Elsevier

Applied Surface Science

Volume 625, 15 July 2023, 157061
Applied Surface Science

Full Length Article
Electron beam irradiation as a straightforward way to produce tailorable non-biofouling poly(2-methyl-2-oxazoline) hydrogel layers on different substrates

https://doi.org/10.1016/j.apsusc.2023.157061Get rights and content

Highlights

  • First report of employing the electron beam irradiation for preparation of poly(2-oxazoline)-based hydrogel coatings.

  • Applied method is independent on surface material (Si wafer, PS plate).

  • Grafting and crosslinking are driven by amount of double bonds essential for the formation of covalent bonds.

  • Presence of the hydrogel layer decreases cell adhesion to a negligible value.

  • The stiffness increases with the degree of crosslinking which denotes the possibility of adjusting the required stiffness by proper selection of the experimental parameters.

Abstract

Uncontrolled accumulation of proteins and cells on implantable materials often leads to failure of their performance in vivo. The idea presented in this paper is the use of electron beam irradiation as a widely applicable, cost-effective, and defined method to produce non-biofouling hydrogel coatings to improve the biocompatibility and in vivo performance of implantable materials. Statistical copolymers poly[2-methyl-2-oxazoline-co-2-(3-butenyl)-2-oxazoline]s were deposited on different substrates and irradiated with beta radiation of different radiation doses (2–100 kGy). In the bulk state experiments, we found that the higher content of crosslinkable 3-butenyl units and a higher radiation dose resulted in more efficient crosslinking. Similarly, the irradiation of coatings demonstrated the high impact of the concentration of 3-butenyl units on crosslinking efficiency. Accordingly, the concentration of crosslinkable double bonds in the copolymer is crucial for the stability and homogeneity of the formed hydrogel layer. Stable and uniform hydrogel layers with thicknesses in the micrometer range were prepared from a 5 wt% copolymer solution. Depending on the preparation conditions, the hydrogel layers showed excellent non-biofouling properties with a low number of adherent cells. In addition, stiffness was dependent on the degree of crosslinking, and can thus be tailored for specific application in living tissue.

Introduction

Poly(2-alkyl-2-oxazolines) (POx), also classified as “polypseudopeptides” are currently intensively studied as promising synthetic polymers for different biomedical applications such as drug delivery [1], [2], gene therapy [3], non-biofouling protective coatings [4], tissue engineering [5], or other therapeutic applications [6], [7]. POx are prepared by living cationic ring-opening polymerization (LCROP), which enables to synthesize polymers with defined molar masses, different polymer architectures, and narrow dispersities [8]. Moreover, various functional and reactive motifs can be introduced as end groups or side chains using functional initiators, terminating agents, or monomers [9], [10], [11]. Hydrophilic/hydrophobic properties can be readily adjusted by using different aliphatic or aromatic substituents in position 2 of 2–oxazoline ring [12]. From the large family of POx, poly(2-methyl-2-oxazoline) (PMeOx) and poly(2–ethyl-2-oxazoline) (PEtOx) represent highly hydrophilic polymers with remarkable cytocompatibility and low immunogenicity [13], [14]. LCROP as a highly controllable polymerization process allows the preparation of POx-based polymeric materials with complex architecture such as block [15], [16], [17], graft [18], [19], gradient copolymers [20], [21], star-shaped polymers [22], [23] as well as hydrogels [24], [25].

Hydrogels can adopt properties similar to living tissues and extracellular matrix; therefore, they are considered an important tool in biomedical applications [26], [27], e.g., controlled delivery of bioactive molecules [28], [29], [30], for the encapsulation of living cells [31], or as implantable materials for regenerative medicine [32], [33]. Various synthetic routes for the preparation of covalently crosslinked hydrogels from 2-oxazolines have been described in the literature [34]. The simplest method is one-pot copolymerization of monofunctional 2-oxazolines with bis(2-oxazoline) as a crosslinker resulting in the formation of a crosslinked network [25], [35]. Christova et al. prepared segmented networks from POx macromonomers and acrylates [36]. Hydrogels with controlled swelling and mechanical properties have been prepared by POx-containing double bonds in the side chain crosslinked via the photoinduced thiol-ene “click” reaction of dithiols with different structure [37], [38], [39]. Such POx-based hydrogels exhibit in vitro [39], [40] as well as in vivo [41] biocompatibility, which along with adjustable stiffness and bio-inertness provide high bio-applicable potential [42], [43], [44].

