full papers Biocatalytic Coatings
Biocatalytic Polymer Coatings: On-Demand Drug Synthesis and Localized Therapeutic Effect under Dynamic Cell Culture Conditions Betina Fejerskov, Najah B. S. Jensen, Boon M. Teo, Brigitte Städler* and Alexander N. Zelikin*
Biocatalytic surface coatings are prepared herein for localized synthesis of drugs and their on-demand, site-specific delivery to adhering cells. This novel approach is based on the incorporation of an enzyme into multilayered polymer coatings to accomplish enzyme-prodrug therapy (EPT). The build-up of enzyme-containing multilayered coatings is characterized and correlations are drawn between the multilayer film assembly conditions and the enzymatic activity of the resulting coatings. Therapeutic effect elicited by the substrate mediated EPT (SMEPT) strategy is investigated using a prodrug for an anticancer agent, SN-38. The performance of biocatalytic coatings under flow conditions is investigated and it is demonstrated that EPT allows synthesizing the drugs on-demand, at the time desired and in a controllable amount to suit particular applications. Finally, using cells cultured in sequentially connected flow chambers, it is demonstrated that SMEPT affords a site-specific drug delivery, that is, exerts a higher therapeutic effect in cells adhering directly to the biocatalytic coatings than in the cells cultured “downstream”. Taken together, these data illustrate biomedical opportunities made possible by engineering tools of EPT into multilayered polymer coatings and present a novel, highly versatile tool for surface mediated drug delivery.
1. Introduction Delivering drugs specifically to the site of their action comprises a major step towards an ideal therapeutic intervention. This opportunity is realized almost perfectly using a suit of techniques engineered into implantable biomaterials.[1,2] B. Fejerskov, N. B. S. Jensen, A. N. Zelikin Department of Chemistry Aarhus University Aarhus, 8000, Denmark E-mail: [email protected] B. M. Teo, B. Städler Interdisciplinary Nanoscience Centre (iNANO) Aarhus University Aarhus, 8000, Denmark E-mail: [email protected] DOI: 10.1002/smll.201303101
1314
wileyonlinelibrary.com
Biodegradable polymer matrices,[3] embolizing beads,[4] hydrogels paved within arteries[2] and other approaches have progressed to various stages of (pre)clinical development and many examples are routinely used in patient treatment. Recently, surface mediated drug delivery (SMDD) has evolved from its advent into a mature paradigm[5,6] with particular successes documented in delivering nucleic acid cargo for localized gene therapy.[7–9] Of the various techniques in SMDD, sequential deposition of polymers (layer-by-layer technique, LbL) received the most widespread attention.[10] This approach presents virtually no limit on the nature of the underlying substrate, thickness of the deposited thin film, type of constituting building blocks (polymers, nanoparticles, liposomes, micelles, etc.), yielding a broad variety of (bio) chemical and physical properties of the film, all of which have proven useful in implementing LbL films in SMDD.[10,11] However, while successes of LbL films in delivery of gene[5,12] and protein[13–15] cargo are sound, achievements in SMDD
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, 10, No. 7, 1314–1324
Biocatalytic Polymer Coatings: On-Demand Drug Synthesis and Localized Therapeutic Effect
for small molecule drugs significantly lag behind and at present are minor. By design, multilayered polymer thin films are ill suited to provide controlled retention and release of small drugs. Existing solitary successful approaches,[16–21] powerful in their own right, fail to be generalized to accommodate therapeutics with diverse structure, hydrophobicity, charge, and solubility. Arguably the most advanced design of LbL coatings for delivery of small drugs exploits drug deposits in the form of cyclodextrins as drug depots.[22] However, this approach relies on a highly variable affinity of drugs to cyclodextrins and is likely to suit only individual drugs and applications. Further significant shortcoming of multilayered polymer thin films and an overall majority of techniques in SMDD lies in limited opportunities for drug release ondemand,[20,23] at the time it is needed, with a precise dose to achieve the desired therapeutic effect. In this report, we specifically address the above mentioned limitations and present nano-engineered coatings based on multilayered polymer thin films for on-demand, localized delivery of therapeutics and/or imaging agents and localized delivery to the adhering cells. Key to our approach is the design of biocatalytic coatings, i.e., multilayered thin films containing active enzymes immobilized within the polymer layers. We hypothesized that when immobilized into the multilayered thin film, enzymes can be used for a localized, in situ synthesis of drugs and/or imaging reagents using externally administered prodrug(s). This approach, termed “substrate mediated enzyme prodrug therapy” (SMEPT) and initially introduced by us using hydrogel biomaterials,[24] is developed herein to make use of the high permeability of LbL coatings to small solutes and the latter is essential to ensure fast exchange of pro(drug)s between the coating and the external phase. Through variation of the constituting polymers, deposition conditions, and/or post-assembly chemical crosslinking, multilayered polyelectrolyte coatings can be engineered to be stiff or soft, dense or gel-like.