- Email: [email protected]
The investigation of cell adhesion on nano-patterned biointerfaces of block copolymer films by reactive microcontact printing approach
Abstracts / Journal of Controlled Release 152 (2011) e192–e269
Fig. 2 shows the polymer mass loss over time from chemically crosslinked PEG-PLA hydrogels, incubated in PBS at 37 °C. No polymer mass loss is observed during the first 10 days. In the following 20 days the networks loose approximately 30% of polymer mass, most likely as a result of hydrolysis of ester groups in the PLA domains. The degradation of the networks is accompanied by an increase in swelling (Fig. 2). The ongoing swelling is probably due to PLA degradation, upon which chemical and physical crosslinks are lost, resulting in a less densely crosslinked network. The results indicate that the chemically crosslinked PEG-PLA hydrogels possess excellent in vitro stability.
Fig. 2. Polymer mass (left) and degree of swelling (right) versus time for chemically crosslinked PEG-PLA hydrogels in PBS at 37 °C.
Conclusion Chemically crosslinked PEG-PLA hydrogels were prepared by a Michael addition reaction between PEG-(SH)8 and PEG-(NHCO)(PLLA11)8-ACR macromonomers in PBS. Rheological measurements showed that the hydrogels form in situ and possess robust mechanical properties. Degradation studies indicated that the hydrogels exhibit a good in vitro stability with 30% mass loss after 30 days. These PEG-PLA hydrogels hold promise for use as injectable systems for biomedical applications such as the controlled delivery of active agents. References [1] N.A. Peppas, A.R. Khare, Preparation, structure and diffusional behavior of hydrogels in controlled-release, Adv. Drug Deliv. Rev. 11 (1993) 1–35. [2] E. Ruel-Gariepy, J.C. Leroux, In situ-forming hydrogels - review of temperature-sensitive systems, Eur. J. Pharm. Biopharm. 58 (2004) 409–426. [3] C. Hiemstra, L.J. van der Aa, Z. Zhong, P.J. Dijkstra, J. Feijen, Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition, Biomacromolecules 8 (2007) 1548–1556. [4] A. Goessl, N. Tirelli, J.A. Hubbell, A hydrogel system for stimulus-responsive, oxygensensitive in situ gelation, J. Biomater. Sci. - Polym. Ed. 15 (2004) 895–904. [5] S.J. Buwalda, P.J. Dijkstra, L. Calucci, C. Forte, J. Feijen, Influence of amide versus ester linkages on the properties of eight-armed PEG-PLA star block copolymer hydrogels, Biomacromolecules 11 (2010) 224–232.
doi:10.1016/j.jconrel.2011.09.007
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spatially directed pronounced elongated shapes on the nanopatterned platforms in a controlled way. Keywords: Polymer, Biointerfaces, Pattering, Cell culture Introduction Patterned biofunctional interfacial architectures are currently at the focal point of sensor and cell biology research [1]. For a wide range of possible applications in the life sciences, in particular, the investigation of controlled cell-surface interactions, (bio)chemical patterning on multiple length scales down to the sub-100 nm level is required. The relevant distances and length scales of ligand clustering in multivalent recognition and those in protein clustering in focal adhesion of certain types of cells on the one hand [2], and the size of biological entities such as bacteria or cells span an enormous range [3]. In this communication we introduce a new reactive microcontact printing (μCP) strategy to fabricate well-defined functional micro- and nanostructured biointerfaces. Central to our new approach is the exploitation of selective surface chemistry on a reactive block copolymer film by using a volatile and highly diffusive reactant (trifluoroacetic acid) delivered via an elastomeric stamp (Fig. 1) [4]. Experimental methods Materials. Polystyrene-block-poly(tert-butyl acrylate) (PS690-bPtBA1210) diblock copolymers were purchased from Polymer Source Company (Dorval, Canada). Amino end-labeled PEG (PEG5000-NH2) was purchased from Nektar UK Company, fluoresceinamine and fibronectin were purchased from Molecular Probes, Inc. (Shanghai). Thin films were prepared as reported previously by spin-coating filtered polymer solutions in toluene [5] and reactive microcontact printing on the films according to a procedure published earlier [6]. Characterization. Fluorescence microscopy images were recorded at room temperature on a Zeiss LSM 510 confocal laser scanning microscope. Optical microscopy was performed using an Olympus BX 60 (standard setup). The contact-mode AFM measurements were carried out with a NanoScope III multimode AFM [7]. Cell culture. Liver cells Hepg2 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal calf serum (FCS), 5% horse serum and 2 mM L-glutamine at 5% CO2. Results and discussion The PS690-b-PtBA1210 block copolymer film platform utilized in this study comprises reactive tert-butyl ester moieties at the film surface (skin layer thickness ca. 8 nm) and microphase-separated glassy polystyrene domains in the film interior that provide excellent thermal and processing stability. The volatile and highly diffusive reactant TFA is delivered locally in the (sub-)microcontacts between an elastomeric stamp and a reactive block copolymer film (Fig. 1).
