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Aust. J. Chem. 2003, 56, 1035–1038
Biomimetic Honeycomb-Structured Surfaces Formed from Block Copolymers Incorporating Acryloyl Phosphorylcholine Martina H. StenzelA,B and Thomas P. DavisA A
Center for Advanced Macromolecular Design (CAMD), School of Chemical Engineering and Industrial Chemistry, University of New South Wales, Sydney 2052, Australia. B Author to whom correspondence should be addressed (e-mail:
[email protected]).
We report the preparation of biomimetic honeycomb-structured porous films. These regular arrays were obtained by casting block copolymers composed of polystyrene and poly(acryloyl phosphorylcholine), so that they mimicked a cell membrane. The size of the pores and regularity of the hexagonal array is strongly dependent on the block length. The block copolymers were prepared via RAFT (reversible addition fragmentation transfer) polymerization leading to well-defined products with a good control over block sizes. Manuscript received: 9 May 2003. Final version: 3 July 2003.
The interaction between the surface of a biomaterial and its biological environment is one of the main foci when designing material for biomedical applications. Inflammatory reaction can often be observed when using conventional polymers as biomaterials. Increasing the hydrophilicity of the polymer is one approach to minimize adverse protein and cellular interactions.[1] Phosphorylcholine, a constituent of phospholipids in biomembranes, has successfully been introduced into polymers to impose a better cytocompatibility.[2,3] There is evidence that this functionality assists in the reduction of protein adsorption and cell–material interaction. Porous polymeric materials have attracted increasing attention as materials for biomedical applications such as tissue engineering.[4] Microporous films can be fabricated in a number of ways. A very simple method has been reported by François et al.[5] who obtained highly regular honeycombstructured porous films by casting a polymer solution under humid conditions. This approach involves dissolving welldefined polymers in a highly volatile solvent. The solvent is then driven off using a humid airflow. The fast evaporation induces the temperature of the solvent to decrease. Consequently, water droplets condense and grow on the cold solution surface while organizing themselves into a hexagonal pattern. The polymer starts precipitating around the water droplets forming a solid envelope. Solvent and water then evaporate leaving a highly organized honeycomb-structured film behind. The water droplets therefore act as templates for the pores.[6–9] The polymers used to obtain honeycomb-structured films include star polymers, comb polymers, and block copolymers, as well as a variety of other polymers.[9] In this communication, we report the formation of regular hexagonal arrays of pores prepared from a biocompatible
HO
Cl Cl
P
CH2Cl2
Cl
Cl
1/2 O2
O P
+
Cl
P O
O
HO
+ HCl
O
OH
O O O
O O
N(C2H5)3 THF
O− P
O
N(CH3)3
O O
O
CH3CN
N+
O
P O O
Scheme 1.
block copolymer based on polystyrene and poly(acryloyl phosphorylcholine) as a biomimicking material. These welldefined block copolymers could easily be prepared using RAFT polymerization.[10,11] Results and Discussion Acryloyl phosphorylcholine has been prepared according to the method described by Ishihara et al.[12] and Seo et al.[13] (Scheme 1). This monomer was used to prepare block copolymers with styrene. A very versatile way to obtain block copolymers is the RAFT process.[14] RAFT polymerization allows the preparation of block copolymers with facile control over the block sizes. Furthermore, the polymerization leads to products with a narrow size distribution (narrow polydispersity).
