Towards a biocompatible artificial lung: Covalent functionalization of poly(4-methylpent-1-ene) (TPX) with cRGD pentapeptide

Article abstract of DOI:10.3762/bjoc.9.33

Covalent multistep coating of poly(methylpentene), the membrane material in lung ventilators, by using a copper-free "click" approach with a modified cyclic RGD peptide, leads to a highly biocompatible poly(methylpentene) surface. The resulting modified membrane preserves the required excellent gas-flow properties while being densely seeded with lung endothelial cells.

Full text of DOI:10.3762/bjoc.9.33

Towards a biocompatible artificial lung: Covalent
functionalization of poly(4-methylpent-1-ene) (TPX)
with cRGD pentapeptide
Lena Möller1, Christian Hess2, Jiří Paleček1, Yi Su1, Axel Haverich2,
Andreas Kirschning*1 and Gerald Dräger*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 270–277.
1Institut für Organische Chemie und Biomolekulares Wirkstoffzentrum
(BMWZ) der Leibniz Universität Hannover, Schneiderberg 1B, 30167
Hannover, Germany; Fax: (+49) 511-762-3011 and 2Leibniz Research
Laboratories for Biotechnology and Artificial Organs (LEBAO)
Hannover Medical School (MHH), 30625 Hannover, Germany
doi:10.3762/bjoc.9.33
Received: 22 September 2012
Accepted: 21 December 2012
Published: 08 February 2013
Email:
Associate Editor: P. J. Skabara
Andreas Kirschning* - andreas.kirschning@oci.uni-hannover.de;
Gerald Dräger* - gerald.draeger@oci.uni-hannover.de
© 2013 Möller et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
click chemistry; growth factor; nitrenes; plasma chemistry;
poly(4-methylpent-1-ene); surface modification
Abstract
Covalent multistep coating of poly(methylpentene), the membrane material in lung ventilators, by using a copper-free “click” ap-
proach with a modified cyclic RGD peptide, leads to a highly biocompatible poly(methylpentene) surface. The resulting modified
membrane preserves the required excellent gas-flow properties while being densely seeded with lung endothelial cells.
Introduction
Respiratory failures are a significant health-care problem with Therefore, lung-support systems that perform the gas exchange
several hundred thousand adult patients each year [1]. Besides extracorporeal can provide an alternative. They are connected to
medical treatment, the use of mechanical ventilators that the patient via two cannulas in an arterio-venous circuit instead
provide breathing support while the lungs recover, is often of an endotracheal aditus. These devices are based on poly-
indispensable. This treatment is conducted when patients meric hollow-fiber membranes that serve as an interface
respond inadequately to medical therapy. However, invasive between blood and gas streams (Figure 1). The material must
mechanical ventilation can damage the lungs physically by allow adequate gas exchange thus providing CO2 removal and
overpressurizing lung tissue or due to inflammation. This may O2 delivery for patients with respiratory or ventilatory failure.
lead to exacerbation of lung dysfunction or even to multiple- The thermoplastic polymer poly(4-methylpent-1-ene) (TPX; 2)
organ failure [2,3]. The morbidity and mortality associated with complies with this requirement. It is currently used as a poly-
these problems still remains high.
meric hollow-fiber membrane material in lung-support systems
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Beilstein J. Org. Chem. 2013, 9, 270–277.
Figure 1: Schematic presentation of the artificial lung (A) and concept of membrane functionalization (B) with cyclic RGD 1b.
due to its low density of 0.835 g/cm3 and therefore its high gas bioactive cRGD domain [15]. As the final coupling strategy, we
permeability, which allows an unhindered gas flow [4]. chose the Huisgen-type azide–alkyne-“click”-chemistry for
However, the artificial polymer surface leads to the formation which connecting elements 5 and 6 were coupled to polymer
of blood clots, which prevent long-term gas exchange and derivative 4. Classically, copper catalysts are required for these
enforce the replacement of the device. This intervention is asso- kinds of 1,3-dipolar cycloadditions, but their potential cyto-
ciated with undesired risks for the patient, such as bleeding and toxic properties limit the usability in biomedical applications
infections, as well as increased costs for the treatment.
