Aptamer supported in vitro endothelialization of poly(ether imide) films

Abstract

Implantation of synthetic small-diameter vascular bypass grafts is often associated with an increased risk of failure, due to thrombotic events or late intimal hyperplasia. As one of the causes an insufficient hemocompatibility of the artificial surface is discussed. Endothelialization of synthetic grafts is reported to be a promising strategy for creating a self-renewing and regulative anti-thrombotic graft surface. However, the establishment of a shear resistant cell monolayer is still challenging. In our study, cyto- and immuno-compatible poly(ether imide) (PEI) films were explored as potential biomaterial for cardiovascular applications. Recently, we reported that the initial adherence of primary human umbilical vein endothelial cells (HUVEC) was delayed on PEI-films and about 9 days were needed to establish a confluent and almost shear resistant HUVEC monolayer. To accelerate the initial adherence of HUVEC, the PEI-film surface was functionalized with an aptamer-cRGD peptide based endothelialization supporting system. With this functionalization the initial adherence as well as the shear resistance of HUVEC on PEI-films was considerable improved compared to the unmodified polymer surface. The in vitro results confirm the general applicability of aptamers for an efficient functionalization of substrate surfaces.

Keywords

Aptamer endothelialization physical functionalization poly(ether imide) films shear resistance

1 Introduction

Cardiovascular syndromes due to atherosclerosis are the major cause of premature death worldwide and remain one of the biggest global health problems [1]. Especially in advanced coronary artery disease, a vascular replacement is often necessary. Auto-grafts taken from the left internal thoracic artery or from the saphenous vein are currently the gold standards for coronary artery bypass grafting [2]. However, e.g. coronary bypass grafting needs small calibre synthetic prostheses, due to the limited supply of auto-grafts (in 5–30% of patients no suitable veins/arteries are available due to previous use or diseased vein wall [3]). Currently clinically used synthetic vascular grafts (e.g. polyethylene terephthalate (PET, Dacron) and expanded polytetrafluoroethylene (ePTFE)), have achieved excellent results in bypassing large-diameter arteries; however, they do not perform well in low-flow or small-diameter vessels (Ø<6 mm), for example, in coronary arteries. Reasons for this failure are material thrombogenicity and inadequate endothelialization [4]. Therefore, a rapid adherence of endothelial cells (EC) to the substrate and a shear-resistant binding of the EC are of utmost importance [5] because EC are the ideal antithrombotic surface and actively support vessel homeostasis [6]. Unfortunately, EC do not spontaneously form a confluent monolayer on many polymers and/or detach under shear stress, which in vivo results in graft failure due to thrombosis [7 –10]. One strategy to overcome the low seeding efficiency and the poor shear resistance in vivo is the in pre-implant seeding of EC.

A potential candidate material for this approach are poly(ether imide) (PEI) films, which were shown to be hemo- and immuno-compatible compared to clinically used polymers like PET or PTFE [11]. In addition, in vitro HUVEC were able to form a functionally-confluent monolayer within nine days [12]. However, the HUVEC monolayer formation was delayed because of the hydrophobic material surface, resulting in a low cell density after initial adherence and, in addition, the HUVEC monolayer did not completely resist venous shear forces. Therefore, the concept of this study was focused on the improvement of the HUVEC initial adherence and adherence strength on PEI-films by an aptamer based EC supporting system. PEI-binding aptamers from a previous study in combination with cRGD-peptides were used as EC binding ligands for non-covalent surface functionalization [13]. Comparable approaches for non-covalent surface functionalization of polymeric biomaterials like polystyrene (PS), tissue culture plate (TCP), polyethylene (PE), PTFE or silicone exist in the form of peptide based coatings, also known as peptide-tags or interfacial biomaterials (IFBMs), with material surface binding ligands derived from in vitro selection (phage display), silk or by mimicking mussel adhesives with poly dopamine derivatives [14 –20]. However, mussel adhesives do not have the potential for a material or cell selective binding, as it is given with ligands derived from in vitro selection. Peptide based IFBMs could be shown to be useful for the in vitro immobilization of bioactive ligands enhancing cell adherence and proliferation or improving hemocompatibility [17 , 22]. Nevertheless, there is an increased risk of unexpected reactions from biological systems by using these peptides in vivo. They could unintentionally mimic cell recognition or recruiting motifs with unpredictable influences on cell behavior or immunologic activation [23, 24]. From natural nucleic acid based aptamers interfering cell biological reactions are less likely, because no considerable immunological response of biological systems is known to date. Furthermore, denaturation of aptamers is reversible, enabling the application of less expensive sterilization methods for a functionalized surface, without jeopardizing the ligands functionality. The use of polymer binding aptamers additionally offers the advantage that an alteration of beneficial polymer surface properties can be approximately excluded. One should have in mind that the surface properties of the PEI-films guaranteed the low thrombogenicity [25] as well as the ability of the HUVEC to form a functionally-confluent EC monolayer. Thus, an alteration of the material’s surface properties could directly influence the HUVEC adhesion, migration, proliferation and functionality [26].

