Protein adsorption on thermoplastic elastomeric surfaces: A quantitative mass spectrometry study

https://doi.org/10.1016/j.ijms.2013.08.007Get rights and content

Highlights

  • Protein adsorption on tissue engineering surfaces quantified by mass spectrometry.

  • Quantities in the range of 0.1–400 pmol/cm2 were detected by MALDI-ToF MS.

  • Adsorption on nonpolar surfaces increases with protein and surface hydrophobicity.

  • Amount adsorbed maximizes at the isoelectric point and decreases with protein size.

  • Electrospun surface morphologies adsorb more protein than molded surfaces.

Abstract

Protein interaction with an implant material is the key event for subsequent cell and tissue growth and ultimately determines the biocompatibility of the material. In this study, a dendritic poly(isobutylene-b-styrene) (D_IBS) block copolymer developed for soft tissue engineering was processed with electrospinning and compression molding to create a fibrous mat and a flat surface, respectively. Proteins (insulin, ubiquitin and lysozyme) were incubated with these surfaces at various pH levels, and the protein adsorption capability of the surfaces was quantified using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS). A reference material, polystyrene (PS), was processed like the D_IBS polymer to elucidate the influence of surface morphology and polymer chemistry on protein adsorption. Water contact angle (WCA) measurements demonstrated the efficacy of electrospinning to produce fibers with superior surface hydrophobicity and surface area-to-volume ratio, as compared to the flat surfaces obtained by compression molding, which in turn led to a significantly higher protein adsorption performance across all pH levels; the three-dimensionality of the fiber surface also played a role in the increased protein adsorption. Compared to the PS fiber mat, the D_IBS fiber mat surface adsorbed far more proteins, in spite of a lower hydrophobicity (based on WCA data), due to the segregation of a thin polyisobutylene (PIB) layer to the fiber surface. Protein adsorption was also found to depend on the protein's isoelectric point (pI). Binding affinity peaked at a pH close to the pI, at which the protein carries no net charge and, thus, can maximize hydrophobic interactions with the surface; at a lower or higher pH, proteins become charged, shielding off their hydrophobic sites and limiting hydrophobic interactions with a surface. The size and hydrophobicity of a protein also affected the binding to a surface; insulin, the smallest molecule with the highest proportion of hydrophobic amino acids, was adsorbed in larger quantities than the bigger and more hydrophilic lysozyme.

Introduction

When cells interact with the environment, cell adhesion to a foreign surface marks the first important step to begin the cycle of migration, proliferation, differentiation and apoptosis [1]. This cell life cycle is crucial to the success of in vitro tissue growth or tissue engineering, where a scaffold made of a biocompatible material is employed as a substrate to initiate cell growth and serve as the structural support for sufficient cell accumulation and the eventual tissue formation. Cell adhesion to foreign surfaces is regulated by the adhesion receptor proteins in the cell membrane, known as integrins [2], [3]. These integrins specifically bind to adhesion proteins, like fibronectin, vitronectin and fibrinogen [3]. The protein adsorption capability of a surface is therefore a key factor for healthy cell growth and tissue colonization [4], [5], [6], [7] and needs to be adequately evaluated to assess the surface's overall performance as a tissue scaffold.

To build successful scaffolds for tissue replacement, the materials of choice should be at least biocompatible to avoid rejection by the human body, possess the right balance of porosity and surface area for the transfer of nutrients and growth factors to initiate protein adsorption and cell growth, and finally have the necessary structural properties to withstand the rigors of stresses imposed by the body after implantation. Focusing on soft tissue replacement, the structural requirement on scaffolds is less onerous since soft tissues, like skin, typically have tensile strengths of only ∼3 MPa and an elongation at break of <18% [8].