In addition to the self-standing hydrogels, hydrogel layers attached to different substrates can represent soft, hydrated, biocompatible and protective coatings applicable for different implantable devices. We have prepared the poly(carboxybetaine) hydrogel coatings covalently attached to polyimide substrates as well as crosslinked via a photolabile 4-azidophenyl group, incorporated into the poly(carboxybetaine) chain. Both attachment and crosslinking processes were driven by C–H insertion of highly reactive nitrene groups formed by photodegradation of aromatic azide groups upon UV irradiation [45]. In another study, the fabrication of crosslinked POx-based layers possessing non-biofouling properties was performed by the UV crosslinking of PMeOx macromonomers composed of meth(acrylate) end groups [46]. Another synthetic approach leading to a POx-based hydrogel layer was conducted in the recent work of Rühe’s research group. In their study, PEtOx-bearing photoactive benzophenone moieties in the side chains were employed for the simultaneous grafting and crosslinking. Upon UV irradiation, formed benzophenone diradicals were able to react with any C–H bonds in the vicinity [47]. Due to the relatively low number of papers devoted to the preparation of POx-based hydrogel layers, there are still many possibilities to design a new, simpler, more accessible and effective approach applicable commercially on a large scale.

We have conducted several studies on the radiation stability of various biocompatible polymers including poly(2-alkyl-2-oxazolines) towards electron beam (i.e., β) and γ irradiation in dry bulk state and in aqueous solutions [48], [49]. We found that i) irradiation generally leads to crosslinking, leading to insoluble crosslinked materials in an aqueous milieu, forming hydrogels in high yield; ii) the irradiation in an aqueous milieu is significantly more efficient than in a dry state and iii) the β irradiation is significantly more efficient than γ irradiation at the same radiation dose delivered. Relevant to POx, the sensitivity to irradiation is dependent on the monomeric unit, PEtOx readily undergoes crosslinking to hydrogels in the aqueous milieu (for homopolymer crosslinking in a dry state was much less efficient), while poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline) is extremely radio-resistant in both dry and wet (aqueous solution) states.