Former aptamer based strategies concerning the endothelialization of artificial implant materials were focused on the use of aptamers as selective cell binding ligands, whereby the oligonucleotides were covalently bound to the substrate surface [27, 28]. In the field of generating HUVEC binding aptamers for in vitro endothelialization or in vivo capturing of endothelial progenitor cells (EPC) on artificial implant materials, the research strongly developed in recent years. An example is capturing of EPC on the surface of intracoronary stents, which was tried in vitro and in vivo with certain aptamer based coatings [29, 30].

The aim of this study was the development of an endothelialization supporting system, which should improve the initial EC adherence and shear resistance (Fig. 1). In contrast to previous applications, the aptamers were used as the polymer binding component, whereby a cyclic RGD-peptide (cRGD) served as the cell binding ligand. RGD-peptides as a coating for cell adhesion were extensively explored in the past, promoting the cell adhesion and growth for several cell types [31 –35]. By connecting the single components via high affine biotin-streptavidin interaction, the whole system was build modular enabling the exchange of each binding component at any time.

Fig.1

(A) Most abundant PEI binding sequences of the enriched PEI-SELEX aptamer library. (B) Molecular structure and interactions of the components for the endothelialization supporting system (* exemplarily shown from PEI 2 sequence) (C) Schematic representation of the concept for non-covalent surface functionalization of PEI films with the aptamer-based endothelialization supporting system.

2 Material and methods

2.1 Preparation of PEI-films

PEI-films were prepared from a solution (20 wt.-% in dichloromethane) of commercially available poly(ether imide) (PEI, ULTEM^® 1000, General Electric, NY, NY, USA), sterilized and characterized as described previously [12]. The polymer was used as received from the manufacturer without further purification. For aptamer binding experiments PEI-film discs (Ø=4 mm) were stamped out by using sterile biopsy punches (SmithKline Beecham Ltd., Berks, UK). For cell culture experiments in static and dynamic in vitro systems, PEI was used as 13 mm discs in 24 well plates (TPP, Techno Plastic Products AG, Trasadingen, Switzerland) or in the form of 30 x 30 mm squares, respectively. Sample preparation and pre-incubation was performed as described in previous studies [12].

2.2 Cell culture

Commercially available primary human umbilical vein endothelial cells (HUVEC, Lonza, Cologne, Germany) were used for no longer than four passages, cultivating them under static cell culture conditions (37 °C, 5 vol.-% CO₂) in polystyrene-based cell culture flasks (TPP, Techno Plastic Products AG, Trasadingen, Switzerland) with endothelial basal medium EBM-2 supplemented with EGM-2 Single Quots^® kit and 2 vol.-% FBS to EGM-2 full medium (Lonza, Cologne, Germany). For cell culture experiments HUVEC were harvested by trypsin/EDTA treatment (0.25% v/v Trypsin and 0.53 mM EDTA in PBS (–/–), PAN-Biotech GmbH, Aidenbach, Germany) and seeded on PEI-films and controls (tissue culture plate (TCP) or glass) with an initial density of 6 · 10⁴ cells/cm² in the EGM-2 or the EBM-2 K1 cell culture system. EGM-2 was the full medium and EBM-2 K1 medium lacked fetal bovine serum (FBS), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and the insulin-like growth factor (IGF). Experiments concerning the initial cell adherence and shear resistance were performed after eight hours of static culturing. The initial adhesion of HUVEC was evaluated by light microscopy. Cell viability and density were analyzed with a fluorescent-based live/dead staining (see 2.5). Morphological adherent (spread) and weak adherent (spherical) cells were distinguished for the determination of the cell density.

2.3 Rheological measurements

The exposure of HUVEC to physiological shear forces was performed with a cone-plate rheometer (Smard-CAD Deutschland GmbH, Neu-Ulm, Germany). The EC were exposed to shear rates of 3 dyn/cm² for two hours. Documentation of the cell density and morphology was carried out before and after the shear stress exposure by taking pictures with a QImaging Retiga™ 4000 R digital camera (3 fields of view per sample with n = 6 samples; Retiga™ 4000 R, QImaging, Surrey, British Columbia, Canada).