© CSIRO 2003
10.1071/CH03124
0004-9425/03/101035
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M. H. Stenzel and T. P. Davis
A crucial step in the preparation of block copolymers using RAFT polymerization is the preparation of a suitable macroRAFT agent, which represents the first block of the copolymer. The macroRAFT agent results from the RAFT polymerization of a monomer in the presence of a suitable RAFT agent. Block copolymers are formed by the polymerization of a second monomer leading to the transfer of the growing macroradical onto the macroRAFT agent and subsequently leading to a block copolymer (Scheme 1).[15] Firstly, we prepared the polystyrene block in the form of a polystyrene macroRAFT agent. The second step proved to be more difficult as the disparate solubilities of the styrene and phosphorylcholine blocks created problems in maintaining homogeneity. Finally, a mixture of N,N-dimethylformamide (DMF) and methanol was used, which was prepared by dissolving the macroRAFT agent and 2,2 -azoisobutyronitrile (AIBN) in DMF and mixing it into a solution of 2-acryloyloxy ethyl phosphorylcholine in methanol. We tried to achieve a broad block copolymer composition with varying block sizes of polystyrene and poly (2-acryloyloxy ethyl phosphorylcholine). Therefore, we prepared two polystyrene macroRAFT agents with different molecular weights and restarted the macroRAFT agents with 2-acryloyloxy ethyl phosphorylcholine (ACP) in different ratios to obtain PACP blocks of different sizes (Table 1). Because of the low monomer concentration used, the reaction rate was low, specifically the conversion after 24 h at 60◦ C was below 30% (Table 1). The conversion was determined using NMR spectroscopy by dissolving the polymer in a solvent mixture (deuterated chloroform and methanol). This leads to signals of both building blocks, while pure chloroform result in the loss of phosphorylcholine signals. To confirm this result and to purify the polymer from the residual monomer, a known amount of dried polymer and monomer mixture was dialyzed against water. Both conversion results were found to be in accord. The conversion of 2-acryloyloxy ethyl phosphorylcholine was found
to increase with decreasing macroRAFT agent concentration. This has been explained by inhibition and retardation phenomena, which occur during the RAFT polymerization using dithiobenzoates.[16] The resulting (co)polymeric products were clearly soluble in hydrophobic solvents such as chloroform indicating that no poly(2-acryloyloxy ethyl phosphorylcholine) homopolymer was generated as this would have precipitated. The theoretical molecular weight, as well as the theoretical composition and the resulting mole fraction of PACP of the polystyrene-block-poly(acryloyl phosphorylcholine) (PS-PAPC) block copolymer can be calculated from the conversion and the RAFT agent concentration. As expected, the calculated molecular weight deviates from the experimental molecular weight. This difference can easily be explained by the altered hydrodynamic volume after adding several units of the ionic monomer. The well-defined PS-PAPC block copolymers were all soluble in carbon disulfide although the polymers with a higher mole fraction of PAPC seemed to form a rather colloidlike solution. Casting these polymers from carbon disulfide (10 mg of polymer in 1 mL of carbon disulfide) under a humid tangential airflow resulted in honeycomb-structured porous films of varying quality (Table 1 and Figure 1). With increasing APC block size the disorder of the film increases and the pore size distribution broadens. The highest regularity was therefore obtained with small APC blocks (Fig. 1). The average pore size remains unaffected by the APC block length, while with increasing PS block size the pore size increases. For comparison, the macroRAFT agent was cast under similar conditions resulting in a porous film with less regularity. By consideration of the casting mechanism, involving water droplets interacting with the polymer solution, we can hypothesize that the hydrophilic polymer block mediates with the phase interface. We predict, therefore, that the surface of the pores is enriched with the PAPC blocks, which should also be concentrated at the surface of the
Table 1. Preparation of polystyrene/poly(2-acryloyloxy ethyl phosphorylcholine) block copolymer from polystyrene macroRAFT agentsA with different macroRAFT agent concentrationB Sample PS1-APC 1 PS2-ACP 1 PS1-APC 2 PS2-ACP 2 PS1-APC 3 PS2-ACP 3 PS1-APC 4 PS2-ACP 4 PS1-APC 5 PS2-ACP 5 PS-RAFT
PS-Raft [mol L−1 ]
ConversionC [%]
Units PS-APC
Mole fraction ACP [%]
Mn (calculated)D [Da]
Mn (GPC, THF) [Da]
PDI
Pore sizeE [µm], order of array
3.906 × 10−4
22 24 25 22 23 22 18 19 17 20
308-38 78-51 308-28 78-34 308-20 78-26 308-12 78-18 308-10 78-15 308-0
10.9 39.5 8.3 30.3 6.1 25.0 3.7 18.7 3.2 16.1
46 123 22 308 42 658 17 617 39 499 15 339 36 551 13 235 34 719 12 179
37 800 14 000 36 200 13 500 33 900 12 900 35 500 11 000 36 800 10 800 32 000
1.05 1.08 1.07 1.09 1.13 1.1 1.10 1.08 1.09 1.02 1.05
4.5, disordered 3, disordered 4.7, disordered 3.2, disordered 4.5, disordered 2.7, ordered 4.5, disordered 2.8, ordered 4.5, slightly disordered 2.7, ordered 3–8
5.859 × 10−4 7.813 × 10−4 9.766 × 10−4 1.172 × 10−3
PS1: Mn 32 000, PDI 1.06; PS2: Mn 8 100, PDI 1.05. [APC] = 8.93 × 10−2 mol L−1 in methanol/DMF, 14 : 86%; [AIBN] = 6.1 × 10−4 mol L−1 . C The conversion was obtained from GPC and the resulting block copolymer compositions and mole fractions of PACP in the block copolymer was calculated. D The calculated molecular weight (M (calculated) = [ACP]/[Raft] × conversion × MW n ACP + MWPSmacroRAFT ) was compared with the molecular weight and the molecular-weight distribution obtained from GPC. E The pore sizes of the resulting honeycomb-structured films were obtained after casting from carbon disulfide solution (10 g polymer per litre). A B
Biomimetic Honeycomb-Structured Surfaces Formed from Block Copolymers Incorporating Acryloyl Phosphorylcholine
honeycombs. This will be further investigated and reported. The initial X-ray photoelectron spectroscopy (XPS) studies of a porous film (PS2-ACP5) with the calculated C, N, O, P composition of 84, 0.72, 5.5, and 1.6 wt.-% are shown in Figure 2. The XPS data show a high carbon content of the cast film indicating a surface enriched with polystyrene. Removal of the surface with adhesive tape reveals the honeycomb structure underneath. The surface is now composed of a higher oxygen content indicating an increased concentration of ACP units around the pores.