These promising results led us to the idea to employ electron beam irradiation as a widely useable low-cost defined way to coat biomedical devices with a tailorable non-biofouling poly(2–alkyl–2–oxazoline) hydrogel layer for biocompatibilization. Our approach is based on the electron beam irradiation of poly[(2-methyl-2-oxazoline)–co-(2-(3-butenyl)-2-oxazoline)]-based (PMeOx-co-EnOx) coatings on the selected substrates in a dry state. The reason for using a small content of 2–(3-butenyl)-2-oxazoline monomeric unit is to enhance crosslinking efficacy, which is otherwise insufficient for pure PMeOx in a dry state (and with irradiation in solution, drying/flowing would lead to irreproducible results due to technical and handling reasons) [48]. High-energy electron beam irradiation (in our case from microtron source, which is cheap, readily available, fast, reproducible and can be easily scaled-up) offers several advantages over published methods of LCROP or UV–irradiation crosslinking of hydrogel layers [25], [35], [36], [37], [39]: I) Polymer precursors can be stored as a long shelf-life kit for custom coating of various surfaces and can be exposed to light, unlike the UV-cured polymers containing unstable (meth)acrylate or azidobenzene moieties. There is also no need for the addition of crosslinking agents (e.g., dithiols), there are no side products, and crosslinking does not require anhydrous conditions (typically essential for the LCROP of 2-oxazolines). II) The procedure is very fast, including the solution coating, drying, irradiation (taking tens of minutes depending on the dose required), and washing. Properties of the coating are tailorable in a wide range. III) For UV-irradiation, curing surfaces that are not flat or even porous is very tricky as the dose of irradiation is inherently spatially inhomogeneous. For betas, the radiation dose is delivered homogeneously (the energy decrease per path for electrons is linear except at the very end nearing the full stop – the Bragg peak) up to low depth, not dependently on the UV-transparency of the matrix. The maximum penetration depth of electrons ranges from ca 4 mm/MeV energy for low density materials (organic polymers, water, tissue), through ca 2 mm/MeV energy for intermediate density materials (glass, aluminium), to ca 0.5 mm/MeV for heavy metals such as lead, assuring the homogeneous irradiation of thicker objects with the herein-employed 10 MeV beam (the highest energy not causing photonuclear activation, i.e. just below the threshold of the (β,n) reaction), where penetration into the above-mentioned materials is several cm [50]. In addition, the penetration of electrons in gases is sufficient (meters to tens of meters depending on initial energy) not to cause technological obstacles for irradiation. IV) Deep-volume sterilization not dependent on UV-transparency and with no need of heating is assured with the radiation doses considered (as typical sterilization doses are within this applied range).

Section snippets

Materials

4-Pentenoyl chloride, methyl 4-nitrobenzenesulfonate, 2-chloroethylamine hydrochloride, Rhodamine123, and calcium hydride were purchased from Sigma-Aldrich (Steinheim, Germany) and used as received. Potassium hydroxide was purchased from Microchem (Slovakia) and used as received. 2-Methyl-2-oxazoline (MeOx) was purchased from Sigma-Aldrich (Steinheim, Germany), dried over KOH for 48 h, distilled over CaH2 under reduced pressure, and stored under argon. Acetonitrile (J.T. Baker, Netherlands,

Polymer preparation and characterization

In this study, we employ electron beam radiation as an easy, fast and low-cost method for the preparation of POx-based hydrogel layers covalently attached to selected substrates and crosslinked in one step. In our previous experiment, poly(2-methyl-2-oxazoline) (PMeOx) was found to be a polymer resistible to beta irradiation (data not published). Therefore, introducing a double bond to the structure of PMeOx should enable crosslinking of PMeOx–based polymers and control over crosslinking

Conclusion

Here we present an easily performable and inexpensive method for coating various surfaces with non-biofouling poly(2-methyl-2-oxazoline)-based (POx) hydrogel films using beta radiation crosslinking. First, the ability of beta radiation to yield self-standing hydrogels from POx copolymer containing 2-(3-butenyl)-2-oxazoline (EnOx) unit with a double bond in the side chain was demonstrated. The higher content of EnOx comonomer as well as the higher radiation dose led to more efficient radiation

CRediT authorship contribution statement

Petra Šrámková: Writing – original draft, Methodology, Investigation, Writing – review & editing, Funding acquisition. Jan Kučka: Investigation, Methodology, Writing – review & editing. Zuzana Kroneková: Methodology, Writing – review & editing. Volodymyr Lobaz: Methodology, Writing – review & editing. Miroslav Šlouf: Methodology, Writing – review & editing. Matej Mičušík: Methodology, Investigation. Josef Šepitka: Methodology, Writing – review & editing. Angela Kleinová: Investigation. Dušan

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

J. Kučka and M.Hrubý acknowledge financial support from the Czech Science Foundation (grant # 21-01090S) and from the Ministry of Education, Youth and Sports of the Czech Republic (National Sustainability Program NPU I, project POLYMAT # LO1507 and grant # EATRIS-CZ LM2023053 ERIC). This work was supported by the Czech Academy of Sciences within the Strategy AV21 framework (VP26-Breakthrough future technologies) and by the Ministry of Education, Youth and Sports of the Czech Republic

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