2.4 Cell viability assessment

HUVEC initial adhesion and viability were examined in the static system after culturing eight hours by fluorescence staining. Vital cells were stained with fluorescein diacetate (FDA, 25 μg/ml, Invitrogen, Carlsbad, CA, USA) in green colour, whereas propidium iodide (PI, 2 μg/ml, Sigma, Taufkirchen, Germany) staining in red colour indicated dead cells. Documentation was realized by taking three pictures from different areas of each sample, using the confocal laser scanning microscope (cLSM, LSM 510 META, Zeiss, Oberkochen, Germany) with a 10x primary magnification.

2.5 Synthetic oligonucleotides (aptamers)

All oligonucleotides for the generation of PEI-film binding aptamers, using the SELEX method, were purchased from IBA GmbH (Göttingen, Germany). In the experiments the enriched library from a previous in vitro selection against the PEI-film surface was used as polymer binding ligand [13]. The PEI binding aptamer motifs were dominated by pyrimidines with a preference for thymine. The single stranded SELEX starting DNA-library with the sequence 5^′-GGG AGA AAT TCC GAC CAG AAG -(N)₅₀- GAT GGA CGA ATA TCG CTC CC-3^′ and negligible material affinity served always as the control for unspecific background binding.

2.6 Quantification of PEI-film bound aptamers by quantitative RealTime PCR (qPCR)

Binding experiments were performed with 20 pmol of the enriched library from the PEI-SELEX round 11 on 4 mm polymer discs under selection conditions [Selection buffer: PBS+/+(Biochrom AG, Berlin, Germany), 1.5 mM MgCl₂;RT] and 30 min incubation time for establishing the aptamer-polymer binding. For comparative binding experiments, elution of polymer bound aptamers was performed after incubation by heat denaturation (95 °C, 10 min) and subsequent isolation of the eluate. For determination of unbound aptamers after incubation in selection buffer or cell culture medium, supernatants were obtained prior to heat denaturation. Amounts of polymer eluted ssDNA or aptamers remaining in the supernatant without elution were quantified in relation to a decadal dilution series of the starting library in the range between 100 pg/μl – 1 fg/μl. Thereby samples were quantified by using the SYBR^® Green PCR Master Mix and the StepOnePlus™ Real-Time PCR system both from Applied BioSystems (Foster City, CA, USA).

2.7 Visualization of polymer bound Streptavidin (SAv) and dsDNA probe by cLSM

Surface binding of PE labelled Streptavidin (PE-SAv, BD Biosciences, Franklin Lakes, NJ, USA) as well as the 5’-AlexaFlour647/5’-Biotin modified dsDNA probe was visualized by the cLSM. Untreated PEI and the polymer incubated with the starting library served as references to determine the background fluorescence from the material as well as unspecific background binding of ssDNA.

2.8 The aptamer-cRGD based endothelialization supporting system

Stepwise development of the endothelialization supporting system with biotinylated polymer binding aptamers, SAv and finally the biotin modified cRGD-peptide was performed by functionalization of the PEI-films layer by layer. For preliminary binding experiments 20 pmol of 5’-biotinylated aptamers were incubated 30 min in selection buffer at ambient temperature with 4 mm PEI-film discs. After washing two times in selection buffer, SAv coating was realized with 20 pmol on the aptamer treated PEI-films for the same timeframe and subsequent washing. The biotinylated cRGD-peptide (c[RGDfK (Biotin-PEG-PEG)], Peptides International Inc., Louisville, KY, USA) was applied by incubating the aptamer-SAv coated PEI-film with 20 pmol cRGD for 30 min followed by two times washing. For in vitro cell adhesion experiments in the static and dynamic test system amounts of aptamers, SAv and cRGD were adjusted to the particular PEI-film surface area (Ø=15.6 mm for 24 well plates in the static system and Ø=24 mm for rheological sample holder in the dynamic conditions).

2.9 Statistics

All data are reported as arithmetic mean±standard deviation (SD) for continuous variables and were analyzed by a two-sided ANOVA with subsequently Holm-Sidak’s post hoc test. A p value of less than 0.05 was considered significant.

3 Results

3.1 Aptamer material binding under cell culture conditions

Binding experiments were carried out for determining the time dependent aptamer binding stability in selection buffer and cell culture conditions (Fig. 2). Under selection conditions the aptamer binding remained constant for up to 20 h. The material binding was averaged over time with 2.22±0.31 pmol more than 100 times higher compared to the starting library with 19.45±0.10 fmol.