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Conclusions RAFT polymerization was adopted for the preparation of extremely amphiphilic block copolymers. The block copolymers obtained are soluble in nonpolar solvents, such as carbon disulfide, provided the acryloyl phosphorylcholine block is restricted in size. Casting these polymers from carbon disulfide solution leads to honeycomb-structured porous films with varying quality. We believe that these substrates may prove valuable in studying cell patterning. Experimental
(a)
(b)
(c)
(d )
2-Chloro-1,3,2-dioxaphospholane A five-necked, 2 L reaction vessel was equipped with a thermometer, mechanical stirrer, pressure equalizing dropping funnel, dry nitrogen gas flow, and a condenser. The condenser was connected to the water pump reservoir to adsorb the large quantities of HCl produced. The reaction vessel was charged with 2.46 kg of phosphorous trichloride and 1 L of anhydrous dichloromethane. The solution was mixed and 1.11 kg (17.9 mol) of anhydrous ethylene glycol was added dropwise over a period of 2 h. The endothermic reaction caused the temperature to decrease to around 8◦ C and the reaction stopped when the reaction mixture stabilized at room temperature. Dichloromethane was removed by rotary evaporation under a reduced pressure. The crude 2-chloro1,3,2-dioxaphospholane was purified by distillation (40–43◦ C/22 mbar) and obtained in 65% yield as a colourless fuming liquid. δH (CDCl3 ) 4.3–4.5. δC (CDCl3 ) 65. 2-Chloro-2-oxo-1,3,2-dioxaphospholane
Fig. 1. Honeycomb structured porous films prepared from polymer solution in carbon disulfide (10 g L−1 ). (a) Polystyrene macroRAFT agent (Mn 32 000). (b) PS2-PACP5. (c) PS1-PACP4. (d) PS1-PACP2.
A five-necked 2 L reaction vessel was equipped with a thermometer, mechanical stirrer, condenser, Drierite (Fluka) filled drying tube, sintered glass tipped tube for oxygen gas flow, pressure equalizing flask and glycerine gas flow indicator. The vessel was charged with 858.7 g (6.8 mol) of 2-chloro-1,3,2-dioxaphospholane and 1 L of dry toluene. The mixture was flushed with dry oxygen and the temperature of the mixture rose sharply. The oxygen flow was continued until the mixture remained at room temperature even with an increase in the flow rate of oxygen. Toluene was removed under a reduced pressure. The crude 2-chloro-1,3,2-dioxaphospholane was distilled under vacuum (86–91◦ C/0.3 mbar) and obtained in 59% yield.The pure 2-chloro-1,3,2dioxaphospholane solidified when cooled with ice. δH (CDCl3 ) 4.3–4.9. δC (CDCl3 ) 66.7.
Side view
C: 90% O: 7.5% P: 0.8% N: 0.8%
Fig. 2.
C: 79.6% O: 14.1% P: 1.5% N: 1.3%
XPS analysis of the porous film before and after the removal of the surface of the film using adhesive tape.
1038
2(2-Oxo-1,3,2-dioxaphospholoyl) Ethyl Acrylate A 1.5 L five-necked reaction vessel was equipped with a mechanical stirrer, pressure equalizing dropping funnel, thermometer, condenser, drying tube, and dry air flow to introduce oxygen and minimize the polymerization side reaction. The vessel was charged with 23.25 g (0.20 mol) of 2-hydroxyethyl acrylate, 22.4 g (0.22 mol) of anhydrous triethylamine, and 300 mL of dry tetrahydrofuran (THF). The reaction mixture was then cooled to −30◦ C with a dry ice/acetone bath and 28.74 g (0.20 mol) of 2-chloro-2-oxo-1,3,2-dioxaphospholane in 150 mL of THF was added dropwise over a period of 2 h. After the addition of the 2-chloro-2-oxo-1,3,2-dioxaphospholane, the mixture was allowed to heat up to room temperature and then heated at 40◦ C for a further 2 h. Finally, the reaction mixture was cooled with an ice/water bath to
