Rgd-functionalization of Poly(2-oxazoline)-based Networks for Enhanced Adhesion to Cancer Cells

Poly(2-oxazoline) networks with varying swelling degrees and varying hydrophilicity can be synthesized from 2-ethyl-2-oxazoline, 2-nonyl-2-oxazoline, 2-9'-decenyl-2-oxazoline and 2,2'-tetramethylene-bis-2-oxazoline in one-pot/one-step strategies. These gels can be loaded with organic molecules, such as fluorescein isothiocyanate, either during the polymerization (covalent attachment of the dye) or according to post-synthetic swelling/deswelling strategies (physical inclusion of the dye). Surface functionalization of ground gels by thiol-ene reactions with cysteine-bearing peptides exhibiting the arginine-glycine-aspartic acid (RGD) motif yields microparticles with enhanced recognition of human cancer cells compared to healthy endothelial cells.


Introduction
Biocompatible and, in particular, biodegradable hydrogels [1][2][3] are in use in a broad range of medical and medicinal applications, such as tissue engineering [4] and drug delivery [5].Drugs can be released from hydrogels by diffusion, degradation of the hydrogels or combined strategies [6].Crosslinking of poly(2-oxazoline)s can be realized either in situ (during the copolymerizations themselves) or after the copolymerizations (so-called polymeranalogous strategies) [7].Poly(2-oxazoline)-based hydrogels are interesting candidates for various biomedical applications [2,8].Like other synthetic or natural polymers, poly(2-oxazoline)s have been functionalized with peptide structures in order to enhance cell adhesion, concomitant with increased biocompatibility and cell-specific drug delivery characteristics.However, reports on that specific topic are rather scarce [9].The functionalization of polymers with peptide units of arginine-glycine-aspartic acid (RGD) has been under extensive research in recent years [10][11][12][13][14], and combinations of polymer-based materials with constrained pentacyclic peptides, like c(RGDfC) [15,16], have been reported [10,17].The mechanism of RGD motif recognition and subsequent cell attachment is based on the presence of integrins on cell surfaces.In particular, the α v β 5 -integrin and its α v β 3 congener have been reported to show strong interaction with the cyclic RGD motif [18,19].Due to the known overexpression, as well as the high activity of integrins in tumor cells [20,21], research started to focus on the preparation of RGD-functionalized hydrogels for enhanced cancer imaging and for the development of targeted strategies for cancer treatment [9,22].Nonetheless, the investigation of these phenomena is still in its infancy.
Instrumentation.Microwave-assisted hydrogel syntheses were performed in sealed vials in the single-mode microwave reactor, Biotage Initiator 8.All reactions were carried out in temperature-controlled modes; the reaction temperature was monitored by a non-invasive IR pyrometer.Removal of excess solvent during the determination of swelling degrees was performed in a Chromabond solid-phase extractor (Macherey Nagel, Dueren, Germany).The hydrogels were ground in a Retsch S1000 planetary ball mill as aqueous suspensions (100 mg/15 mL) for 24 h.Particle sizes were determined with a CILAS 1180 Particle Size Analyzer (CILAS, Orleans, France).Thiol-ene photoreactions were performed with Hg/Xe lamps of the EFOS Novacure (EFOS, Mississauga, Canada) from EXFO equipped with a light guide for the transfer of the UV light in close vicinity to the suspensions to be illuminated (8-cm distance).Transmission light and green fluorescence microscopy (ex/em ~495 nm/~510 nm) were monitored with a fluorescence microscope from Zeiss (Axioskop-2; Jena, Germany), digitalized with a Kappa DX-4 camera (Kappa, Gleichen, Germany) and stored and analyzed with Kappa camera control software (version 1.4.0.8053).Scanning electron microscopy energy dispersive X-ray (SEM-EDX) measurements were performed using a Tescan Vega 3 scanning electron microscope with an energy dispersive X-ray spectrometer (EDX Oxford Instruments INKAx-act, Abington, United Kingdom) attached.Electron energy levels were set to 20 kV.
Hydrogel Syntheses and Purifications.Hydrogels were synthesized under microwave irradiation from EtOx, NonOx, Dc = Ox and TMBO with MeOTs as the initiator using anhydrous dichloromethane as the solvent.The ratios of monofunctional 2-oxazolines were chosen from = 100:0:50:30:1 was mixed from 1.33 g (13.5 mmol, 100 eq) EtOx, 1.41 g (6.7 mmol, 50 eq) Dc = Ox, 0.792 g (4 mmol, 30 eq) TMBO and 0.025 g (0.13 mmol, 1 eq) MeOTs.The mixtures were placed in microwave vials under argon flow, which were sealed with a septum.Microwave irradiation was applied for 1 h at 140 °C.The yellowish products were subjected to several swelling-recovery-drying cycles in dichloromethane for purification.The yields of the purified gels upon a weight constant were equal to or higher than 95%.
Swelling Degrees.Samples of 0.3 g of each of the purified and carefully dried hydrogels were swollen in excess amounts of deionized water, ethanol or dichloromethane, respectively.The swelling degrees were determined gravimetrically after 24 h in duplicate (referred to as SD1 and SD2), preceded by the removal of excess solvent.The swelling degrees were calculated as the relative differences of masses according to (m swollen − m dry )/m dry (Tables 1-5 and Figure 1).Dye Inclusion.(i) In Situ Inclusion: FITC was added in 0.2 wt% amounts to the corresponding reaction mixture, yielding 2 g of hydrogel of the composition pEtOx 100 -pDc = Ox 50 -pTMBO 30 -FITC 0.1 ; (ii) Post-Synthetic Strategy: Hydrogels of the composition pEtOx 100 -pDc = Ox 50 -pTMBO 6 (0.5 g) were swollen in a suspension of 50 mg FITC in dichloromethane/ethanol (1.5:0.5 mL) for 24 h.After recovery from the clear solutions, the hydrogels were dried and washed multiple times with water.According to gravimetric analysis, the dye was absorbed completely, yielding a hydrogel of the composition pEtOx 100 -pDc = Ox 50 -pTMBO 6 (FITC) 2.8 .
Thiol-ene Photoreactions.Fifty milligrams of ground hydrogel of the composition pEtOx 100 -pDc = Ox 50 -pTMBO 30 -FITC 0.1 were suspended in 7.5 mL of water and mixed with 2.0 mg of c(RGDfC) and 10 µL of photoinitiator Lucirin TPO-L prior to polychromatic irradiation with 4500 mW•cm −2 at a distance of 8 cm to the light source (light guide) and irradiated for 45 min.After completion of the illumination, the suspension was decanted from the insoluble photoinitiator residues/decomposition products and used for the cell tests.
Cell Tests.For microscopic evaluation, cells were seeded on glass cover slips (approximately 20.000/cover slip).After 24 h of adherence, cells were incubated with 2 mL of a hydrogel-containing medium (1:100 dilution of hydrogel stock solutions with 6.7 mg hydrogel/mL) for 3 or 24 h, respectively.Subsequently, the cover slips were gently rinsed in phosphate-buffered saline (PBS) in order to remove non-adhered hydrogel particles.Cover slips were mounted on glass slides and evaluated by transmission light and green fluorescence microscopy.
Comparably low degrees of crosslinking, such as 150:3 (50:1), yielded crosslinked copoly(2-oxazoline)s, which dissolved in dichloromethane during purification.Hence, this study was limited to networks with the lowest crosslinking degrees of 150:6.Concomitantly, a network library of 5 × 5 = 25 members was obtained from one-pot/one-step strategies analogous to a previously developed recipe [30].The gels were recovered as pre-swollen networks of a yellowish color, dried under reduced pressure and subjected to repeated swelling-recovery-drying cycles in dichloromethane, until a weight constant of the dried gels was achieved.Yields of the colorless to yellowish purified hydrogels were 95% or higher.
Due to its functional groups, FITC can act as a terminating agent in cationic ring-opening polymerizations (Scheme 1): The addition of small amounts of FITC to the initial reaction mixture yielded networks with covalently bound FITC, which was validated on the example of the network, pEtOx 100 -pDc = Ox 50 -pTMBO 30 -FITC 0.1 .Correspondingly functionalized hydrogels could be ground in a ball mill in order to yield microparticles (see the section "Milling Experiments").

