However, considering the application of the Co-loaded micelles as a therapeutic agent, the investigation of these polymers in an aqueous environment is of more importance. The solution of Co-containing polymers could not directly be transferred to an aqueous solution because brown precipitate was formed upon the addition of water. The chloroform solvent was therefore evaporated and redissolved in DMF followed by dialysis against water. The solubility in DMF is limited, but enough to achieve concentrations, which are needed for cell testing. The subsequent dialysis of the DMF solution against water resulted in an orange-red aqueous solution, which was stable for several days. Only after 4 days at ambient temperature was the formation of a brown precipitate observed. It is therefore important that aqueous solutions are freshly prepared for further analysis. The Co-complexed P(PAMA) homopolymer and Co2(CO)6(propargyl alcohol) are not water-soluble and were therefore not investigated any further. To determine the amount of cobalt in the micelle, a small fraction of the sample was freeze-dried and investigated via thermogravimetric analysis. For the un-cross-linked block copolymer, a theoretical weight fraction of cobalt of 6.5% was calculated. The remaining weight fraction after the sample was heated up to 600 °C (see ESI) was found to be only 5%. It seems, therefore, that during the procedure of

3224


Figure 7. Distribution of hydrodynamic diameter Dh as obtained using dynamic light scattering (DLS) in water before and after complexation with Co2(CO)8 (1 mg mL-1).

redissolving of the Co-loaded polymer in DMF and subsequent dialysis some cobalt has been lost. The Co-loaded cross-linked and un-cross-linked micelles were then investigated using DLS. In contrast to a chloroform solution, the block copolymer formed micelles in aqueous solution caused by the inherent amphiphilic character in water. Cross-linked micelles already had a core-shell structure in chloroform, which was maintained in a water environment. As listed in Table 1 and depicted in Figure 7, the block copolymer and cross-linked block copolymer form core-shell nanoparticles with hydrodynamic diameters of less than 20 nm. With the formation of Co2(CO)6(alkyne) pendant side groups, the core-shell nanoparticles swell inside and double approximately in size. The Co-loaded particles can be investigated using TEM without additional staining because the presence of cobalt can easily be identified. The underlying block copolymer needed negative staining with phosphotungstic acid to be visible and reveal its round shape. The cobalt-loaded micelle, in contrast, discloses the location of cobalt without additional treatment. As displayed in Figure 8, spherical features with a diameter of 15 ( 3 nm are clearly visible outlining the core of the micelle. The presence of cobalt can further be confirmed by energy dispersive spectroscopy. The inset in Figure 8 shows the presence of cobalt at different energy levels.

Withey et al.

Prior to the cell study, the reductive cleavage of cross-linker B was studied. The reductive sensitive cross-linker is known to degrade within a short time in lab experiments in the presence of reducing agents such as dithiothreitol (DTT), which cleaves the disulfide bridge to thiols. This reaction is sufficiently fast with DTT and occurs often within minutes in micelles that have been cross-linked using disulfide bridges.53 It is believed the same reaction can take place within the cell where the reduced form of glutathione (GSH) will take on the role of DTT cleaving the disulfide bridge to thiols, thus, degrading the cross-linker of the micelle.54 The micelle was therefore dissolved in DMAc, which is a good solvent for both blocks, and stirred at room temperature and at 25 and 70 °C with 6.5 mM DTT. The reaction was monitored using DLS and GPC. While DLS only reveals a decline in scattering intensity, which is equivalent to the breakdown of the cross-linked micelle, the GPC results allow a more quantitative analysis. Samples were injected into the GPC system after 30 min reaction time. At 70 °C, 65% of the sample consisted of free block copolymer (or a products with twice the size of the first peak), which is similar in size to the block copolymer before cross-linking. The remaining 35% of material are still captured in cross-linked structures (see ESI). At 25 °C, in contrast, less than 10% of block copolymer has been released. It seems therefore that the degradation process in this system is rather slow in contrast to earlier reports where the micelle has been cleaved into unimers in less than 30 min.53 To test the cytotoxicity of Co-loaded micelles, similar cobalt concentrations (5.0 × 10-7 M, 1.0 × 10-6 M, 5.0 × 10-6 M) to an earlier study using low molecular weight acetylenehexacarbonyldicobalt complexes, including the gold standard displayed in Scheme 1, were employed. The studies were carried out up to 10 days showing cytotoxicity depending on the type of Co ligand.55 The polymers in aqueous solution, the Co-loaded block copolymer without cross-linking and cross-linked either with A or B, were exposed to fibroblast L929 cells. In addition to Co-loaded polymers, the block copolymer P(PAMA)-bP(PEGMA) was also tested (Figure 9). Earlier studies showed that micelles with P(PEGMA) shells are easily taken up by cells independent from the content of the interior.27 Slight variations in the rate of uptake can be found depending on