[25,26] For an overall majority of designs and specifically for the gel-like examples, multilayered coatings present a weak barrier for diffusion of small molecule solutes through the film. This phenomenon facilitates the use of LbL coatings with immobilized enzymes in flow-through biocatalytic membranes,[27] encapsulated micro-reactors with external activation of the enzymatic conversion,[28–30] etc. We note that proteins have been previously incorporated into the LbL coating as therapeutic cargo[13] or for biosensing.[27,31] Recently, enzymes were incorporated into multilayered films for external activation of enzymatic conversion using mechanical deformation of the film.[32,33] However, to the best of our knowledge, there are no prior examples of use of immobilized enzymes for in situ conversion of prodrugs and localized delivery of the synthesized product to the adhering cells. The choice of a suitable enzyme for this undertaking was guided by extensive prior knowledge of existing enzyme prodrug therapies, specifically antibody- and gene directed EPTs.[34–38]β-Glucuronidase (β-Glu) stands out as an enzyme with extensive prior characterization[39] and availability of the substrates (prodrugs) to yield therapeutics with a wide spectrum of action, many of which are available commercially. This includes drugs to treat cancer,[24,40] small 2014, 10, No. 7, 1314–1324
inflammation,[41,42] hypertension, viral diseases,[41] as well as agents for imaging[43,44] – all due to the fact that glucuronides are natural metabolic products of commercial drugs and therefore undergo stringent analysis for regulatory approval and to define the scope and utility of therapies. The above cited literature reports describe examples of glucuronide substrates to yield therapeutic molecules to treat a magnitude of diseases as well as imaging reagents and this illustrates that the same enzyme, β-Glu, can serve to produce a panel of diverse products, independent of their structure, function, solubility and charge. Once engineered into implantable devices for SMEPT, this provides for a unique opportunity that the same implant will be capable of synthesizing diverse drugs with diversity of function being limited only by the availability of prodrug molecules. Such opportunities have no precedent in the field of implantable biomaterials. This work represents the first major step towards engineering SMEPT into therapeutic implants, specifically into surface coatings comprised of multilayered polymer thin films. For the guidance in design of polyelectrolyte films, we rely on extensive literature on the subject and vast opportunities as far as candidate materials and properties of resulting films.[10,11,45] Depending on the envisioned application, synthetic and natural polymers are similarly attractive as building blocks for LbL films; biodegradable and ultimately stable polymer coatings find use in biomedicine, and all of the above could equally benefit from enhanced opportunities in drug delivery, such as EPT. In this initial study, we build on success of our recent report on the use of liposome-containing multilayered polyelectrolyte films for drug delivery to adhering hepatocytes and myoblasts.[46] As a positively charged polymer, we used a synthetic polypeptide with extensive use in the field of LbL coatings, poly-L-lysine (PLL). As negatively charged counterparts, we used polymers with different origin, structure, and biodegradation behavior: alginate (Alg, polysaccharide), poly-L-glutamic acid (PGA, synthetic polypeptide), poly(methacrylic acid) (PMA, synthetic polyanion), and a cholesterol functionalized PMA, PMAc. We have shown that these combinations of polyelectrolytes can be successfully used in the assembly of multilayered coatings, support adhesion of mammalian cells, and sustained drug delivery to adhering cells, specifically mediated by immobilized liposomal drug deposits.[46] Herein, we investigate films assembled from these polymers as reservoirs for β-Glu and analyze effectiveness of the films as EPT-functionalized coatings. A significant phenomenological challenge to the performance of SMEPT is a requirement for the prodrug to gain access to the enzyme in the structure of the coating and afford concentration of the product in physiologically relevant range. Blood, lymph, and/or interstitial fluids create dynamic flow around the implants thus, contributing to the performance of biocatalytic systems via mixing and replenishing the prodrug pool and at the same time working against the feasibility of EPT through decreased co-localization time of the enzyme and its substrate. The above reasons are speculative and as such, drug delivery under dynamic, flow conditions is an emerging field with research findings reported only in the nearest past. Studies appearing to date largely relate to the aspects of