[16,17]. For overcoming this problem, several copper-free liga-
tion methods were developed. In this work we pursue both
Previously, experiments showed that the negative side effects of options, i.e., the copper-mediated as well as the copper-free
the artificial surface can be significantly reduced by seeding version [18].
endothelial cells (ECs) onto a heparin/albumin-coated TPX
Results and Discussion
surface [5]. Although these endothelialized membranes showed
improved hemocompatibility, the cells were easily detached Plasma-activation and dip coating of TXP
from the membrane due to the water-soluble protein coating, We found that chemical modification of the rather inert TPX
which is necessary for the cell attachment.
membrane 2 was only possible in polar solvents because of the
hydrophilic nature of the pegylated precursor 3. Unfortunately,
In this paper, we disclose a strategy to strongly attach ECs to dip coating of TPX 2 with a methanolic solution of nitrene
TPX 2 membranes by covalent functionalization of the chemi- precursor 3, and then drying followed by UV irradiation (Hg
cally rather inert material with a cyclic peptide, containing the lamp, 254 nm) initially did not lead to surface modification. The
RGD (arginine-glycine-asparagine) amino-acid sequence. We presence of F and N on the TPX membrane was not detectable
chose cRGD pentapeptide 1b derived from lysine precursor 1a, by XPS analysis. The photoinduced reaction of azide 3 with
as cRGD’s intensively studied by the Kessler group [6-9] are cyclohexane served as a model reaction and provided the
well-established cell-recognition motifs that can trigger inte- expected tetrafluoro-substituted arylcyclohexyl amine in 47%
grin-mediated cell adhesion [9]. Because of the high potential to yield, proving the viability of the functionalization concept (see
stimulate this, these RGD peptides have successfully been used Supporting Information File 1, section 2D). We assumed that
to generate biocompatible materials [10,11].
the hydrophobic nature of TPX 2 inhibited the bedabbling of the
polymer with linker 3. Therefore, TPX 2 was pretreated with
Conceptually, we planned to first functionalize the hydro- different types of plasma under conditions that resulted in lower
carbon TPX 2 via UV-mediated nitrene insertion to form contact angles (see below). We chose atmospheric-pressure
polymer derivative 4 (Scheme 1) [12-14]. Therefore, nitrene plasma, which simplifies the procedure because the plasma
precursor 3 was modified with an oligo(ethylene glycol) linker, chamber does not need to be evacuated [19]. Importantly, the
which served as a spacer unit between the polymer and the plasma had to be optimized (with respect to the gas composi-
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Scheme 1: Functionalization of poly(4-methylpent-1-ene) (TPX) 2.
tion, time of treatment, distance of the plasma to the polymer insertion of the nitrene moiety. Irradiation with UV light at
surface, and electronic parameters; see Supporting Information 312 nm clearly showed absorption generated from the π-system
File 1) in order not to damage the membrane by creating holes of the fluorinated phenyl ring (Figure 2). In contrast to this,
or altering the shape of the material. This optimization finally nonfunctionalized foils did not show any absorption.
resulted in the use of a low-energy forming-gas plasma
(10% H2). The use of plasma for altering the surface properties X-ray photoelectron spectroscopy (XPS) analysis of functional-
of polymers is well established. It can be expected that only the ized TPX membrane 4 was used to determine the elements on
surface is partially oxidized without affecting the integrity of the polymer surface (Figure 3a). The strongest peak with a
the material [20-22].
Next, the TPX membrane 2 (see below; bars are given in cm)
was treated with a solution of azide 3 as described above and
UV-irradiation gave covalently functionalized polymer 4. For
practicability reasons, flat TPX membranes instead of hollow
fibers were used.
Physicochemical analysis of modified TPX
foils
Analysis of all materials, including starting material TPX 2, was
achieved with different methods described in the following.
Contact-angle measurements showed an increase of about 60%
compared to the plasma-treated material, which is a diagnostic
for the coverage of the surface with linker 3. The IR spectrum
of 4 displayed carbonyl vibrations at 1640 cm−1. The IR vibra-
tion for the azido moiety at a wavelength of 2120 cm−1 was
absent, indicating that no adsorbed linker molecules were
Figure 2: Analyses of functionalized TPX membranes with UV light at
312 nm; left: derivative 4; right: nonfunctionalized material as negative
control (scale bar equals 1 cm).
present on the TPX surface (see Supporting Information File 1,
Figure S1). Additionally, UV served to prove successful CH
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Beilstein J. Org. Chem. 2013, 9, 270–277.
Figure 3: Analyses of functionalized TPX 4 by XPS spectroscopy using hν = 1250 eV; (a) overview, (b) F 1s core level, (c) N 1s core level.