Fig.2

Stability of the aptamer functionalization on PEI-films over time under selection and cell culture conditions. Stable aptamer binding on PEI under selection conditions for 20 h. Under cell culture conditions (EGM-2 full medium, 37 °C) the aptamer functionalization got lost within the first hour (Quantification by qPCR; arithmetic mean ± standard deviation; n = 4).

After evaluation of a stable aptamer-PEI binding under selection conditions, the functionalization stability under cell culture conditions was investigated. For this purpose, PEI-films were initially incubated under selection conditions with the aptamers to establish the aptamer-material binding (time point: 0 h). Thereafter, PEI-films were incubated for up to 20 h at 37 °C in EGM-2 culture medium. The results demonstrated that cell culture conditions had a marked influence on the aptamer-PEI binding stability. The amount of surface bound aptamers decreased dramatically from 1.68±0.09 pmol to 0.11±0.01 pmol within the first hour of incubation. Over time a small amount of bound DNA on the PEI-film surface was always detectable, which was in average with 0.07±0.01 pmol still seven times higher compared to the unselected library.

To analyze how the aptamer binding was influenced by the cell culture system and to clarify disturbing factors within the full EGM-2 medium, additionally binding experiments were performed with the EBM-2 basal medium, which is the basic medium of EGM-2 without any supplements. Here the aptamer-material binding remained stable on the polymer surface (Fig. 3). In the following binding experiments single supplements of EGM-2 were tested in EBM-2. Fetal bovine serum (FBS) and the fibroblast growth factor (FGF) strongly influenced the aptamer affinity. By adding these supplements to the EBM-2 basal medium nearly no aptamers remained on the PEI-films. Other supplements like the vascular endothelial growth factor (VEGF) and the insulin-like growth factor (IGF) slightly affected the aptamer binding. However, the aptamers were not degraded. In case of FBS and FGF nearly the full amount of aptamers were detected in the supernatant by qPCR.

Fig.3

Aptamer binding on PEI after four hours of incubation in EBM-2 medium added with single supplements at 37 °C. Compared to the protein free aptamer binding control (light grey) and the EBM-2 control without supplements the aptamer binding got strongly influenced by FBS and FGF and less disturbed by VEGF and IGF. (Quantification by qPCR; arithmetic mean ± standard deviation; n = 6).

3.2 Adaption of the cell culture system suitable for aptamer-polymer functionalization

With the knowledge that the proteinogenic supplements FBS, FGF, VEGF and IGF had an adverse effect on the aptamer-polymer interaction two medium compositions based on the EBM-2 basal medium were created. The first contained all usual supplements of the full EGM-2 medium except of the four above mentioned, named in the following as EBM-2 K1. EBM-2 K2 additionally contained the moderate disturbing supplements VEGF and IGF. With these medium compositions aptamer-PEI binding experiments were performed (Fig. 4). EBM-2 K2 still exhibited a strong adverse influence on the aptamer binding affinity, where only 10% of the ssDNA was detectable on the polymer surface after incubation, compared to baseline value at 0 h. EBM-2 K1 also showed an effect on the aptamer binding. However, this effect was less marked and after incubation more than 50% of the initially bound aptamers could be detected.

Fig.4

Stability of the aptamer-material binding after 4 h at 37 °C in EBM-2 K1 and K2 medium. Strong influence on the aptamer binding with EBM-2 K2, but moderate with EBM-2 K1. (Quantification by qPCR; arithmetic mean ± standard deviation; n = 4).

The assessment of cell adhesion and morphology of HUVEC on TCP using EGM-2 and EBM-2 K1 as culture medium revealed the formation of a dense HUVEC monolayer in the full medium within the first eight hours (Fig. 5). After 24 h the HUVEC monolayer was optically confluent. In EBM-2 K1 medium the initial adherence of the HUVEC was reduced and small cell clusters were formed after eight hours. In these clusters the HUVEC morphology was mostly spindle shaped and an increased formation of pseudopodia was visible. Within 24 h most of the HUVEC lost adherence and swam with a spherical shape in the supernatant. In this cell culture system no closed cell monolayer developed.

Fig.5

Adhesion and morphology of primary HUVEC after confluent seeding on TCP in the EBM-2 K1 compared to the EGM-2 cell culture system. Initial HUVEC adherence was strongly influenced by EBM-2 K1 medium. (Pictures taken by light microscopy in phase contrast mode with 10x primary magnification; scale bar: 100 μm; n = 3).