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.small-journal.com
1315
full papers
B. Fejerskov et al.
antibody-assisted targeting,[47–50] or non-specific association of nanoparticles[51,52] or polyplexes[53] with endothelial cells under flow conditions. In terms of therapeutic response in the presence of shear stress, enhanced transfection efficiency,[54,55] or increased cytotoxicity of 50 nm mesoporous nanoparticles[56] have been demonstrated. Interesting approaches on the drug release from aggregated nanoparticles[57] and lenticular vesicles[58] under high shear stress as a concept to treat strokes and embolisms have recently been reported. Recently, we provided a systematic investigation of drug delivery using nanoscopic drug carriers to myoblasts and hepatocytes under shear stress[59,60] and revealed fundamental differences in cell/ drug carrier interaction depending on the presence of shear stress. In particular, positively charged liposomes showed enhanced association with cells under flow conditions and this translated into improved therapeutic response, specifically in terms of efficacy of cytotoxic drugs and efficiency of nucleic acid delivery.[59] Further, PEGylated carrier exhibited cell association characteristics significantly differed depending on the used cell type, exposure times and shear stress.[60] Particularly, in the presence of shear stress, we found that the association of non-PEGylated carriers with hepatocytes compared to the PEGylated ones was significantly higher after 30 min, while for the myoblasts, the difference only became obvious after 4 h. These data illustrate that flow of media surrounding cells is an important parameter to consider for the in vitro characterization of drug carriers. In this work, we investigate the performance of enzymeequipped LbL thin films under flow conditions and use this tool to illustrate unique opportunities associated with the EPT approach in the context of SMDD (Scheme 1). Specifically, in this work we i) present the assembly of biocatalytic surface coatings based on multilayered polymer thin films, ii) demonstrate that the EPT strategy engineered into surface coatings affords physiologically relevant concentrations of drugs, and does so under flow conditions, iii) illustrate that biocatalytic surfaces provide opportunity for on demand, “on-off” synthesis of the drug, and iv) reveal that this mode of drug delivery is site specific. We believe that these accomplishments have no prior precedent and contribute significantly to the development of drug eluting coatings for engineering of therapeutic implants.
Scheme 1. Enzyme-functionalized, biocatalytic surface coatings based on multilayered polymer thin films are investigated herein as tools for localized, on-demand drug delivery under flow conditions mimicking dynamic conditions encountered in vivo.
1316 www.small-journal.com
Figure 1. Frequency changes Δf of QCM-D crystals upon sequential deposition of PLL/PGA multilayers and the incorporation of β-Glu into the polymer films. Polymer deposition was conducted from 1 g/L solutions of PLL and PGA in HEPES buffer, pH 7.4 supplemented with 0.15 M NaCl. Adsorption of the enzyme was conducted from buffers with pH 5–8.5.
2. Results and Discussion 2.1. Assembly of the biocatalytic films Assembly of polymer multilayers was quantitatively monitored using QCM-D. In the first experiment, we used one of the most well-studied pair of polyelectrolytes, synthetic polypeptides PLL and PGA. This pair has been studied towards fundamental understanding of LbL deposition of polymers[61] and also used in biomedical applications for e.g. controlled cell adhesion and proliferation[62,63] as well as drug delivery.[17,62,64–66] As is typical for “weak” polyelectrolytes, deposition of these polymers can be controlled via the pH of the solution as well as the presence of low molecular weight electrolytes such as NaCl. These aspects of materials engineering are well documented in the state of art and herein, we used widely accepted deposition conditions, 1 g/L concentration of each of the polymers prepared using 10 mM HEPES buffer with pH 7.4 and supplemented with NaCl to 0.15 M. Deposition of each polymer layer was reflected by a decrease in resonance frequency (Δf) of the QCM crystals, Figure 1. Following deposition of 5 polymer layers, the thin films were exposed to a solution of β-Glu (20 μg/mL) in aqueous buffers with pH values from 5 to 8.5. This pH range was monitored herein in an attempt to optimize the adsorption of the protein through a variation of its ionization (isoelectric point pI 4.8). Surprisingly, exposure of the polymer coated crystals to β-Glu afforded a rather small Δf (
Biocatalytic Polymer Coatings: On-Demand Drug Synthesis and Localized Therapeutic Effect under Dynamic Cell Culture Conditions Betina Fejerskov, Najah B. S. Jensen, Boon M. Teo, Brigitte Städler* and Alexander N. Zelikin*
