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Biodegradable Open Cell Foams of Telechelic Poly(E-caprolactone) Macroligand with Ruthenium (II) Chromophoric Subunits via Sub-Critical CO2 Processing A. Victoria Nawaby,*,† Abdiaziz A. Farah,‡ Xia Liao,† William J. Pietro,§ and Michael Day† Institute for Chemical Process and Environmental Technology, National Research Council Canada, 1200 Montreal Rd, Ottawa, Ontario, Canada K1A 0R6, and Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3 Received March 24, 2005; Revised Manuscript Received June 14, 2005
This work reports on the effect of CO2 at subcritical conditions and the gaseous state on the telechelic poly(-caprolactone) polymers. The tested polymers are semicrystalline in nature and thus the effect of functional groups and their overall contribution to foaming and formation of microstructures with open-cell morphollogy is discussed. Introduction Many biocompatible aliphatic polyesters such as polylactones, polylactides, and polyglycolides have been used in biomedical studies because of the possibility to design components for implants with a wide range of structures with active or passive surfaces.1 Moreover, they can be tailored to have mechanical properties that match bio-structured needs in tissue reconstruction and regeneration. Among these polyesters, poly(-caprolactone) (PCL) offers good resorbable properties. The biodegradability of poly(-caprolactone) has been evaluated against many clinical domains for use in tissue engineering, drug delivery, and target bone imaging.2 Poly(-caprolactone) is unique for its miscibility with a variety of polymers,3 thus extending the properties of PCL and its use in different applications.4 Although for uses in tissue engineering and drug delivery many forms and morphologies of poly(-caprolactone) such as hydrogels,5 blends or composites anchored with other amphiphilic compounds have been produced,6 none of the matrices generated is ideal for cell culturing with concurrent low cellular development in vitro.7 For tissue engineering scaffolds, a macroporous open cell structure is required with pore sizes of 100-300 µm for cell penetration. Several techniques have so far been developed for the fabrication of porous biodegradable polymers for cell transplantation. Solvent casting/salt bleaching,8 phase separation,9 emulsion freeze-drying10 thermally induced phase separation,11 and foaming with benign gases such as carbon dioxide (CO2) are among the reported studies.12 Although many of the developed methods are capable of producing porous matrices, they either lack in degree of * To whom correspondence should be addressed. Telephone: (613) 9939698. Fax: (613) 991-2384. E-mail:
[email protected]. † National Research Council Canada. ‡ Present address: National Research Council Canada (NRC), Ottawa, Ontario, Canada. § York University.
interconnectivity between the cells or use solvents that limit their end use application.13 The use of nontoxic solvents in the generation of porous bio scaffolds is a field of interest for investigation with considerable commercial application.14 It is well-known that compressed gases such as CO2 and N2 can dissolve in polymers at high concentrations to nucleate cells upon a sudden thermodynamic change and instability inducing a phase separation in the polymer-gas solution.15 This will thus result in the escape of the gas from the matrix leaving behind a cellular morphology. Generally, the cellular morphologies with cell diameter in the range of 1-100 µm and cell density of about 108 cells/cm3 are termed as microcellular foams. Plastic morphologies with cell diameter in the range 0.01-1 µm and cell density in excess of 1012 cells/cm3 are termed as ultramicrocellular foams. For biomedical and industrial applications, CO2 is an attractive choice due to its plasticization effect on polymers, reduction in viscosity, and ease in processing such as polymer impregnation.16 The type of morphologies produced by this gas is very much dependent on the degree of solubility and temperatures at which the instability in the system is induced. Provided that the right processing conditions are available, the cell membranes can rupture during the foaming process with no foam collapse leaving behind an open cell morphology. The interaction of CO2 with polymers does not always have a plasticization effect and in some cases upon depressurization a more compact structure in terms of polymer chain packing is obtained. This effect has been demonstrated in the poly(caprolactone)-CO2 system. 17 The generation of intriguing polymeric morphologies is not only dependent on the amount of gas sorbed but also the types of modifications applied to polymer chains. The incorporation of transition metal complexes into a polymeric matrix offers a unique approach for the preparation of processable materials with tunable electrical, electrochemical, magnetic, and optical properties associated with the metal.18 Very often metal polymeric complexes might also act as
10.1021/bm050223n CCC: $30.25 © 2005 American Chemical Society Published on Web 07/16/2005
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Figure 1. Chemical structures of the telechelic poly(-caprolactone) macroligand (1) and the metallopolyester (2).