kinetic energy of 967 eV was assigned to the 1s core level of according to Brea et al. [15] Membrane 7a, synthesized from
the carbon polymer backbone. In addition to carbon, oxygen polymer 4 and amino acid 5, was coupled with RGD peptide
(O 1s at Ekin.= 720 eV) could be monitored on the surface, 1b, bearing an azido group, by the use of Huisgen-type “click”
which can be ascribed to the oxidative pretreatment with the chemistry (Scheme 2). cRGD pentapeptide 1b was prepared in
forming-gas plasma. Fluorine and nitrogen were also detected sufficient amounts by solution-phase chemistry [23]. Because of
on TPX derivative 4 (Figure 3b and 3c) by XPS measurements. the disadvantage associated with copper-mediated 1,3-cycload-
The fluorine peak (F 1s at Ekin.= 562 eV) was undoubtedly ditions of alkynes with azides in biological or biomedical appli-
related to the inserted linker because unmodified TPX cations we alternatively pursued a copper-free approach that
membranes did not show any absorption at 562 eV. Nitrogen relied on the oxanorbornadiene strategy of Rutjes [24-28]. This
(N 1s at Ekin.= 846 eV) was already present on the surface after type of specific conjugation most likely proceeds by a 1,3-
plasma pretreatment but its percentage significantly increased dipolar cycloaddition/retro-Diels–Alder cascade. By incubating
after the insertion process. Importantly, gas permeability tests oxanorbornadiene functionalized membranes 7b with cRGD
showed that the chemical modification of TPX did not alter its pentapeptide 1b the cycloaddition product 8a was formed in the
excellent gas-flow properties (see Supporting Information absence of any additives (Scheme 2). Prior to chemical reac-
File 1, section 8). These analyses clearly demonstrate that tions carried out with modified TPX materials, all reactions
plasma treatment prior to the reaction with nitrene from were first probed in solution (see Supporting Information File 1,
precursor 3 provides the hydrophilization of the polymer, which section 2D).
is a prerequisite for successful dip-coat functionalization.
In order to analyze the outcome of 1,3-dipolar cycloadditions,
With these results on functionalized TPX 4 in hand we the model compound fluoresceinyl azide 9[29,30] was first
continued the synthesis towards RGD functionalized TPX. coupled to TPX derivatives 7a or 7b, under identical conditions
Therefore, simple washing protocols for facile workup were as described for cRGD peptide 1b. The resulting polymers 8c
applied because all reactions were performed on the polymer and 8d were studied by determining the UV absorption peaks of
surface. Thus, TPX derivative 4 was either coupled with alkyne fluorescein between 400 and 600 nm (Figure 4). Absorption
5 or with oxa-norbornadiene 6, by using classical coupling maxima of fluorescein were located at 457 nm and 481 nm,
chemistry, which resulted in polymers 7a and 7b, respectively which can be ascribed to the presence of two isomeric forms
(Scheme 1). The alkyne-containing amino acid 5 was prepared (lacton versus carboxylate) of fluorescein [31]. These measure-
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Beilstein J. Org. Chem. 2013, 9, 270–277.
Scheme 2: Copper-catalyzed and copper-free azide–alkyne “click“ reaction between functionalized TPX membranes 7a or 7b and cRGD peptide 1b
or with fluorescein R5N3 9 for analytic purposes.
about 515 nm (dotted line versus dashed line) compared to
starting TPX 7a.
In contrast, the copper-free method led to intense fluorescein
staining (polymer 8d, continuous line) of the TPX membrane
with an increased factor for the absorption intensity of about 65
compared to 8c. This remarkable result may be rationalized if
one assumes that copper is not ideally distributed during the
course of the 1,3-dipolar cycloaddition reaction, as the pres-
ence of the heterogeneous TPX foils hampers continuous stir-
ring. When functionalized polymers 8c and 8d were washed by
soxhlet treatment (six hours in methanol) no change of the color
of the polymer membranes or the extracts was encountered. It
must be noted that neither TPX 2 nor functionalized analogues
4, 7a or 7b have an absorption maximum between 400 and
Figure 4: UV spectra of TPX 8c and 8d functionalized with fluorescein
600 nm. In addition, plasma-treated unfunctionalized TPX
membrane 2, lacking an alkyne functionality, was treated with
azide 9 in order to determine the degree of physisorption of
fluorescein. Washing of the membrane, as routinely done in this
revealing the efficiency of the 1,3-dipolar cycloaddition reactions on
modified TPX membranes. Continuous line: 8d; dotted line: 8c; dashed
line: substrate for Huisgen-type “click” reaction 7a.
ments clearly revealed the successful covalent functionalization study, provided a material that did not reveal absorption
of TPX with fluorescein by 1,3-dipolar cycloaddition. The maxima between 440–600 nm. These results demonstrate that
copper-catalyzed method provided material 8c, which showed a the 1,3-dipolar cycloaddition protocols gave covalently linked
small but significant absorption maximum for fluorescein at fluorescein–TPX membrane adducts.
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Beilstein J. Org. Chem. 2013, 9, 270–277.