Biocatalytic surface coatings are prepared herein for localized synthesis of drugs and their on-demand, site-specific delivery to adhering cells. This novel approach is based on the incorporation of an enzyme into multilayered polymer coatings to accomplish enzyme-prodrug therapy (EPT). The build-up of enzyme-containing multilayered coatings is characterized and correlations are drawn between the multilayer film assembly conditions and the enzymatic activity of the resulting coatings. Therapeutic effect elicited by the substrate mediated EPT (SMEPT) strategy is investigated using a prodrug for an anticancer agent, SN-38. The performance of biocatalytic coatings under flow conditions is investigated and it is demonstrated that EPT allows synthesizing the drugs on-demand, at the time desired and in a controllable amount to suit particular applications. Finally, using cells cultured in sequentially connected flow chambers, it is demonstrated that SMEPT affords a site-specific drug delivery, that is, exerts a higher therapeutic effect in cells adhering directly to the biocatalytic coatings than in the cells cultured “downstream”. Taken together, these data illustrate biomedical opportunities made possible by engineering tools of EPT into multilayered polymer coatings and present a novel, highly versatile tool for surface mediated drug delivery.
1. Introduction Delivering drugs specifically to the site of their action comprises a major step towards an ideal therapeutic intervention. This opportunity is realized almost perfectly using a suit of techniques engineered into implantable biomaterials.[1,2] B. Fejerskov, N. B. S. Jensen, A. N. Zelikin Department of Chemistry Aarhus University Aarhus, 8000, Denmark E-mail: [email protected] B. M. Teo, B. Städler Interdisciplinary Nanoscience Centre (iNANO) Aarhus University Aarhus, 8000, Denmark E-mail: [email protected] DOI: 10.1002/smll.201303101
1314
wileyonlinelibrary.com
Biodegradable polymer matrices,[3] embolizing beads,[4] hydrogels paved within arteries[2] and other approaches have progressed to various stages of (pre)clinical development and many examples are routinely used in patient treatment. Recently, surface mediated drug delivery (SMDD) has evolved from its advent into a mature paradigm[5,6] with particular successes documented in delivering nucleic acid cargo for localized gene therapy.[7–9] Of the various techniques in SMDD, sequential deposition of polymers (layer-by-layer technique, LbL) received the most widespread attention.[10] This approach presents virtually no limit on the nature of the underlying substrate, thickness of the deposited thin film, type of constituting building blocks (polymers, nanoparticles, liposomes, micelles, etc.), yielding a broad variety of (bio) chemical and physical properties of the film, all of which have proven useful in implementing LbL films in SMDD.[10,11] However, while successes of LbL films in delivery of gene[5,12] and protein[13–15] cargo are sound, achievements in SMDD
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, 10, No. 7, 1314–1324
Biocatalytic Polymer Coatings: On-Demand Drug Synthesis and Localized Therapeutic Effect
for small molecule drugs significantly lag behind and at present are minor. By design, multilayered polymer thin films are ill suited to provide controlled retention and release of small drugs. Existing solitary successful approaches,[16–21] powerful in their own right, fail to be generalized to accommodate therapeutics with diverse structure, hydrophobicity, charge, and solubility. Arguably the most advanced design of LbL coatings for delivery of small drugs exploits drug deposits in the form of cyclodextrins as drug depots.[22] However, this approach relies on a highly variable affinity of drugs to cyclodextrins and is likely to suit only individual drugs and applications. Further significant shortcoming of multilayered polymer thin films and an overall majority of techniques in SMDD lies in limited opportunities for drug release ondemand,[20,23] at the time it is needed, with a precise dose to achieve the desired therapeutic effect. In this report, we specifically address the above mentioned limitations and present nano-engineered coatings based on multilayered polymer thin films for on-demand, localized delivery of therapeutics and/or imaging agents and localized delivery to the adhering cells. Key to our approach is the design of biocatalytic coatings, i.e., multilayered thin films containing active enzymes immobilized within the polymer layers. We hypothesized that when immobilized into the multilayered thin film, enzymes can be used for a localized, in situ synthesis of drugs and/or imaging reagents using externally administered prodrug(s). This approach, termed “substrate mediated enzyme prodrug therapy” (SMEPT) and initially introduced by us using hydrogel biomaterials,[24] is developed herein to make use of the high permeability of LbL coatings to small solutes and the latter is essential to ensure fast exchange of pro(drug)s between the coating and the external phase. Through variation of the constituting polymers, deposition conditions, and/or post-assembly chemical crosslinking, multilayered polyelectrolyte coatings can be engineered to be stiff or soft, dense or gel-like.