3.3 Design and preparation of the endothelialization supporting system

As described above the aptamer based system for the stabilization of seeded EC was designed consisting of two main parts, which were connected via non-covalent biotin-streptavidin interactions combining material binding aptamers and EC binding cRGD-peptides. For the preparation of this system, each of the steps was consecutively performed and the formation of the multilayer experimentally verified.

The first step was the biotinylation of the aptamers by PCR amplification method and subsequently validation, whether these modified sequences were still able to bind to their target material. DNA quantification from the corresponding binding experiment showed no change in the material binding on PEI-films because of the 5’-biotin modification. The aptamer binding on the PEI-film surface was with 1.31±0.36 pmol more than 500 times stronger compared to the unspecific background binding of the unselected starting library with 2.57±2.42 fmol. In the second step the accessibility of aptamer bound biotin for streptavidin (SAv) was investigated. All initial experiments to detect the specific biotin-SAv binding on PEI-films remained unsuccessful. A closer view to the exact composition of the SAv solution revealed the presence of BSA (1 wt.-%) in the buffer system. Additional quantitative aptamer binding studies with various concentrations of BSA contaminated as well as BSA free SAv were performed in regard to an interfering influence of BSA and the SAv itself on the aptamer-material binding (Fig. 6). The results elucidated a concentration dependent interfering influence of BSA on the aptamer-polymer binding, which was not visible for SAv alone.

Fig.6

Binding of aptamers on PEI in the presence of streptavidin (SAv). Compared to the protein free aptamer binding controls (light grey) the aptamer-PEI binding was strongly influenced in a concentration dependent manner by BSA in the SAv buffer system. (Quantification by qPCR; arithmetic mean ± standard deviation; n = 6).

To confirm the selective binding of streptavidin on the biotinylated 5’-terminus of the material bound aptamers, the protein was visually detected by PE fluorescence labeling and cLSM method. On the PEI-film, which was incubated with the biotinylated polymer binding aptamers, the PE signal was significantly increased both compared to unspecific adsorbed PE-SAv on the untreated PEI and PEI films incubated with the biotinylated starting ssDNA library (Fig. 7A). Finally, the accessibility of the bound SAv for other biotinylated molecules such as the cRGD-peptide was investigated (Fig. 7B). Because there is no RGD specific antibody commercially available for a direct detection of SAv bound biotin-cRGD, the SAv accessibility was validated by using a dsDNA probe, modified on the one 5’-terminus with Alexa647 fluorescent dye and on the other with biotin. The findings from the corresponding binding studies showed that there was a slight background binding of the probe on the surface of both, the PEI-film prior treated with SAv and the film treated with the starting library+SAv. Nevertheless, the probe fluorescence signal was significantly increased on the PEI-films, which were incubated in the first binding step with PEI binding aptamers. Moreover, no fluorescence signal for the probe was detected on such PEI samples, when omitting SAv.

Fig.7

Layer-by-layer development of the aptamer based endothelialization system. (A) Significantly increased fluorescence intensity for PE-SAv binding on aptamer functionalized PEI-films. (B) Increased signal of the DNA probe solely by using a combination of PEI binding biotinylated aptamers and PE-SAv. (Arithmetic mean ± standard deviation;* p < 0.0001; n = 6).

3.4 Aptamer supported endothelialization of PEI-films

In the static in vitro model HUVEC were seeded on untreated as well as on PEI-films functionalized with the aptamer-cRGD system. HUVEC seeded on TCP served as control. The functionality of the endothelialization supporting system was tested in both EGM-2 and the EBM-2 K1 medium. EC adherence clearly differed between both cell culture systems after seeding on TCP. In EGM-2 the cells were extensively spread and uniformly distributed. In contrast, in the EBM-2 K1 medium EC showed a more distinct spindle shape and the formation of cell clusters (Fig. 8). However, total HUVEC density and the ratio between spread and spherical cells did not differ (TCP: total cell density: EGM-2 18,978±2,698 vs. EBM-2 K1 16,920±2,301; spread cells: EGM-2 17,812±2,652 vs. EBM-2 K1 15,460±2,314; spherical cells: EGM-2 1,166±153 vs. EBM-2 K1 1,461±419; for all p > 0.05; n = 6, Fig. 9). On the surface of untreated PEI-films only few spread HUVECs were visible in both cell culture systems and the majority of the inherent HUVEC showed a spherical morphology. No differences between both media concerning cell morphology as well as density were detectable (untreated PEI-films: total cell density: EGM-2 9,486±1,544 vs. EBM-2 K1 8,745±1,343; spread cells: EGM-2 1,989±1,124 vs. EBM-2 K1 2,106±266; spherical cells: EGM-2 7,497±1,157 vs. EBM-2 K1 6,639±1,181; for all p > 0.05; n = 6). However, all evaluated aspects (total EC density, number of spread and spherical cells) were in EGM-2 medium solely for TCP and in EBM-2 medium significantly inferior compared to aptamer-SAv-cRGD coated samples and the TCP reference.