convenient source of metal embedded films and coatings with desirable conformational and morphological properties. This communication reports on our preliminary investigation on fabrication of microcellular biodegradable telechelic poly(-caprolactone) foams with open pore diameters of about 10 µm via subcritical CO2 processing. The effect of different functionality at the poly(-caprolactone) chain end such as the bipyridine with vacant coordinating sites and tris(bipyridine) Ru complex at one termini was studied for its interaction with CO2 gas. Due to the low metal content in the polymer microstructure, we anticipate these open microcellular polymers tagged with luminescent chromophores, not only to function as an ideal cell culture and cell attachment niche for potential application in tissue skin regeneration but also good candidates in diagnostic molecular imaging as optical sensors for biomedical applications. Experimental Section Methods and Materials. The poly(-caprolactone) macroligand (1) and the Ru anchored polyester (2) were obtained and characterized as reported earlier.19 Bone dry CO2 supplied by Air Products was used. The equilibrium solubility of CO2 in the samples were determined using the method described by Arora.20 Polymer films with a thickness of 380 µm were placed in saturation vessels and saturated with CO2 at a pressure of about 60 atm and 25 °C for 24 h. The pressure in the vessel was rapidly released, and the sample was transferred to a balance where mass loss as a function of time was recorded. Thermal analyses of the polyesters and foamed specimens were performed using a TA Instruments 2920 MDSC. All samples were prepared by compression moulding at 100 °C followed by ice water quench. DSC samples were heated from room temperature to 160 °C at 10 °C/min, cooled at room-temperature conditions, and reheated to 150 °C at 10 °C/min all under a nitrogen atmosphere. The first cooling and second heating cycle was used to determine the melting temperature. The X-ray diffraction patterns were measured using a Bruker AXS Inc. instrument at a radiation wavelength of 1.5418 Å, a scan range of 2θ from 1 to 40°, and a scan speed of 0.9°/min. Samples for foaming were prepared by saturating 380 µm thick films with CO2 at 25 °C and pressures up to 60 atm. After rapid depressurization, the samples were foamed for 2 min in the temperature range from room to 58 °C and then cooled in ice water to capture the cellular morphology. Foamed samples were aged at room conditions for at least 2 days, prior to being fractured in liquid nitrogen and surfaces examined by scanning electron microscopy (SEM) using a Hitachi S-4800 field emission
scanning microscope. Foam densities were determined with the total immersion method 21 and the cellular morphology was characterized using the image pro software. Results and Discussion Poly(-caprolactone) macroligand (1) and the Ru tagged polyester (2) of the structures as depicted in (Figure 1) were obtained via insertion-coordination ring-opening polymerization.19 These telechelic polyesters provide macromolecular architectures with two distinct functional groups per polymeric chain. They both possess reactive hydroxyl chain end at one termini and the other termini end-capped with an R,R′diimine ligand or Ru tris(bipyridine) moiety originating from the initiators. These polymers are semicrystalline in nature with bulk density of 1.14 g/cm3, glass transition temperature of -63 °C and crystal melt point of 58 °C. The equilibrium solubility of CO2 in poly(-caprolactone) macroligand (1) and the Ru anchored polyester (2) at 25 °C and 60 atm was found to be about 5.9 and 7.5 wt % respectively. The reported percent mass uptakes were calculated from the mass loss data obtained from desorption studies. The data were ploted against the square root of the desorption time which resulted in linear plots.20 Extrapolation of the lines to zero desorption time results in the uptake of CO2 at the end of the 24 h sorption period. The differential scanning calorimetry of the neat polymers as represented in (Figure 2) have indicated that the poly(caprolactone) macroligand (1) (Figure 2a) has a more uniform crystalline structure than the Ru tagged polyester (2) due to narrower melting peaks obtained (Figure 2b). This change in re-crystallization and degree of order between the two polymers was further supported by X-ray diffraction studies. The poly(-caprolactone) macroligand (1) exhibits higher intensity in the 2θ region between 20 and 25° (Figure 3a). In the same region, the Ru tagged polyester (2) shows a much lower intensity indicating a lower degree of crystallinity and order in the molecule as depicted in (Figure 3b). Moreover, the observed WAXD patterns for both samples have revealed two strong and one weak crystallographic reflections that were indexed satisfactorily to the known poly(-caprolactone) diffraction data.22 In using gases as blowing agents for polymers, the nature of interaction between the gas molecules and the polymer chains affects the rate of plasticization or crystallization and the types of foam morphologies generated. It is well-known in crystalline polymers that the gas does not dissolve in the crystalline portion of the polymer and the gas molecules go around the crystallites in order to move through the amorphous phase. Thus, there will be an impedance of flow
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Figure 2. DSC thermogram of (a) poly(-caprolactone) macroligand (1) and (b) the metallopolyester (2).
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Figure 4. XRD pattern of (a) poly(-caprolactone) macroligand (1) and (b) the metallopolyester (2) both conditioned with CO2 at 60 atm and 25 °C and subsequently foamed at 58 °C.