Dendritic poly(isobutylene-b-styrene) (D_IBS) [9] block copolymers bear great potential for tissue engineering applications because they have very similar structures with linear poly(styrene-b-isobutylene-b-styrene) triblock polymers [10], which were approved by FDA as a drug-eluting coating on TAXUS Express2™ coronary stents in 2004 [11]. D_IBS polymers are synthesized by living cationic polymerization and have a dendritic polyisobutylene (PIB) core with end-blocks of polystyrene (PS) [12], [13], PS derivatives [14] or copolymers of PIB and PS [15]. They exhibit excellent biocompatibility [16], believed to stem from the phase segregation of PIB to form a thin (∼10 nm) layer at the surface [13]. Depending on their PS content, D_IBS polymers can have a tensile strength and elongation at break of up to 8.7 MPa and 1800%, respectively, which exceed those of most soft tissues [12], [17].

Synthetic micro- and nano-sized polymer fibers produced by electrospinning are widely employed for the manufacture of scaffolds [18], [19], as their increased surface area and porosity improves cell incorporation for tissue engineering [20], [21]. Moreover, the morphological similarity of electrospun fiber mats to natural tissues, such as collagen fibrils, promotes biocompatibility and better cellular response [22], [23]. Previous studies have also demonstrated that electrospinning can yield super-hydrophobic surfaces [24], [25], [26], whose self-cleaning and antifouling properties make them particularly useful for biomedical applications [27].

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has become an established technique for the analysis and quantification of biomolecules [28], including proteins [29], [30], oligosaccharides [31] and lipids [32]. Inherent advantages of MALDI-ToF MS are a high sensitivity (femtomole range), a rapid sample preparation protocol and straightforward data interpretation; further, reproducible quantification can be achieved by the addition of a properly selected internal standard to each sample and by maintaining constant laser power and voltages [28]. In this study, MALDI-ToF MS was employed to evaluate for the first time the extent of protein adsorption on surfaces developed for tissue engineering applications. D_IBS compression molded films and electrospun fiber mats were investigated over a range of pH values; for comparison, PS molded films and electrospun fiber mats were also considered to determine the influence of polymer chemistry and surface morphology on the resulting protein adsorption. Three model proteins (insulin, ubiquitin and lysozyme) were selected for in vitro adsorption experiments. The protein adsorption behavior observed for the different surfaces by MALDI-ToF MS was correlated to the corresponding surface morphology and hydrophobicity, as assessed by scanning electron microscopy (SEM) imaging and water contact angle (WCA) measurement, respectively.

Section snippets

Materials

Two polymers, D_IBS and PS, were used to prepare the compression molded sheets and electrospun fiber mats examined in this study. D_IBS with 29.4% PS content was synthesized via living carbocationic polymerization [12] and had a molecular weight (Mn) of 220,300 g/mol and a polydispersity index of 1.87, both established by size exclusion chromatography (SEC). Based on the SEC analysis, Mn of the PS end-block was estimated to be ∼73,600 g/mol. The PS sample was used as received from Americas

Calibration curves

Fig. 1 shows two MALDI-ToF mass spectra used for the construction of calibration curves, obtained from PMS samples containing insulin (I), ubiquitin (U) and lysozyme (L) at 1 and 4 pmol/μL, and the IS cytochrome C (CC, 0.5 pmol/μL). Specific peaks corresponding to the ionized proteins and their dimers are indicated in the spectra. It is evident from these spectra that the height and area of the various protein peaks increases at higher concentration relative to those of the IS whose concentration

Conclusions

In this study, a potentially biocompatible polymer, D_IBS, was investigated for its protein adsorption performance. Two polymer-processing techniques, electrospinning and compression molding, were employed to create a fiber mat surface and a flat surface, respectively, and PS was included as a reference material to investigate the effect of surface morphology and polymer chemistry on protein adsorption. Three model proteins, insulin, ubiquitin and lysozyme, were incubated with the surfaces at

Acknowledgements

We thank the National Science Foundation for generous financial support (grant CHE-1012636 to C.W. and grants DMR-0509687 and DMR-0804878 to J.E.P.). The synthesis of the materials was partially supported by the Austen BioInnovation Institute in Akron.

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    1

    Current address: Amgen, Puerto Rico.

    2

    Current address: Exponent Failure Analysis Associates, Shanghai 200127, PR China.

    3

    Current address: Department of Chemistry and Biochemistry, Texas State University-San Marcos, San Marcos, TX 78666, USA.

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