Fig.8

EC adherence and vitality eight hours after seeding in EGM-2 and EBM-2 K1 medium on untreated compared to aptamer-cRGD functionalized PEI-films and TCP as the control. Vital cells were stained by fluorescein diacetate (FDA, green), whereas propidium iodide (PI, red) staining indicated dead cells. (Pictures taken by cLSM with 10x primary magnification; scale bar: 100 μm).

Fig.9

HUVEC density after eight hours culturing in EGM-2 and EBM-2 K1 medium on untreated compared to aptamer-cRGD functionalized PEI-films and TCP as control. Total cell density is composed of morphologically spread (dark grey) and spherical cells (light grey). (Arithmetic mean ± standard deviation; ^#p total cell number; *p spread cells; ⁺p spherical cells; ^# ,*^, +p < 0.05; n = 6).

The non-covalent functionalization of the PEI-films with the aptamer-cRGD system caused a change in the adherence behavior of the EC in EGM-2 medium. Compared to the untreated polymer more cells were spread and the total number was significantly increased. However, these differences were marginal compared to the control. Differences were much more obvious for EBM-2 K1. The aptamer-cRGD functionalization caused a significant increase of the number of spread EC as well as the total cell count, which was close to the reference (Aptamer-cRGD functionalized PEI-films: total cell density: EGM-2 12,003±996 vs. EBM-2 K1 15,741±2,575; spread cells: EGM-2 4,390±508 vs. EBM-2 K1 13,141±2,804; spherical cells: EGM-2 7,613±1,441 vs. EBM-2 K1 2,599±397; for all p < 0.05; n = 6). Morphologically, the HUVEC were also comparable to TCP, by showing mainly a spindle cell shape and the formation of cell clusters. Nevertheless, the total cell number and the amount of spherically shaped cells were only slightly, but however significantly reduced.

In the dynamic in vitro model, HUVEC were seeded on untreated and aptamer-cRGD coated PEI-films in EBM-2 K1 medium, incubated eight hours under static cell culture conditions and subsequently exposed for two hours to physiological shear rates. The EC morphology before shear stress exposure was comparable to the results of the static conditions. On the aptamer-cRGD functionalized PEI-films and the control, most EC showed spindle shaped morphology and the formation of clusters. In contrast only few spread cells were detectable on the untreated PEI surface. After shear stress exposure a tendentially but never significant decrease in the cell number was visible on each material (Fig. 10). In direct comparison of the EC adherence behavior, no significant difference between the aptamer-cRGD coated PEI-film and the reference was found, while the cell density on the untreated polymer was significantly reduced prior to and after shear stress exposure.

Fig.10

HUVEC density eight hours after seeding in the EBM-2 K1 cell culture system on untreated compared to aptamer-cRGD functionalized PEI-films and glass as the control before and after shear stress exposition. (Arithmetic mean ± standard deviation; shear stress exposure for 2 h at 3 dyn/cm²; * p < 0.05; ** p < 0.005; *** p < 0.0005;**** p < 0.0001; n = 6).

4 Discussion

Former studies with smooth poly(ether imide) films revealed that this material exhibited – compared to PEI-membranes – a strongly reduced thrombogenicity [25]. In addition, HUVEC were able to form a functionally-confluent monolayer within nine days, showing a distinct interaction with the underlying polymer [12]. However, initial EC adherence and proliferation was delayed and the generated EC layer did not sufficiently resist physiological shear rates. To improve the rate of initial EC adherence an in vitro selection method was recently evolved to generate PEI-film binding aptamers for the physical surface functionalization of this biomaterial [13]. In the present study, the suitability of these PEI-film binding aptamers was investigated.

The strategy to use PEI binding aptamers for non-covalent surface functionalization was chosen to prevent a chemical alteration of the PEI-films surface [25]. Covalent immobilization may adversely influence beneficial polymer surface properties as well as the efficiency and stability of bound ligands [36]. In vivo all these factors can increase thrombogenicity or provoke inflammation and loss of cell adherence, possibly leading to a loss of implant function [37 –40].