[25,26] For an overall majority of designs and specifically for the gel-like examples, multilayered coatings present a weak barrier for diffusion of small molecule solutes through the film. This phenomenon facilitates the use of LbL coatings with immobilized enzymes in flow-through biocatalytic membranes,[27] encapsulated micro-reactors with external activation of the enzymatic conversion,[28–30] etc. We note that proteins have been previously incorporated into the LbL coating as therapeutic cargo[13] or for biosensing.[27,31] Recently, enzymes were incorporated into multilayered films for external activation of enzymatic conversion using mechanical deformation of the film.[32,33] However, to the best of our knowledge, there are no prior examples of use of immobilized enzymes for in situ conversion of prodrugs and localized delivery of the synthesized product to the adhering cells. The choice of a suitable enzyme for this undertaking was guided by extensive prior knowledge of existing enzyme prodrug therapies, specifically antibody- and gene directed EPTs.[34–38]β-Glucuronidase (β-Glu) stands out as an enzyme with extensive prior characterization[39] and availability of the substrates (prodrugs) to yield therapeutics with a wide spectrum of action, many of which are available commercially. This includes drugs to treat cancer,[24,40] small 2014, 10, No. 7, 1314–1324
inflammation,[41,42] hypertension, viral diseases,[41] as well as agents for imaging[43,44] – all due to the fact that glucuronides are natural metabolic products of commercial drugs and therefore undergo stringent analysis for regulatory approval and to define the scope and utility of therapies. The above cited literature reports describe examples of glucuronide substrates to yield therapeutic molecules to treat a magnitude of diseases as well as imaging reagents and this illustrates that the same enzyme, β-Glu, can serve to produce a panel of diverse products, independent of their structure, function, solubility and charge. Once engineered into implantable devices for SMEPT, this provides for a unique opportunity that the same implant will be capable of synthesizing diverse drugs with diversity of function being limited only by the availability of prodrug molecules. Such opportunities have no precedent in the field of implantable biomaterials. This work represents the first major step towards engineering SMEPT into therapeutic implants, specifically into surface coatings comprised of multilayered polymer thin films. For the guidance in design of polyelectrolyte films, we rely on extensive literature on the subject and vast opportunities as far as candidate materials and properties of resulting films.[10,11,45] Depending on the envisioned application, synthetic and natural polymers are similarly attractive as building blocks for LbL films; biodegradable and ultimately stable polymer coatings find use in biomedicine, and all of the above could equally benefit from enhanced opportunities in drug delivery, such as EPT. In this initial study, we build on success of our recent report on the use of liposome-containing multilayered polyelectrolyte films for drug delivery to adhering hepatocytes and myoblasts.[46] As a positively charged polymer, we used a synthetic polypeptide with extensive use in the field of LbL coatings, poly-L-lysine (PLL). As negatively charged counterparts, we used polymers with different origin, structure, and biodegradation behavior: alginate (Alg, polysaccharide), poly-L-glutamic acid (PGA, synthetic polypeptide), poly(methacrylic acid) (PMA, synthetic polyanion), and a cholesterol functionalized PMA, PMAc. We have shown that these combinations of polyelectrolytes can be successfully used in the assembly of multilayered coatings, support adhesion of mammalian cells, and sustained drug delivery to adhering cells, specifically mediated by immobilized liposomal drug deposits.[46] Herein, we investigate films assembled from these polymers as reservoirs for β-Glu and analyze effectiveness of the films as EPT-functionalized coatings. A significant phenomenological challenge to the performance of SMEPT is a requirement for the prodrug to gain access to the enzyme in the structure of the coating and afford concentration of the product in physiologically relevant range. Blood, lymph, and/or interstitial fluids create dynamic flow around the implants thus, contributing to the performance of biocatalytic systems via mixing and replenishing the prodrug pool and at the same time working against the feasibility of EPT through decreased co-localization time of the enzyme and its substrate. The above reasons are speculative and as such, drug delivery under dynamic, flow conditions is an emerging field with research findings reported only in the nearest past. Studies appearing to date largely relate to the aspects of
