MATERIALS
A.T. Neffe and A. Lendlein
Institute of Polymer Research, GKSS Forschungszentrum GmbH, Teltow, Germany
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A common approach in polymer synthesis aims at designing monomers and polymerising these, or combinations of them, through a variety of reaction mechanisms into linear (co)polymers. In addition to the selection of (co)monomers and the synthesis procedure, the versatility of polymers results from various options that exist for their processing and surface modification. In this way, polymers are tailored for specific applications. In particular, the introduction of advanced processing techniques, defined chemical functionalisation, and the systematic investigation of interactions between materials and specific biological systems has extended the innovation potential of established polymers for medical applications.
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Figure 1: Polymers described in the text PEI 1; PBI 2; PAN 3 and its (co)monomers N vinylpyrrolidon 4; aminoethylmethacrylate 5; 2-Methyl-2-propene sulphonic acid sodium salt 6; and 2-acrylamido-2-propane sulphonic acid 7.
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This article discusses the tailoring of biofunctionalities of selected polymer systems whose syntheses are established and whose main applications are nonmedical. These functionalities include differences in material-cell interactions determined in cell culture tests with certain cell lines to influence cell adhesion and proliferation.1-7 For example, in co-cultures, reduced adsorption of biological materials (proteins and cells) on the polymer surface,8-14 and increased effectiveness in the separation of specific components in a biological system (apheresis and gas separation) has been achieved.15-16 Figure 1 shows the compounds discussed in this article. The functionality of poly(ether imide) (PEI) 1 was tailored by processing techniques that resulted in defined complex three-dimensional (3D) shapes. Alternatively, PEI 1 can be tailored by blending with other polymers such as poly(benzimidazol) (PBI) 2 to increase the hydrophilicity of the resulting polymer, or by suitable surface modification. The biofunctionality of polyacrylonitrile (PAN) 3 depends strongly on the type of comonomer and this interdependence is discussed. Typical (co)monomers of acrylonitrile (AN) varying charge and hydrophilicity of the AN-based (co)polymer are N-vinylpyrrolidon (NVP) 4, 2-aminoethyl-methacrylate (AEMA) 5, or 2-methyl-2-propene-1-sulphonic acid sodium salt (NaMAS) 6.
Haemocompatiblity and cell adhesion
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Table I: Interaction of PAN and its copolymers with kidney epithelial cells, fibroblasts and blood. Key: (++) highly favourable, (+) favourable, (o) not harmful, (-) unfavourable, a: depending on NVP content, haemocompatibility increases with increasing NVP content, while cell compatibility descreases with increasing NVP content.
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Synthesis of PEI 1 requires several steps: in general nitro displacement of a nitrophthalodinitrile with a bisphenolate, followed by hydrolysis of the nitriles, and imide formation. PEI is commercially available from different providers, for example, GE Plastics (www.geplastics.com ), PolyOne Cooperation (www.polyone.com), and RTP Co. (www.rtpcompany.com). It is amorphous; its glass transition temperature (Tg) is approximately 215 °C and it has a high Young’s modulus (E) of 3-10 GPa. A typical average molecular weight (Mn) for the commercially available material from GE Plastics is in the range of 20000–25000 g·mol-1. PEI is soluble in N-Methyl-2-pyrrolidone (NMP), N,N-dimethylacetamide and meta cresol. Its main applications are in the food industry, aircrafts and automobiles.17 In medical technology, PEI is used for sterilisation containers, pipettes and dental devices. PEI is compatible with many sterilisation methods: steam, gamma radiation, ethylene oxide (EtO) and dry heat sterilisation.
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Figure 2: PEI microparticle formed from a 10 wt.% solution of PEI in NMP–water (96:4) (B) with a spraying/coagulation process. Top: low magnification; bottom: high magnification of the marked section. The microparticles formed under these conditions have an average size of 120 µm and 80 % total porosity.15
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To decrease the hydrophobicity of PEI, it can be blended with other polymers such as PBI 2 to give a more hydrophilic material that supports the growth of fibroblasts and keratinocytes.2 The PEI–PBI blend supported the adhesion of cells to the polymer, however, cell growth was slow after several days and cell agglomerates were released from the polymer after 10–15 days.2 This functionality could be advantageous for the tailored transfer of cells. Cells are grown on this scaffold and are released after a pre-set time to be applied in regenerative therapy.