Figure 8. TEM of micelles obtained from Co-complexed P(PAMA)-b-P(PEGMA) cross-linked using 25% A, including the energy dispersive spectrum showing a high presence of cobalt in the sample (arrows indicate Co signal).



3225

was found to be highly efficient, with an almost complete reaction of all available cross-linker. The reaction of stoichiometric amounts of alkyne groups and Co2(CO)8 leads to the formation of Co-complexed polymers, which are evident by the red-orange color. The reaction was found to be complete in less than a 1 h reaction time. While un-cross-linked micelles have a good antiproliferative effect, cross-linking of the core of the micelle and permanent encapsulation of the Co complex reduces this effect significantly. The cytotoxicity is slightly elevated when a degradable cross-linker has been used.

Figure 9. Cell growth inhibition of L929 fibroblast cells after being exposed to various solutions of Co-containing micelles (including the underlying block copolymer only) at different concentrations for 60 h. The concentration refers to the cobalt concentration, and the error bars are the result of three independent experiments.

the amount of core-cross-linking.56 The block copolymer alone did not show any significant cytotoxicity. Upon complexation with Co2(CO)8, all the samples showed an increased cytotoxicity. The un-cross-linked block copolymer resulted in an inhibition of cell growth of up to 60% at a Co concentration of 5 × 10-6 mol L-1. Both cross-linked (A or B cross-linker) and Co-loaded micelles show a significant reduction in cell toxicity. Cross-linking of the micelle obviously hampers the antiproliferative effect. Because the mode of action of cobalt drugs is unknown, it can only be speculated what causes the reduced toxicity. It seems that the mechanism involves the direct contact of the Co complex to down- or up-regulate certain functions. This pathway is prevented when the Co complex is hidden in the core of the micelle, while cross-linking prevents any dynamics of the micelle such as the unimers-micelle equilibrium. A slightly higher toxicity is observed with the degradable cross-linker B. The cleavage of the cross-linker can explain the increased toxicity of the Co-loaded micelle cross-linked with B. As discussed earlier, the cleavage of the cross-linker in the current system is rather slow, and there might not be any significant breakdown within the cell interior after 72 h to cause a significantly different toxicity compared to the permanently cross-linked micelle. However, a more detailed study is necessary to follow the degradation of the micelle in a surrounding typically found in the interior of a micelle, including potential deactivation of the Co complex by thiols.57 This preliminary cell study showed that the micelles prepared have an antiproliferative effect, and a future study will investigate parameters such as the stability of the micelle in various cell environments and the uptake of the Co-loaded micelle by the cell, which are important aspects to understand the mode of action.

Conclusion The aim of this study was to develop a core-shell carrier for the delivery of cobalt pharmaceuticals. The core-shell nanoparticle was created by self-assembly of the block copolymer based on poly(propargyl methacrylate)-block-poly(polyethylene glycol methyl ether methacrylate). The pendant alkyne groups could hereby take on two functions, as a reactive functional group for cross-linking via click chemistry and as a ligand for the reaction with Co2(CO)8 leading to the complex Co2(CO)6(alkyne). The click reaction to cross-link the micelle