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.small-journal.com
1315
full papers
B. Fejerskov et al.
antibody-assisted targeting,[47–50] or non-specific association of nanoparticles[51,52] or polyplexes[53] with endothelial cells under flow conditions. In terms of therapeutic response in the presence of shear stress, enhanced transfection efficiency,[54,55] or increased cytotoxicity of 50 nm mesoporous nanoparticles[56] have been demonstrated. Interesting approaches on the drug release from aggregated nanoparticles[57] and lenticular vesicles[58] under high shear stress as a concept to treat strokes and embolisms have recently been reported. Recently, we provided a systematic investigation of drug delivery using nanoscopic drug carriers to myoblasts and hepatocytes under shear stress[59,60] and revealed fundamental differences in cell/ drug carrier interaction depending on the presence of shear stress. In particular, positively charged liposomes showed enhanced association with cells under flow conditions and this translated into improved therapeutic response, specifically in terms of efficacy of cytotoxic drugs and efficiency of nucleic acid delivery.[59] Further, PEGylated carrier exhibited cell association characteristics significantly differed depending on the used cell type, exposure times and shear stress.[60] Particularly, in the presence of shear stress, we found that the association of non-PEGylated carriers with hepatocytes compared to the PEGylated ones was significantly higher after 30 min, while for the myoblasts, the difference only became obvious after 4 h. These data illustrate that flow of media surrounding cells is an important parameter to consider for the in vitro characterization of drug carriers. In this work, we investigate the performance of enzymeequipped LbL thin films under flow conditions and use this tool to illustrate unique opportunities associated with the EPT approach in the context of SMDD (Scheme 1). Specifically, in this work we i) present the assembly of biocatalytic surface coatings based on multilayered polymer thin films, ii) demonstrate that the EPT strategy engineered into surface coatings affords physiologically relevant concentrations of drugs, and does so under flow conditions, iii) illustrate that biocatalytic surfaces provide opportunity for on demand, “on-off” synthesis of the drug, and iv) reveal that this mode of drug delivery is site specific. We believe that these accomplishments have no prior precedent and contribute significantly to the development of drug eluting coatings for engineering of therapeutic implants.
Scheme 1. Enzyme-functionalized, biocatalytic surface coatings based on multilayered polymer thin films are investigated herein as tools for localized, on-demand drug delivery under flow conditions mimicking dynamic conditions encountered in vivo.
1316 www.small-journal.com
Figure 1. Frequency changes Δf of QCM-D crystals upon sequential deposition of PLL/PGA multilayers and the incorporation of β-Glu into the polymer films. Polymer deposition was conducted from 1 g/L solutions of PLL and PGA in HEPES buffer, pH 7.4 supplemented with 0.15 M NaCl. Adsorption of the enzyme was conducted from buffers with pH 5–8.5.
2. Results and Discussion 2.1. Assembly of the biocatalytic films Assembly of polymer multilayers was quantitatively monitored using QCM-D. In the first experiment, we used one of the most well-studied pair of polyelectrolytes, synthetic polypeptides PLL and PGA. This pair has been studied towards fundamental understanding of LbL deposition of polymers[61] and also used in biomedical applications for e.g. controlled cell adhesion and proliferation[62,63] as well as drug delivery.[17,62,64–66] As is typical for “weak” polyelectrolytes, deposition of these polymers can be controlled via the pH of the solution as well as the presence of low molecular weight electrolytes such as NaCl. These aspects of materials engineering are well documented in the state of art and herein, we used widely accepted deposition conditions, 1 g/L concentration of each of the polymers prepared using 10 mM HEPES buffer with pH 7.4 and supplemented with NaCl to 0.15 M. Deposition of each polymer layer was reflected by a decrease in resonance frequency (Δf) of the QCM crystals, Figure 1. Following deposition of 5 polymer layers, the thin films were exposed to a solution of β-Glu (20 μg/mL) in aqueous buffers with pH values from 5 to 8.5. This pH range was monitored herein in an attempt to optimize the adsorption of the protein through a variation of its ionization (isoelectric point pI 4.8). Surprisingly, exposure of the polymer coated crystals to β-Glu afforded a rather small Δf (