The investigation of cell adhesion on nano-patterned biointerfaces of block copolymer films by reactive microcontact printing approach Chuan-Liang Feng1, Di Zhang1, Holger Schönherr2 State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China 2 Siegen University, Physical Chemistry, Adolf-Reichwein-St. 2, AR-F0103, 57076 Siegen, Germany E-mail address: [email protected] (C.-L. Feng). 1
Abstract summary A new reactive microcontact printing strategy to fabricate welldefined functional micro- and nanostructured biointerfaces is introduced. Hepg2 liver cells were observed to spread in highly
Fig. 1. Schematic of new reactive microcontact printing strategy and functionalization on block copolymer films.
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Abstracts / Journal of Controlled Release 152 (2011) e192–e269
Micropatterns fabricated according to the reactive μCP procedure shown in Fig. 1 were proved by fluorescence microscopy (Fig. 2a). The thickness of passivating PEG5000-NH2 layers grafted via directtransfer μCP was assessed by ellipsometry (Fig. 2b). The fluorescence microscopy image shows a homogeneous coverage of the covalently coupled dye and a very sharply defined pattern. Interestingly, the thickness of PEG5000 grafted by direct transfer μCP is 20-25% larger than that of PEG5000-NH2 coupled from solution (Fig. 2b). This result indicates that the high local concentration (and pressure) under the solventless conditions of μCP improve the coupling efficiency.
were observed to spread and grow in highly spatially directed, pronounced elongated shapes on nanopatterned platforms exposing 400 nm wide lines of fibronectin and 700 nm wide lines of PEG5000, thus indicating that tailored surfaces that allow one to acquire important insight into cell behavior at surfaces are now readily accessible. Acknowledgements This work has been financially supported by the MESA + Institute for Nanotechnology of the University of Twente, the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO). Financial support came from Special Researcher of Shanghai Jiaotong University, Chinese Program for New Century Excellent Talents in University, Shanghai Natural Science Foundation (10ZR1415600), and Shanghai Pujiang Program. References
Fig. 2. (a) Fluorescence microscopy image and (b) PEG grafting thickness using different way.
The sharp line-edge definition observed in the friction-mode AFM images as well as the pronounced friction force contrast between the hydrophilic and hydrophobic areas shown in Fig. 3a and b suggest the occurrence of a laterally homogeneous and well-defined deprotection reaction. On the nanopatterned substrates exposing 400 nm wide lines of fibronectin and 700 nm wide lines of PEG5000, the cells were observed to spread in highly unusual very elongated shapes after 60 h culture. While almost no cells showed an elongated morphology on unpatterned fibronectin with culturing, the cells were found to be elongated on the nanopatterns and the cells were proliferating along the nanopattern direction. In Fig. 3c an alignment for the elongated cells on the nanopatterns were observed compared with nearisotropic orientation on the unpatterned films. These observations indicate that the cells are indeed forced to stretch along the submicrometer-sized pattern of cell adhesive (fibronectin) and start to grow in the confined environment and nonadhesive (PEG5000) stripes and can differentiate the in-plane directionality of the anisotropic surface chemistry. As a consequence, the cells spread out in the direction of the highest fibronectin coverage.
Fig. 3. (a,b) AFM morphology and friction image acquired on a PS690-b-PtBA1210 film after local hydrolysis by reactive μCP (c) Optical microscopy image of Hepg2 cells on a pattern of fibronectin (400 nm line width) and PEG5000-NH2 (700 nm line width) on a block copolymer film prepared.