PAN 5 and its copolymers are synthesised by free radical polymerisation of acrylonitrile and comonomers in N,N-dimethyl formamide (DMF). The reaction can be performed so that the resulting copolymers have a random sequence structure of repeating units. PAN is commercially available from Aldrich (www.sigmaaldrich.com); PAN fibres are available from Bayer (www.bayerindustry.com). The Mn of the polymer is influenced by the reaction conditions and the type and ratio of the (co)monomer(s), and is typically in the range of 20000–100000 g·mol-1. PAN is an amorphous solid with a Tg of 105 °C, which is influenced by the kind and ratio of (co)monomer. The mechanical properties are strongly influenced by the (co)monomers, for example, poly(acrylonitrile-co-styrene) has an E modulus of 3.9 GPa. PAN is soluble in DMF and NMP. PAN is mostly used nowadays for textiles and the preparation of carbon fibres. Membranes of PAN have been used in dialysis.
The comonomer has a strong influence on the compatibility with blood or certain cell lines as listed in Table I.3,4 Incorporation of polar neutral or acidic groups increases the haemocompatibility, and basic groups reduce haemocompatibility compared with the homopolymer PAN. However, basic groups such as the amino groups in poly(AEMA-co-AN) favour adhesion of kidney epithelial cells and, to some respect, fibroblasts. Hepatocytes5 prefer an amphoteric polymer such as the material obtained from copolymerisation of acrylonitrile with 2-acrylamido-2-methylpropane sulphonic acid 7. The tailoring of PAN and its copolymers for certain cell lines can beneficially be applied in cell/tissue culture systems and biohybrid organs. EtO and gamma irradiation have been successfully used in the sterilisation of PAN.
3D processing
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Figure 3: SEM morphology of a PEI hollow fibre membrane A formed from a solution consisting of 25 wt.% PEI, 55 wt.% NMP, and 20 wt.% g-butyrolactone in a triple spinneret. a) cross-section, b) morphology at the inner and c) at outer edge.16
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A challenge in the formation of 3D structures is the synthesis of particles with tailored size and porosity. A promising procedure for the preparation of highly porous microparticles is a spraying/coagulation process. This process is described below for PEI 1.15 PEI was dissolved in either NMP, NMP–dimethylsulphoxide, or NMP–water. The solution was pressed through a spraying spinneret of 50 µm in diameter to form small droplets. The size of the droplets was predominantly controlled by the gas flow during the spraying process and the spinneret size. However, other factors such as viscosity and concentration of the solution also have an influence on droplet size. The droplets then fell through an air gap into a water coagulation bath as nonsolvent, which induced a phase inversion and formation of the highly porous structure of the PEI particles. Depending on the exact conditions, particles with a highly homogeneous particle size of 77–120 µm were formed that have a total porosity of 76–81%. Figure 2 shows a scanning electron microscope image of a cut through one of these particles in which the inner porosity is clearly visible. The difference between inner and outer porosity of the particles formed in this process is especially noteworthy. These particles from PEI are suitable for application in apheresis; they combine mechanical and thermal stability with a highly porous structure. As will be demonstrated later, the PEI microparticles can potentially be surface modified by covalently binding ligands. This could be utilised to design particles for specific adsorption processes.
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Figure 4: PEI modified with linear poly(ethylene imine). A poly(ethylene imine) with an average molecular weight of approximately 600 g·mol-1 was used, thus m (number of repeating units in polymer chain) was approximately 13.1
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A processing technique for the synthesis of complex 3D structures from a polymer solution is the use of a triple spinneret in the formation of highly asymmetric hollow fibres. This procedure has been demonstrated for PEI.16 The inner orifice of the triple spinneret was fed with water as coagulation fluid. The PEI solution in NMP/y-butyrolacton or NMP/DMSO was transported through the middle orifice; and the outer orifice was used for the transportation of NMP and NMP–water mixtures. Therefore, during the coagulation the two sides of the membrane forming polymer were exposed to two different solvents, which resulted in a hollow fibre material with highly asymmetric inner and outer surface structure. An example is shown in Figure 3. In this way, hollow fibres can be formed that have an open outer shell structure. These hollow fibres have greatly improved permeation capabilities while retaining their separation profile (a mean pore diameter of 3.9–6 nm and cut-off of 5.6–8 nm) and are potentially useful in bioreactors, dialysis and gas separation applications.
Surface modification
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Figure 5: Visualisation of keratinocytes by vital staining with fluorescein diacetate after 8 days (left), 11 days (middle), and 13 days (right) of culture on a poly(ethylene imine) modified PEI membrane. Each picture represents 330 µm.1
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PEI itself is a hydrophobic material and the support of cell growth can be enhanced by introducing a more hydrophilic surface. This can be achieved, for example, by reaction with poly(ethylene imine) (Figure 4).1 The reaction is performed in water/2-propanol at a ratio of 1:1 at 70 °C and is finished within 30 min. Excess reagent can be removed by washing with water. Therefore, this method constitutes a fast and simple method of modification. This approach is usable with other amines as well, thereby the potential to introduce a wide range of chemical groups, for example, acidic or basic groups, or compounds with biological activity. This gives the manufacturer a further tool for tailoring PEI for a specific biological environment or process. This can potentially be used in tailoring the apheresis particles from PEI for the removal of a specific compound as mentioned above. The derivatised material is suitable as cell support for keratinocytes, which showed a faster proliferation on it than on unmodified PEI (Figure 5). The combination of modified PEI with keratinocytes can potentially be applied as artificial skin substitute.