Acknowledgment. The authors thank Dr. Guy Clentsmith from the School of Chemistry, UNSW, for helpful discussions. M.S. would like to acknowledge the Australian Research Council (ARC) for funding. Supporting Information Available. Kinetics of PEGMA polymerization, TEM of un-cross-linked micelle, NMR, GPC, and UV/vis information of TMS deprotection, cross-linking density vs hydrodynamic radius, solubility of P(PAMA)24Co2(CO)6 and P(PAMA)24-b-P(PEGMA)40-Co2(CO)6, and GPC data of cross-linking micelle before and after reduction with DDT. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Guo, Z.; Sadler, P. J. Angew. Chem., Int. Ed. 1999, 38, 1512. (2) Hall, M. D.; Failes, T. W.; Yamamoto, N.; Hambley, T. W. Dalton Trans. 2007, 3983. (3) Ott, I.; Koch, T.; Kircher, B. J. Med. Chem. 2005, 48, 622. (4) Ott, I.; Schmidt, K.; Kircher, B.; Schumacher, P.; Wiglenda, T.; Gust, R. J. Med. Chem. 2005, 48, 622. (5) Uhrich, K. E.; Canizarro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. ReV. 1999, 99, 3181. (6) Matsumura, Y.; Maeda, H. Cancer Res. 1986, 46, 6387. (7) Stolnik, S.; Illum, L.; Davis, S. S. AdV. Drug DeliVery ReV. 1995, 16, 195. (8) Zhang, L.; Nguyen, L. T. U.; Bernard, J.; Davis, T. P.; BarnerKowollik, C.; Stenzel, M. H. Biomacromolecules 2007, 8, 2890. (9) O’Reilly, R. K.; Hawker, C. L.; Wooley, K. L. Chem Soc ReV. 2006, 35, 1068. (10) O’Reilly, R. K.; Joralemon, M. J.; Hawker, C. J.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 5203. (11) Read, E. S.; Armes, S. P. Chem. Commun. 2007, 3021. (12) O’Reilly, R. K.; Joralemon, M. J.; Hawker, C. J.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 5203. (13) Ievins, A. D.; Wang, X.; Moughton, A. O.; Skey, J.; O’Reilly, R. K. Macromolecules 2008, 41, 2998. (14) Thurmond, K. B.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1996, 118, 7239. (15) Li, Y.; Lokitz, B. S.; McCormick, C. L. Macromolecules 2006, 39, 81. (16) Jaturanpinyo, M.; Harada, A.; Yuan, X.; Kataoka, K. Bioconjugate Chem. 2004, 15, 344. (17) Joralemon, M. J.; O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L. J. Am. Chem. Soc. 2005, 127, 16892. (18) O’Reilly, R. K.; Joralemon, M. J.; Hawker, C. J.; Wooley, K. L. New J. Chem. 2007, 31, 718. (19) Jiang, X.; Zhang, J.; Zhou, Y.; Xu, J.; Liu, S. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 860. (20) O’Reilly, R. K.; Joralemon, M. J.; Hawker, C. J.; Wooley, K. L. Chem.sEur. J. 2006, 12, 6776. (21) Huisgen, R.; Moebius, L.; Mueller, G.; Stangl, H.; Szeimies, G.; Vernon, J. M. Chem. Ber. 1965, 98, 3992. (22) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057. (23) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596. (24) Evans, R. A. Aust. J. Chem. 2007, 60, 384. (25) Lutz, J. F. Angew. Chem., Int. Ed. 2007, 46, 1018. (26) Binder, W. H.; Sachsenhofer, R. Macromol. Rapid Commun. 2007, 28, 15.

