3 Development of Microstructuring Technologies
3.5 Chemical Functionalization of Polycarbonate
Rapidly growing interest for discovering new materials and surfaces for biomedical application has attracted various scientific disciplines for understanding cell mate-rial interactions [26]. Literature of biomatemate-rial research has shown that the arti-ficial extracellular microenvironment so-called scaffolds play a pivotal role in the process of regenerating tissues [27]. For tissue engineering applications, mostly the bulk properties of the material are considered for use as implant for a particular tissue type. On the other hand, it is the surface properties of polymer material and not the bulk properties that tune the biocompatibility. Many of the polymers are
Figure 3.6 (left) Scaffold with sharp thin corners; (right) Scaffold made using the improved mold.
1
X Y Z
Thermoforming of a cavity with friction
0 .356E-04.713E-04.107E-03.143E-03.178E-03.214E-03.249E-03.285E-03.321E-03 PLOT NO. 1 NODAL SOLUTION
STEP=3 SUB =200 TIME=15 /EXPANDED USUM (AVG) RSYS=0 DMX =.321E-03 SMX =.321E-03
1
X Y Z
Thermoforming of a cavity without friction
0 .367E-04.734E-04.110E-03 .147E-03.183E-03.220E-03.257E-03.294E-03.330E-03 PLOT NO. 1 NODAL SOLUTION
STEP=3 SUB =500 TIME=15 /EXPANDED USUM (AVG) RSYS=0 DMX =.330E-03 SMX =.330E-03
Figure 3.7 Simulated thermoformed cavity of the scaffold (rotationally symmetric): (left) with friction, (right) without friction.
1
YZX
Thermoforming of a step 1mm
.101E-04.145E-04.189E-04.234E-04.278E-04.322E-04.367E-04.411E-04.455E-04.499E-04 PLOT NO. 1 AVG ELEMENT SOLUTION
STEP=5 SUB =250 TIME=30 S(AVG) S E N K C I H TTOP DMX =.001123 SMN =.101E-04 SMX =.499E-04
1
YZ X
Thermoforming of a step 0.5mm
.257E-04.284E-04.311E-04 .338E-04.365E-04.392E-04.419E-04.446E-04.473E-04.499E-04 PLOT NO. 1 AVG ELEMENT SOLUTION
STEP=5 SUB =250 TIME=30 S(AVG) S E N K C I H TTOP DMX =.537E-03 SMN =.257E-04 SMX =.499E-04
Figure 3.8 Simulation model of a quarter of the mold; (left) the initial state, (right) optimized result.
82 Handbook of Polymers for Pharmaceutical Technologies
qualifying physical and mechanical properties but fail to provide desired biocom-patibility. The chemical nature and morphology of the polymer surface mostly gov-ern the biological response. In order to solve this difficult problem a hybrid material is required which possesses the advantages of both. The complex tissue- or cell cul-ture-derived extracellular matrix (ECM) is used as surface coating for seeding cells, but the composition and structures of these naturally derived materials is not well defined. Alternatively, proteins or other molecules have been adsorbed on the mate-rial surface to see their interaction with growing cultures. The disadvantage of these surfaces is that the coated layer can be dissociated from the substrate during the experiment.
The advantage of the naturally derived surfaces is their inherent biocompatibility.
However, inconsistent purity arising from lot-to-lot variability and potential contami-nation of pathogens in cases where the material is obtained from a non-human source are disadvantages. On the other hand, synthetic materials are better to reproduce but they lack biological cues found in natural extracellular matrix. Therefore significant efforts have been made to search for polymer blend with functional groups that can be used to couple bioactive species on the surface.
Due to the emerging concepts of engineering, microscale topographies to control cell-substrate interface [28] and reasonably sensitive post-functionalization methods are required. There are various methods through which surface functionalization of the polymer materials can be achieved with covalent linkage, among them are grafting copolymerization, plasma/laser irradiation and chemical functionalization. Obviously, during fabrication of different topographies the surface chemistry of the material in question cannot be protected. Therefore, it is reasonable to opt for post-functional-ization methods to make sure of the available chemical environment on the scaffold.
Chemical methods with mild reaction condition are best suited for this purpose due to their having the least influence on the micro- and nanoscopic topographies and homogenous distribution over the surface.
Among the various useful polymer materials, recent years have witnessed a strong rise in the use of polycarbonates as a material of choice in biomedical applications. Lee et al. examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surface with different micropore sizes (200 nm–8.0 µm) [29]. Welle et al. described electrospun aliphatic polycarbonate as tailored tissue scaffold, where the photochemical bulk modification indicates the possibility of spatial control of the biodegradation rate [30]. In an earlier section we mentioned the use of track-etched polycarbonate membranes that have been introduced as substrate for perfused cell cul-ture in 3D format [31]. The microscopic cavities of the polymer scaffold provide three-dimensionality and nanoscopic pores provide nourishment to the cell culture from all around. Therefore, it is interesting to develop polycarbonate chemistry so that the desired functional groups and molecules can be introduced to the surface for obtaining cell substrate response.
The functionalized polycarbonates are mostly prepared by premodification of the monomers or block and copolymerization with other monomers [32–34]. For instance, tyrosine-derived polycarbonate (TyrPCs) are versatile polymer platform that can be tuned to different substrate types by copolymerization with other monomer types [35,36].