To improve the stability of the surface modifications, PEI surfaces modified with poly(ethylene imine) have been stabilised by reaction with poly(methacrolein-co-vinylpyrrolidon).18 The blood compatibility was improved by coating the PEI-poly(ethylene imine) surfaces with brominated polyvinylpyrrolidon.7 Modification of PEI membranes with amines can also be utilised to systematically influence the pore structure of the membrane by a partial and controlled degradation of the surface.19,20
Next steps for industry
PEI and copolymers based on acrylonitriles are examples of established polymer systems that allow tailoring with respect to their biofunctionality. Knowledge of their chemistry, processing, sterilisability and biointeractions makes them interesting candidates for biomedical applications, which should enable the industry to efficiently translate these technologies into new products. Potential areas of application include particles for apheresis, membranes and scaffolds for bioreactors, and tissue engineering.
References
1. C. Trimpert et al., “Poly(ether imide) Membranes Modified with Poly(ethylene imine) as Potential Carriers for Epidermal Substitutes,” Macromol. Biosci., 6, 274–284 (2006).
2. G. Altankov et al., “On the Tissue Compatibility of Poly(ether imide) Membranes: An In Vitro Study on Their Interaction With Human Dermal Fibroblasts and Keratinocytes,” J. Biomat. Sci. Polym. Ed., 16, 23–42 (2005).
3. T. Groth et al., “Interaction of Human Skin Fibroblasts With Moderate Wettable Polyacrylonitrile-Copolymer Membranes,” J. Biomed. Mater. Res., 61, 290–300 (2002).
4. T. Groth et al., “Development of Membranes With Improved Haemocompatibility for Biohybrid Organ Technology,” Clin. Hemorheol. Microcirc., 32, 129–143 (2005).
5. M.H. Grant et al., “The Viability and Function of Primary Rat Hepatocytes on Polymeric Membranes Developed for Hybrid Artificial Liver Devices,” J. Biomed. Mater. Res., Part A, 73A, 367–375 (2005).
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12. I. Taniguchi et al, “Macromonomer Purification Strategy for Well-Defined Polymer Amphiphiles Incorporating Poly(ethylene gylcol) Monomethacrylate,” Macromol., Rapid Commun., 27, 631–636 (2006).
13. J.Y. Park et asl., “Polysulfone-Graft-poly(ethylene glycol) Graft Copolymers For Surface Modification of Polysulfone Membranes,” Biomat., 27, 856–865 (2006).
14. J. Groll et al., “A Novel Star PEG-Derived Surface Coating For Specific Cell Adhesion,” J. Biomed. Mater. Res. A 74A, 607–617 (2005).
15. W. Albrecht et al., “Development of Highly Porous Microparticles From Poly(ether imide) Prepared By a Spraying/Coagulation Process,” J. Memb. Sci., 273, 106–115 (2006).
16. W. Albrecht et al., “Preparation of Highly Asymmetric Hollow Fiber Membranes From Poly(ether imide) by a Modified Dry–Wet Phase Inversion Technique Using a Triple Spinneret,” J. Memb. Sci., 262, 69–80 (2005).
17. Ultem Profile Brochure, GE Plastics 2001.
18. W. Albrecht et al., “Preparation of Novel Composite Membranes: Reactive Coating on Microporous Poly (ether imide) Support Membranes,” J. Memb. Sci., 269, 49–59 (2006).
19. W. Albrecht et al., “Preparation of Aminated Microfiltration Membranes By Degradable Functionalization Using Plain PEI Membranes With Various Morphologies ,” J. Memb. Sci., 292, 1–2, 145–157 (2007).
20. R. d’Aggostino, “Process Control and Plasma Modification of Polymers,” J. Photopolym. Sci. Technol., 18, 245–249 (2005).
Axel T. Neffe is Head of Department Biomimetic Materials and Andreas Lendlein is the Director of the Institute of Polymer Research, GKSS Forschungszentrum GmbH, Kantstrasse 55, D-14513 Teltow, Germany, tel. +49 3328 352 450, email: andreas.lendlein@gkss.de, http://biomaterialien.gkss.de.