3226


(27) Zhang, L.; Bernard, J.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H. Macromol. Rapid Commun. 2008, 29, 123. (28) Tsarevsky, N. V.; Matyjaszewski, K. Macromolecules 2005, 38, 3087– 3092. (29) Oh, J. K.; Siegwart, D. J.; Matyjaszewski, K. Biomacromolecules 2007, 8, 3326–3331. (30) Li, Y. T.; Armes, S. P. Macromolecules 2005, 38, 815. (31) Oh, J. K.; Siegwart, D. J.; Lee, H.; Sherwood, G.; Peteanu, L.; Hollinger, J. O.; Kataoka, K.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 5939. (32) Li, Y.; Lokitz, B. S.; Armes, S. P.; McCormick, C. L. Macromolecules 2006, 39, 2726. (33) Greenberg, S.; Clendenning, S. B.; Liu, K.; Manners, I.; Aouba, S.; Ruda, H. E. Macromolecules 2005, 38, 2023. (34) Minea, L. A.; Sessions, L. B.; Ericson, K. D.; Glueck, D. S.; Grubbs, R. B. Macromolecules 2004, 37, 8967. (35) Sessions, L. B.; Minea, L. A.; Ericson, K. D.; Glueck, D. S.; Grubbs, R. B. Macromolecules 2005, 38, 2116. (36) Favier, A.; Charreyre, M.-T. Macromol. Rapid Commun. 2006, 27, 653. (37) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2005, 58, 379. (38) Perrier, S.; Takolpuckdee, P. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 5347. (39) Barner-Kowollik, C.; Davis, T. P.; Heuts, J. P. A.; Stenzel, M. H.; Vana, P.; Whittaker, M. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 365. (40) Stenzel, M. H. Chem. Commun. 2008, 3486. (41) Oes, S.; Yagihara, T.; Okabe, T. Tetrahedron 1972, 28, 3203. (42) Gujadhur, R.; Venkataraman, D.; Kintigh, J. T. Tetrahedron Lett. 2001, 42, 4791. (43) Ladmiral, V.; Mantovani, G.; Clarkson, G. J.; Cauet, S.; Irwin, J. L.; Haddleton, D. M. J. Am. Chem. Soc. 2006, 128, 4823.

Withey et al. (44) Chen, G.; Tao, L.; Mantovani, G.; Geng, J.; Nystrom, D.; Haddleton, D. M. Macromolecules 2007, 40, 7513. (45) Quemener, D.; Le Hellaye, M.; Bissett, C.; Davis, T. P.; BarnerKowollik, C.; Stenzel, M. H. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 155–173. (46) Stenzel, M. H. Macromol. Rapid Commun. 2009, DOI: 10.1002/ marc.200900180. (47) Sternberg, H. W.; Greenfield, H.; Friedel, R. A.; Wotjz, J.; Markby, R.; Wender, I. J. Am. Chem. Soc. 1954, 76, 1457. (48) Greenfield, H.; Sternberg, H. W.; Friedel, R. A.; Wotiz, J. H.; Markby, R.; Wenders, I. J. Am. Chem. Soc. 1956, 78, 120. (49) Tirpak, M. R.; Wotiz, J. H.; Hollingsworth, C. A. J. Am. Chem. Soc. 1958, 80, 4265. (50) Atkin, A. J.; Williams, S.; Sawle, P.; Motterlini, R.; Lynam, J. M.; Fairlam, I. J. S. Dalton Trans. 2009, 3653. (51) Pauson, P. L.; Khand., I. U. Ann. N.Y. Acad. Sci. 1977, 295, 2. (52) Dickson, R. S.; Fraser, P. J. In AdVances in organometalic chemistry; Gordon, F., Stone, A., Eds.; Academic Press: New York, 1974; p 336. (53) Zhang, L.; Liu, W.; Lin, L.; Chen, D.; Stenzel, M. H. Biomacromolecules 2008, 9, 3321. (54) Kakizawa, Y.; Harada, A.; Kataoka, K. Biomacromolecules 2001, 2, 491. (55) Schmidt, K.; Jung, M.; Keilitz, R.; Schnurr, B.; Gust, R. Inorg. Chim. Acta 2000, 306, 6. (56) Duong, H. T. T.; Nguyen, U. T. L.; Stenzel M. H. Polymer Synth. 2010, in press. (57) Ott, I.; Gust, R. BioMetals 2005, 18, 171.

BM901050X

Macromolecular Cobalt Carbonyl Complexes ... - ACS Publications

Recommend Documents