Conclusion In summary, we have shown that highly localized, selective deprotection chemistry and efficient grafting reactions can be carried out in the microcontact region between an elastomeric stamp and a reactive PS690-b-PtBA1210 diblock copolymer film. Hepg2 liver cells
[1] C.L. Feng, A. Embrechts, I. Bredebusch, J. Schnekenburger, W. Domschke, G.J. Vancso, H. Schönherr, Reactive microcontact printing on block copolymer films: exploiting chemistry in microcontacts for sub-micrometer patterning of biomolecules, Adv. Mater. 19 (2007) 286–290. [2] R. Willmann, C. Fuhrer, Neuromuscular synaptogenesis: clustering of acetylcholine receptors revisited, Cell. Mol. Life Sci. 59 (2002) 1296–1316. [3] A. Curtis, C. Wilkinson, Topolgraphical control of cells, Biomaterials 18 (1997) 1573–1583. [4] H. Gaussier, H. Morency, M.C. Lavoie, M. Subirade, Replacement of trifluoroacetic acid with HCl in the hydrophobic purification steps of pediocin PA-1: a structural effect, Appl. Environ. Microbiol. 68 (2002) 4803–4808. [5] C.L. Feng, G.J. Vancso, H. Schönherr, Interfacial reactions in confinement: kinetics and temperature dependence of the surface hydrolysis of polystyrene-b-poly(tert-butyl acrylate), Langmuir 21 (2005) 2356–2363. [6] H. Hillborg, N. Tomczak, A. Oláh, H. Schönherr, G.J. Vancso, Nanoscale hydrophobic recovery: a chemical force microscopy study of uv/ozone-treated cross-linked poly (dimethylsiloxane), Langmuir 20 (2004) 785–794. [7] E. Tocha, H. Schönherr, G.J. Vancso, Quantitative nanotribology by AFM: a novel universal calibration platform, Langmuir 22 (2006) 2340.
doi:10.1016/j.jconrel.2011.09.008
Nitric oxide release from polycarbonate-urethane films containing copper(ii) complexes Yakai Feng1,2,3, Haiyang Zhao1, Jian Lu2, Jintang Guo1,2,3 1 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China 2 Tianjin University-GKSS Research Centre, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, Tianjin 300072, China 3 Kantstr. 55, 14513 Teltow, Germany E-mail addresses: [email protected] (J. Guo). Abstract summary NO is a well known potent antiplatelet agent to prevent the adhesion of platelets on artificial blood vessel walls. Polycarbonateurethane (PCU) films with NO releasing function were prepared by blending with Cu(II)-complex. The PCU film could release NO continually over 2 d, which will provide a promising approach to enable endogenous NO generation on the surface of medical devices. The generation of biologically active levels of NO at the blood/ polymer interface can reduce the risk of implant related thrombosis. Keywords: Polycarbonate-urethanes, Cu(II)-complex, Hemocompatibility, NO releasing, Blend Introduction Polycarbonate-urethane (PCU), a new type of biostable polyurethane with excellent mechanical properties, has been developed in recent years [1]. PCU has been used in various biomedical applications such as catheters, vascular grafts, blood bags, and artificial
Fig. 2 shows the polymer mass loss over time from chemically crosslinked PEG-PLA hydrogels, incubated in PBS at 37 °C. No polymer mass loss is observed during the first 10 days. In the following 20 days the networks loose approximately 30% of polymer mass, most likely as a result of hydrolysis of ester groups in the PLA domains. The degradation of the networks is accompanied by an increase in swelling (Fig. 2). The ongoing swelling is probably due to PLA degradation, upon which chemical and physical crosslinks are lost, resulting in a less densely crosslinked network. The results indicate that the chemically crosslinked PEG-PLA hydrogels possess excellent in vitro stability.
Fig. 2. Polymer mass (left) and degree of swelling (right) versus time for chemically crosslinked PEG-PLA hydrogels in PBS at 37 °C.
Conclusion Chemically crosslinked PEG-PLA hydrogels were prepared by a Michael addition reaction between PEG-(SH)8 and PEG-(NHCO)(PLLA11)8-ACR macromonomers in PBS. Rheological measurements showed that the hydrogels form in situ and possess robust mechanical properties. Degradation studies indicated that the hydrogels exhibit a good in vitro stability with 30% mass loss after 30 days. These PEG-PLA hydrogels hold promise for use as injectable systems for biomedical applications such as the controlled delivery of active agents. References [1] N.A. Peppas, A.R. Khare, Preparation, structure and diffusional behavior of hydrogels in controlled-release, Adv. Drug Deliv. Rev. 11 (1993) 1–35. [2] E. Ruel-Gariepy, J.C. Leroux, In situ-forming hydrogels - review of temperature-sensitive systems, Eur. J. Pharm. Biopharm. 58 (2004) 409–426. [3] C. Hiemstra, L.J. van der Aa, Z. Zhong, P.J. Dijkstra, J. Feijen, Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition, Biomacromolecules 8 (2007) 1548–1556. [4] A. Goessl, N. Tirelli, J.A. Hubbell, A hydrogel system for stimulus-responsive, oxygensensitive in situ gelation, J. Biomater. Sci. - Polym. Ed. 15 (2004) 895–904. [5] S.J. Buwalda, P.J. Dijkstra, L. Calucci, C. Forte, J. Feijen, Influence of amide versus ester linkages on the properties of eight-armed PEG-PLA star block copolymer hydrogels, Biomacromolecules 11 (2010) 224–232.