Investigating how the binding behavior of the polymer binding aptamers was influenced by cell culture conditions was the first step to establish an aptamer supported endothelialization system. Therefore, the aptamer functionalization stability was investigated for the time period necessary for initial EC adherence. Because of the quantitative higher binding of the enriched SELEX library compared to single aptamers, found out in our previous study [13], for this prove of concept not one single aptamer, but rather the enriched library from selection round 11 of the SELEX against the PEI-film surface was used for the setup of the endothelialization system. Due to the higher amount of aptamers on PEI, possible interactions between the polymer and the EC should be maximized in this proof of concept by the concurrently increased number of cell binding ligands, which should further stabilize the whole system. The maximization of binding ligands was particularly important, because we previously showed that the aptamers tend to form aggregates in the nm range on the PEI-film surface [13]. HUVEC adhesion would probably happen on cRGD-peptide clusters, present every few nm on the polymer surface. Recent publications concerning the distribution of endothelialization supporting ligands such as RGD proved that the spatial microstructure of the ligands in the order from nano- to micrometers influenced directly the initial EC adherence on polymeric surfaces [41 –43]. The cellular spreading area and the formation of focal adhesions were directly dependent on the RGD density [44]. In consideration of the suspected cRGD-peptide density on the aptamer coated PEI-films in the nanometer range and the natural spreading area of an EC in the range between 1–6 × 10³ μm² [45], there were plenty possible contact points for an distinct interaction of the HUVEC surface integrin clusters with the underlying aptamer-cRGD coated PEI-film available.

The results from the binding experiments showed that the aptamer binding on the PEI-films was stable for 20 h under standard in vitro conditions. Within this timeframe no evidence for a degradation of the oligonucleotides was detected. Thus, the basis for further endothelialization studies was given. However, following experiments demonstrated that the aptamer binding got lost immediately in a standard HUVEC culture system [46]. The aptamers were displaced from the PEI-film surface by certain proteinogenic supplements in the full medium and dissociated mainly intact into the supernatant. In particular FBS and FGF eliminated the aptamer binding nearly completely. Other supplements like VEGF and IGF interfered moderately with the aptamers. The loss of the aptamer binding was most likely caused by a concentration dependent displacement, attributable to the coexisting proteins. Proteins, which are present in all biological fluids, tend to adsorb to hydrophobic surfaces [47, 48]. The amount of surface bound aptamers was compared to the surrounding proteins vanishingly low. The aptamer binding was additionally aggravated by the fact, that the interaction between DNA and polymer surface was most likely mediated to a major contribution by hydrophobic interactions, the same binding forces acting for unspecific protein adsorption [49, 50]. Thus, there was a displacement of bound aptamers from the PEI-film surface, because the equilibrium was postponed in favour of the adsorbed proteins. By elimination of the interfering components from the full medium, a stable aptamer binding could be achieved.

The development of the aptamer based endothelialization supporting system consisting of non-covalent via biotin-streptavidin interactions coupled PEI-film binding aptamers and RGD was carried out layer-by-layer, whereby each working step was ascertained individually. As cell binding component a cyclic RGD-peptide (cRGD) was chosen as model ligand, because of its higher stability compared to the linear form [51] and an increased EC affinity, most likely because of a more native conformation [52]. However, it was also described that RGD affected the activity of thrombocytes, whereby the findings were contrary. On the one hand an increased adhesion of platelets on RGD functionalized polymeric surfaces was observed, which would be similar to a lesion of the endothelium, which would cause a thrombogenic response of the thrombocytes [53]. However, cRGD is described to act as a selective antagonist of the platelet integrin α_IIβ₃, inhibiting platelet mediated thrombus formation [54]. For the in vitro validation of the general aptamer-cRGD system functionality, this point was not of vital importance, because the accessibility of RGD for thrombocytes would be no longer given after complete endothelialization. Furthermore, the modular structure of the endothelialization supporting system enabled the possibility to easily replace one of the binding ligands by another.

In the initial step of the setup it was proven that the modification of the aptamers with biotin on the 5’-terminus had no influence concerning the binding affinity to the PEI-film surface. Therefore it seems that this modification did not alter the folding of the oligonucleotides, which might be crucial for the binding. During the evaluation of the biotin accessibility of polymer bound aptamers for streptavidin (SAv), the displacement of the aptamer functionalization by interfering with proteinogenic molecules was reconfirmed. The first tested SAv contained BSA (1 wt.-%) in the buffer system, which caused an almost complete binding loss of the aptamers from the PEI surface. This inhibiting effect depended directly on the protein concentration. The proof that not SAv itself competes with the aptamers was made by comparative binding studies on PEI-films, by using SAv buffered only in PBS. Within the last step it was proven that the already bound SAv was accessible for other biotinylated molecules like the cell binding cRGD-ligand. A direct detection of SAv bound cRGD was not realizable, because no RGD binding antibody is commercially available and quantitative protein detection assays are not sensitive enough to detect few pmol of surface bound peptides. For that reason the accessibility of the PEI surface bound SAv was proven by the use of a dsDNA probe, which was modified on both 5’-termini with biotin and a fluorescence dye, respectively. With this probe the accessibility of SAv could be demonstrated by a strong fluorescence signal over the whole polymer surface. Slight background signals from SAv and the dsDNA probe were most likely descended from a minor unspecific adsorption. Nevertheless, the fluorescence intensity of the probe was on the aptamer treated PEI-films significantly higher and confirmed the complete setup of the aptamer-cRGD based endothelialization supporting system. Additionally by omitting the SAv in between PEI bound aptamers and the probe, no binding of the probe was detectable.