Similarly, pendant amino groups were incorporated into PC chains by polymerization
Establishing Advanced Cell Cultivation Systems 83 of the cyclic carbonate monomer, (2-oxo-[1,3]-dioxan-5-yl)carbamic acid benzyl ester, followed by removal of the protective benzyloxycarbonyl groups [33,34]. A bioerodible poly(hydroxyalkylene carbonate), which is an example of water soluble polycarbon-ate, was first reported by Acemoglu et al. This polycarbonate is sufficiently stable to be derivatized by means of its pendant hydroxyl groups [37]. Another example of water soluble polycarbonate is a lipase-catalyzed ring-opening reaction of 5-methyl-5-ben-zyloxycarbonyl-1,3-dioxane-2-one. In this case, after removal of benzyloxy group, it furnishes polycarbonate possessing pendent carboxyl groups on the main chain [38].
Vandenberg et al. have reported the Bis(hydroxymethyl)polcarbonates (HPC) as crys-talline high melting polymer. This hydroxyl pendent polycarbonate was synthesized by the polymerization of the cyclic carbonate of 2,2-dimethyl-5,5-bis(hydroxymethyl)-1,3-dioxane [39]. In 2003, Ray et al. reported a poly(carbonate-ester)s of glycerol and L-lactic acid that possess hydrolyzable backbone and functionalized with free hydroxyl groups. These free hydroxyl groups were derivatized with 4-isobutylmethylphenylace-tic acid, which is a common nonsteroidal anti-inflammatory drug [40]. By designing a synthetic cyclic carbonate monomer 5-methyl-5-(succinimide-N-oxycarbonyl)-1,3-dioxane-2-one (MSTC), Zhou et al. were able to synthesize the copolymer of MSTC with caprolactone (CL). Further, amido-amine pendent groups were obtained by ami-onolysis of this copolymer with ethylenediamine [41]. A morpholine-functionalized polycarbonate has been synthesized by copolymerization of 2-(morpholine-4-yl)ethyl substituted six membered carbonate with polyethylene oxide linker [42].
However, these chemical methodologies involve bulk modification and are not suit-able for functionalizing the polymer scaffolds that are predesigned using micro engi-neering approaches. This drawback can be overcome by the post-modification chemical strategies that functionalize the surface only, without altering the bulk and majority of topography. From the chemical point of view, polycarbonate has the advantage of bear-ing carbonate functional group that can be used as intrinsic chemical entry which pos-sesses the possibility to react with other nucleophilic groups. Of course, other grafting methods that are generally used for the chemical functionalization of the surface of polymer are equally valid. Following is a summation of the possible post-modification chemical methods for the functionalization of polycarbonate surface.
Maquieira et al. functionalized the polycarbonate surface through nitration of the phenyl ring at 60°C, followed by reduction with NaBH4, for producing aminated poly-mer surface. Using this method a chemical derivatization of compact disc with oligonu-cleotides [43] and hapten [44] was achieved for developing microimmunoassays. This was further modified to thiol and aldehyde. Another method to functionalize polycar-bonate was originally developed by Li et al., where 3-aminopropyltriethoxysilane was reacted with carbonate groups of PC chain by forming urethane linkages [45]. Recently, Jankowski et al. have utilized this approach in order to tune the hydrophobicity of the polycarbonate in microfluidic channels [46]. In order to synthesize amino-function-alized polycarbonate surface a layer-by-layer deposition of polyelectrolyte multilayer (PEM) method was used, where poly(allyamine hydrochloride) (PAH)13 or branched polyethyleneimine14 was deposited on PC. Using this method the functionalization of the polycarbonate with proteins has been realized [47]. Hydrophilic polycarbon-ate channels for generation of oil-in-wpolycarbon-ater and wpolycarbon-ater-in-oil-in-wpolycarbon-ater emulsion have been reported by Jankowski et al. for treatment of the PC surface with a solution of
84 Handbook of Polymers for Pharmaceutical Technologies
tin(II) chloride [48]. In another instance, poly-(L-lysine) was coupled with polycar-bonate membrane using urethane coupling and the thus produced aminated surface is used to immobilize glucose oxidase [49]. Recently, Yang et al. developed a method to functionalize polycarbonate surfaces by grafting PEG and zwitterionic polymers with a multicomb structure. Such a modified surface has shown a very low platelet adsorp-tion, indicating that this material can become a preferred candidate for blood contact materials [50]. Another example of hemocompatible polycarbonate urethane has been reported by Feng et al., were they grafted heparin on the polycarbonate urethane sur-face by using diamino-poly(ethylene glycol) as linker [51]. Interestingly, multifunc-tional praseodymium-coordinated polycarbonate films have been formed by direct coordination of Pr3+ to the oxygen of the polycarbonate [52]. This kind of modification can help in tuning the physical and chemical properties of the material.
Recently, our group has developed a structured polymer scaffold which is made of perforated polycarbonate thin film. The scaffold provides a three-dimensional micro-environment to cell culture and is continuously perfused with the nutrient medium [31]. Therefore, polycarbonate chemistry is of particular interest for our applications with a special focus on the mild reaction conditions. Moreover, most of the methods described above are for post modification of the surface of polycarbonate using aggres-sive reaction conditions. In this chapter we describe a mild and efficient method for the functionalization of polycarbonate using terminal diamines.