doi:10.1016/j.jconrel.2011.09.007
e201
spatially directed pronounced elongated shapes on the nanopatterned platforms in a controlled way. Keywords: Polymer, Biointerfaces, Pattering, Cell culture Introduction Patterned biofunctional interfacial architectures are currently at the focal point of sensor and cell biology research [1]. For a wide range of possible applications in the life sciences, in particular, the investigation of controlled cell-surface interactions, (bio)chemical patterning on multiple length scales down to the sub-100 nm level is required. The relevant distances and length scales of ligand clustering in multivalent recognition and those in protein clustering in focal adhesion of certain types of cells on the one hand [2], and the size of biological entities such as bacteria or cells span an enormous range [3]. In this communication we introduce a new reactive microcontact printing (μCP) strategy to fabricate well-defined functional micro- and nanostructured biointerfaces. Central to our new approach is the exploitation of selective surface chemistry on a reactive block copolymer film by using a volatile and highly diffusive reactant (trifluoroacetic acid) delivered via an elastomeric stamp (Fig. 1) [4]. Experimental methods Materials. Polystyrene-block-poly(tert-butyl acrylate) (PS690-bPtBA1210) diblock copolymers were purchased from Polymer Source Company (Dorval, Canada). Amino end-labeled PEG (PEG5000-NH2) was purchased from Nektar UK Company, fluoresceinamine and fibronectin were purchased from Molecular Probes, Inc. (Shanghai). Thin films were prepared as reported previously by spin-coating filtered polymer solutions in toluene [5] and reactive microcontact printing on the films according to a procedure published earlier [6]. Characterization. Fluorescence microscopy images were recorded at room temperature on a Zeiss LSM 510 confocal laser scanning microscope. Optical microscopy was performed using an Olympus BX 60 (standard setup). The contact-mode AFM measurements were carried out with a NanoScope III multimode AFM [7]. Cell culture. Liver cells Hepg2 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal calf serum (FCS), 5% horse serum and 2 mM L-glutamine at 5% CO2. Results and discussion The PS690-b-PtBA1210 block copolymer film platform utilized in this study comprises reactive tert-butyl ester moieties at the film surface (skin layer thickness ca. 8 nm) and microphase-separated glassy polystyrene domains in the film interior that provide excellent thermal and processing stability. The volatile and highly diffusive reactant TFA is delivered locally in the (sub-)microcontacts between an elastomeric stamp and a reactive block copolymer film (Fig. 1).
The investigation of cell adhesion on nano-patterned biointerfaces of block copolymer films by reactive microcontact printing approach Chuan-Liang Feng1, Di Zhang1, Holger Schönherr2 State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China 2 Siegen University, Physical Chemistry, Adolf-Reichwein-St. 2, AR-F0103, 57076 Siegen, Germany E-mail address: [email protected] (C.-L. Feng). 1
Abstract summary A new reactive microcontact printing strategy to fabricate welldefined functional micro- and nanostructured biointerfaces is introduced. Hepg2 liver cells were observed to spread in highly
Fig. 1. Schematic of new reactive microcontact printing strategy and functionalization on block copolymer films.
e202
Abstracts / Journal of Controlled Release 152 (2011) e192–e269
Micropatterns fabricated according to the reactive μCP procedure shown in Fig. 1 were proved by fluorescence microscopy (Fig. 2a). The thickness of passivating PEG5000-NH2 layers grafted via directtransfer μCP was assessed by ellipsometry (Fig. 2b). The fluorescence microscopy image shows a homogeneous coverage of the covalently coupled dye and a very sharply defined pattern. Interestingly, the thickness of PEG5000 grafted by direct transfer μCP is 20-25% larger than that of PEG5000-NH2 coupled from solution (Fig. 2b). This result indicates that the high local concentration (and pressure) under the solventless conditions of μCP improve the coupling efficiency.
were observed to spread and grow in highly spatially directed, pronounced elongated shapes on nanopatterned platforms exposing 400 nm wide lines of fibronectin and 700 nm wide lines of PEG5000, thus indicating that tailored surfaces that allow one to acquire important insight into cell behavior at surfaces are now readily accessible. Acknowledgements This work has been financially supported by the MESA + Institute for Nanotechnology of the University of Twente, the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO). Financial support came from Special Researcher of Shanghai Jiaotong University, Chinese Program for New Century Excellent Talents in University, Shanghai Natural Science Foundation (10ZR1415600), and Shanghai Pujiang Program. References
Fig. 2. (a) Fluorescence microscopy image and (b) PEG grafting thickness using different way.