To prove the functionality of the endothelialization supporting system, EC were seeded under static conditions on untreated and aptamer-cRGD functionalized PEI-films in comparison to TCP as the reference. The cells were analyzed deliberately after a relative short incubation time of eight hours, because the focus of this study was the initial phase of cell adherence. HUVEC adhered within few hours, markedly more HUVEC on the surface with RGD-ligands [55]. Under static conditions the initial adherence of HUVEC could be significantly improved on PEI-films by the aptamer-cRGD functionalization in both cell culture systems. This was true for both, the number of cells with physiological spread morphology as well as for the total number of adherent HUVEC on the polymer surface. The efficiency of the aptamer-cRGD coating differed between the full and the EBM-2 K1 medium. These observations were consistent with the preliminary findings, that the aptamer binding was instable in full medium and consequently only a fractional part of the supporting functionalization was available for cell interaction on the PEI-film surface. The circumstance that the EC in the EBM-2 K1 medium showed a considerable better adherence compared to the EGM-2 system, proved the functionality of the aptamer-cRGD functionalization and excluded the possibility that unspecific adsorbed SAv on the hydrophobic PEI surface was responsible for this effect. In that case the cells would have shown a comparative adhesion behavior in both cell culture systems. After validation of a significantly improved initial HUVEC adherence on aptamer-cRGD functionalized PEI under static in vitro conditions, additional experiments were performed in a dynamic in vitro model. Here, it was reconfirmed that the HUVEC in the EBM-2 K1 medium were not able to form an optically confluent cell monolayer within the short incubation time. Prior to shear stress exposure the morphology of adherent EC on the aptamer-cRGD functionalized PEI-films was basically spindle shaped and the cells were organized in small cell clusters comparable to the EC on the reference material. This observation proved the supporting effect of the aptamer-cRGD system on the initial EC adherence. Compared to the functionalized polymer, only one third of the HUVEC with spread morphology were detected on the surface of the untreated PEI-film. These cells were scattered and no cell clusters were formed. After exposing the adherent cells to physiological shear rates, again a 3-fold higher cell number remained on the functionalized PEI-films. However, a complete shear resistance could not be reached. Independent of the underlying matrix nearly a quarter of the adherent EC detached. After the short incubation period this result was to be expected, because the initial adherence of HUVEC depends on the interaction between membranous integrins and the underlying substrate. For further stabilization by secretion and embedding in their own ECM the cells would need more time. Furthermore, the spindle shaped morphology and the formation of elongated cell clusters provided a larger flow resistance for the laminar flow, than it would be by spread EC.

5 Conclusion

The studies confirmed the in vitro functionality of the aptamer-cRGD system for an improved initial endothelial cell adherence and shear-resistance on a polymer-based biomaterial. However, the aptamer-cRGD system was subjected to a certain limitation. The aptamers were dislocated from the PEI-film surface by competing proteins, typically found in comparable high concentrations in biological fluids, preventing an application of the endothelialization supporting system in vivo. Nevertheless, utilization of the improved initial cell adherence by switching to the protein-rich culture system after the adhesion phase should result in an accelerated cell layer formation. The preservation of the EC shear resistance as well as the formation of a functionally dense monolayer after the transfer in a protein-rich environment will be the objective of future investigations. Therefore, a deeper understanding of the molecular interactions between aptamers and polymers, aptamers and proteins as well as the aptamers among themselves is needed. However, in this study the general applicability of aptamers for the fast and uncomplicated functionalization of polymeric surfaces was confirmed.

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Acknowledgments

This work was financially supported partially by the Helmholtz-Association (program-oriented funding and grant no. VH-VI-423) and the Federal Ministry of Education and Research, Germany (grant no. 0315696A “Poly4Bio BB”).

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