Figure 3. XRD pattern of the neat (a) poly(-caprolactone) macroligand (1) and (b) the metallopolyester (2).
and hence polymers with a higher degree of amorphous fraction are able to dissolve more gas, and as such, more segmental mobility is observed. Previous reports have indicated that the gas dissolved in the amorphous phase has a crystallization effect associated with poly(-caprolactone)CO2 system.17 During this process, the polymer chains after the escape of the gas molecules are capable of packing into a much denser and compact formation. The same effects were observed when Ru tagged polyester (2) is foamed with CO2 at saturation conditions of 60 atm and 25 °C. Under the same saturation conditions the poly(-caprolactone) macroligand (1) exhibited no foaming or cell nucleation. X-ray diffraction patterns obtained for both samples after foaming is shown in (Figure 4). The reflection at (111) plane grew significantly in intensity and two additional weak reflections at 2θ of about 34° and 37° were observed for Ru tagged polyester (2). The effect of thermal treatment and CO2 conditioning of the sample was thus further investigated by DSC studies. As presented in Figure 5, under the same heating rate the melting peak for both samples thermally treated and conditoned with CO2 is broader in comparison with the untreated samples (Figure 2). The lower heat of melting observed in Figure 5 as compared to Figure 2 as well as presence of a shoulder between 45 and 50 °C (Figure 5) indicates the formation of
Figure 5. DSC thermogram of foamed (a) poly(-caprolactone) macroligand (1) and (b) metallopolyester (2) at 58 °C, CO2 saturation conditions of 25 °C and 60 atm.
new crystals from the former amorphous phase and perfection of the existing crystals. Thermally treated specimens without CO2 conditioning as well as CO2 conditioned samples without thermal treatment both revealed broadened peaks and a shoulder between 45 and 50 °C in their DSC thermograms. This finding suggests that both treatments given to the specimens play an important role in the crystal growth. Furthermore, the crystalline melt point for the treated samples shifted and increased by 6 °C. Similar behavior has been reported for poly(-caprolactone) and its morphological changes as a result of treatment with CO2.17
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the rigid-amorphous phase in the polymer and effect of gas on these regions little information is presented in the literature. Since it is not possible to isolate the phases (crystalline, rigid-amorphous, and amorphous), it is difficult to investigate their role in foaming and the types of foams obtained. However, the role of metal-polymeric complexes and the types of end group ligands incorporated into biopolymers and their contribution to foaming and changes in the morphology of the semicrystalline poly(-caprolactone) can be investigated. This finding however points to a new research area leading to fabrication of novel biodegradable polyester morphologies with fluorescent labels, an important goal for many potential applications. Since the design of new ruthenium (II) chemotherapeutic drugs now includes new metallopharmaceuticals for antitumor application, it is thus anticipated that the cellular structures produced to be of low toxicity and to have biomedical applications.23,24 Acknowledgment. We express our gratitude to Dr. Pamela Whitfield for XRD data collection and Dave Kingston for assistance with the SEM analysis. References and Notes Figure 6. SEM microphotographs of CO2 conditioned (a) poly(caprolactone) macroligand (1) and (b) the metallopolyester (2); CO2 saturation conditions of 60 atm and 25 °C and subsequent foaming at 58 °C.
Our observation for the samples under study was further confirmed via measuring the densities of the foamed specimens prepared in the temperature range 25 to 58 °C. The interaction between CO2 and poly(-caprolactone) macroligand (1) resulted in no changes in the density over the entire tested temperature range where as in the case of Ru tagged polyester (2), the density reached a maximum value of 1.7 g/cm3 upon depressurization at room conditions followed by a decrease to 1.5 g/cm3 at about 50 °C. Since the segmental mobility has changed as the result of metal chromophore at one polymer termini, the amorphous fraction in the Ru tagged polyester (2) is comparatively higher than the poly(-caprolactone) macroligand (1). This physical change does have an important effect on the morphology of the foam samples generated even though the polymer does undergo crystallization when in contact with CO2 (Figure 6, parts a and b). At about 50 °C, analysis of the samples indicates that cells indeed can be nucleated in regions of the Ru tagged polyester (2), and as the temperature increases to 58 °C, these regions turn into open microcellular structures. Under the same processing conditions, the poly(-caprolactone) macroligand (1) exhibits no cell nucleation and the sample remain unchanged. In conclusion, this study leads us to believe that the presence of the metal chromophores in the poly(-caprolactone) microstructure as the case of polyester (2) seem to have generated sites within the polymer whereby nucleation of cell is promoted as compared to the neat poly(-caprolactone) macroligand (1). These domains can either be amorphous in nature or rigid-amorphous and the foaming process in the case of amorphous polymers is easy to understand; however, it remains unclear for the case of semicrystalline polymers and the role crystallites play in this process. With respect to
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