The sharp line-edge definition observed in the friction-mode AFM images as well as the pronounced friction force contrast between the hydrophilic and hydrophobic areas shown in Fig. 3a and b suggest the occurrence of a laterally homogeneous and well-defined deprotection reaction. On the nanopatterned substrates exposing 400 nm wide lines of fibronectin and 700 nm wide lines of PEG5000, the cells were observed to spread in highly unusual very elongated shapes after 60 h culture. While almost no cells showed an elongated morphology on unpatterned fibronectin with culturing, the cells were found to be elongated on the nanopatterns and the cells were proliferating along the nanopattern direction. In Fig. 3c an alignment for the elongated cells on the nanopatterns were observed compared with nearisotropic orientation on the unpatterned films. These observations indicate that the cells are indeed forced to stretch along the submicrometer-sized pattern of cell adhesive (fibronectin) and start to grow in the confined environment and nonadhesive (PEG5000) stripes and can differentiate the in-plane directionality of the anisotropic surface chemistry. As a consequence, the cells spread out in the direction of the highest fibronectin coverage.
Fig. 3. (a,b) AFM morphology and friction image acquired on a PS690-b-PtBA1210 film after local hydrolysis by reactive μCP (c) Optical microscopy image of Hepg2 cells on a pattern of fibronectin (400 nm line width) and PEG5000-NH2 (700 nm line width) on a block copolymer film prepared.
Conclusion In summary, we have shown that highly localized, selective deprotection chemistry and efficient grafting reactions can be carried out in the microcontact region between an elastomeric stamp and a reactive PS690-b-PtBA1210 diblock copolymer film. Hepg2 liver cells
[1] C.L. Feng, A. Embrechts, I. Bredebusch, J. Schnekenburger, W. Domschke, G.J. Vancso, H. Schönherr, Reactive microcontact printing on block copolymer films: exploiting chemistry in microcontacts for sub-micrometer patterning of biomolecules, Adv. Mater. 19 (2007) 286–290. [2] R. Willmann, C. Fuhrer, Neuromuscular synaptogenesis: clustering of acetylcholine receptors revisited, Cell. Mol. Life Sci. 59 (2002) 1296–1316. [3] A. Curtis, C. Wilkinson, Topolgraphical control of cells, Biomaterials 18 (1997) 1573–1583. [4] H. Gaussier, H. Morency, M.C. Lavoie, M. Subirade, Replacement of trifluoroacetic acid with HCl in the hydrophobic purification steps of pediocin PA-1: a structural effect, Appl. Environ. Microbiol. 68 (2002) 4803–4808. [5] C.L. Feng, G.J. Vancso, H. Schönherr, Interfacial reactions in confinement: kinetics and temperature dependence of the surface hydrolysis of polystyrene-b-poly(tert-butyl acrylate), Langmuir 21 (2005) 2356–2363. [6] H. Hillborg, N. Tomczak, A. Oláh, H. Schönherr, G.J. Vancso, Nanoscale hydrophobic recovery: a chemical force microscopy study of uv/ozone-treated cross-linked poly (dimethylsiloxane), Langmuir 20 (2004) 785–794. [7] E. Tocha, H. Schönherr, G.J. Vancso, Quantitative nanotribology by AFM: a novel universal calibration platform, Langmuir 22 (2006) 2340.
doi:10.1016/j.jconrel.2011.09.008
Nitric oxide release from polycarbonate-urethane films containing copper(ii) complexes Yakai Feng1,2,3, Haiyang Zhao1, Jian Lu2, Jintang Guo1,2,3 1 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China 2 Tianjin University-GKSS Research Centre, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, Tianjin 300072, China 3 Kantstr. 55, 14513 Teltow, Germany E-mail addresses: [email protected] (J. Guo). Abstract summary NO is a well known potent antiplatelet agent to prevent the adhesion of platelets on artificial blood vessel walls. Polycarbonateurethane (PCU) films with NO releasing function were prepared by blending with Cu(II)-complex. The PCU film could release NO continually over 2 d, which will provide a promising approach to enable endogenous NO generation on the surface of medical devices. The generation of biologically active levels of NO at the blood/ polymer interface can reduce the risk of implant related thrombosis. Keywords: Polycarbonate-urethanes, Cu(II)-complex, Hemocompatibility, NO releasing, Blend Introduction Polycarbonate-urethane (PCU), a new type of biostable polyurethane with excellent mechanical properties, has been developed in recent years [1]. PCU has been used in various biomedical applications such as catheters, vascular grafts, blood bags, and artificial