Endothelialization of TPX foils
with an aqueous solution of EDTA in order to remove all
Based on these promising results, we next tested the biological copper traces adsorbed on the polymer. In contrast, TPX
properties of the new TPX materials 8a and 8b functionalized membranes modified under copper-free “click” conditions were
with cRGD. Particularly, we investigated the growth of homogenously covered with endothelial cells (Figure 5g). This
endothelial cells (ECs) through integrin-mediated binding. ECs difference in surface coverage of ECs can be rationalized either
were seeded at a density of 1 × 104 cells/cm2 on TPX by (a) either the toxicity of the remaining copper traces or (b)
membranes 8a and 8b [5]. Adherence, growth and viability of by the lower amount of surface-bound cRGD peptide on the
cells were monitored and quantified by calcein staining and TPX membrane 8a (see Figure 4). It is worth noting that the
fluorescence imaging (see Supporting Information File 1, experiments were repeated eight times and revealed high repro-
section 2B).
ducibility of biological response for a given mode of chemical
modification.
Unmodified TPX membranes were used as negative control,
while albumin/heparin coated TPX, which are currently used in
clinical applications, were applied to monitor cell viability.
After 48 h cultivation the formation of a confluent endothelial
cell monolayer was observed on both the cRGD as well as
albumin/heparin-modified membranes. In contrast, no adherent
cells could be found on the unmodified membranes 2. To verify
that the observed effect is in fact only RGD-mediated,
membranes obtained during every functionalization step en
route to RGD-modified polymers 8a and 8b were also analyzed.
We found that seeding of cells onto the TPX membranes that
were treated with forming-gas plasma gave cell coverage
comparable to the control with heparin/albumin. This effect is
associated with increased hydrophilicity resulting from the
oxidative plasma conditioning on the surface [32]. The plasma-
mediated surface oxidation yields polar groups including
hydroxy groups, which rapidly vanish, a well-investigated aging
effect of plasma-treated materials. This process is an entropy-
initiated surface reorientation and it hampers direct chemical
modification of these oxidized surfaces [32]. Also our TPX
membrane 2 revealed this aging effect, as judged by changes of
the contact angles. Plasma treatment led to a lower angle (from
107°± 1° to 46° ± 7°), which increased to 70° ± 5° after one
week and further to 75° + 2° within two months, making
plasma-treated TPX 2 not suited for biomedical applications.
Once the TPX-membrane was functionalized with the PEG unit,
a significant reduction of the number of adhered cells was en-
countered (Figure 5c). This effect was even more pronounced
on TPX membranes 7a and 7b, where the ECs were either
exposed to the alkyne or the oxanorbornadiene-functionalized
surfaces, respectively (Figure 5d and 5f). This observation
correlates well with previous reports showing that cells do not
Figure 5: Cell seeding onto TPX derivatives (scale bar equals 500 µm)
(a) 2 as control with heparin/albumin dip coating; (b) 2 treated with
forming-gas plasma; (c) functionalized TPX 4 with PEG; (d) functional-
ized TPX 7a with PEG and alkyne group; (e) functionalized TPX 8a
with cRGD (copper-catalyzed approach); (f) functionalized TPX 7b with
PEG and oxanorbonadiene group; (g) functionalized TPX 8b with
cRGD (copper-free approach).
attach on PEGylated polymers as long as no additional growth Finally, we also repeated the whole synthetic sequence, but this
factors are present [33]. Finally, TPX surfaces 8a and 8b func- time using a PEG unit with a molar-mass distribution of
tionalized with cRGD behaved differently regarding their cell- 3000 g/mol instead of the defined PEG linker 3. This modified
adhesion properties (Figure 5e and 5g). These tests strongly material should reveal the impact of linker length on the effec-
indicate that copper-catalyzed attachment of cRGD pentapep- tivity to grow ECs on TPX membranes. We found that this new
tides only provides a minor increase of biocompatibility, despite TPX membrane 8a showed reduced biological potency to bind
the fact that the functionalized TPX 8a was excessively treated ECs. This observation is in accordance with studies by Beer et
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Beilstein J. Org. Chem. 2013, 9, 270–277.
al. who showed that virtually all RGD binding sites can be (Heilbronn) for providing the TPX membrane employed in arti-
reached by integrins, when the distance between the surface and ficial lungs. The plasma equipment was kindly provided by Dr.
the RGD peptide amounts to 46 Å. Access by integrins again A. Knospe and C. Buske from Plasmatreat GmbH (Steinhagen).
decreases when the spacer is longer and likely folds in such a XPS measurements were carried out by PD Dr. C. Tegenkamp,
way that RGDs become less surface-exposed [34].
T. Langer and Prof. H. Pfnür (Institut für Festkörperphysik,
Leibniz Universität Hannover).
Conclusion
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Finally, cRGD-functionalized TPX membrane surfaces showed
excellent biocompatibility regarding the adhesion of endothe-
lial cells. These studies pave the way for the development of
improved, extracorporeal oxygenators.
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