Swelling Degrees and Polymeranalogous Compound Loading
The swelling degrees were determined in water, ethanol and dichloromethane as test solvents after 24 h (Figure 1).Only an insignificant further increase of the swelling degrees was observed after that period; swelling degrees were determined in duplicate.The gels that were composed exclusively of EtOx as a monofunctional monomer showed fairly comparable swelling in all three test solvents: in the pEtOx 150 -pTMBO x series, swelling degrees only depended on the degree of crosslinking with maximum swelling degrees of eight for crosslinking degrees of 150:6.Due to their comparable swelling degrees in water, ethanol and dichloromethane, these gels may be referred to as amphigels.The networks composed of EtOx and Dc = Ox, as well as those composed of EtOx and NonOx, as well as Dc = Ox exhibited hardly any swelling in water, reasonable swelling in ethanol and high swelling in dichloromethane: due to the reasonable swelling in ethanol, the networks of the compositions pEtOx 100 -pDc = Ox 50 -pTMBO x and pEtOx 50 -pNonOx 50 -pDc = Ox 50 -pTMBO x still may be referred to as amphigels.Their swelling degrees were quasi-constant in water (one or lower) and increased with decreasing crosslinking degrees in the other two test solvents to maximum values of approximately five in ethanol and approximately 13 in dichloromethane.
Networks that did not contain any pEtOx swelled only in dichloromethane and can most adequately be described as lipogels.Maximum swelling degrees in the series pNonOx 100 -pDc = Ox 50 -pTMBO x and pNonOx 150 -pTMBO x were in the range of 20.Notably, insoluble networks in the pNonOx 150 -pTMBO x series were only obtained for crosslinking degrees up to 150:7.5.
In addition to in situ functionalization (Section 3.1.),the poly(2-oxazoline)-based gels could also be modified post-synthetically in order to subsequently act as loaded drug carriers: networks with comparably high swelling in dichloromethane and low or no swelling in water, for example, could be impregnated post-synthetically with organic molecules during swelling in dichloromethane (diffusion-mediated inclusion of the organic molecules into the networks).After recovery and drying of the networks, the organic molecules were not released from dried non-degraded hydrogels in aqueous environments.The loading of hydrogels of the composition pEtOx 100 -pDc = Ox 50 -pTMBO 6 with FITC as the model compound was accomplished from dichloromethane:ethanol = 3:1 v/v suspensions of the fluorescent dye.Within 24 h, the suspension cleared and no solid residues of FITC could be detected.According to gravimetric control, FITC was included in the network (quasi-)quantitatively, yielding a gel of the composition pEtOx 100 -pDc = Ox 50 -pTMBO 6 (FITC) 2.8 .Notably, this strategy paves the way for the loading of the gels with drugs at room temperature, overcoming the risk of the decomposition of the drugs at high temperatures, like those during the polymerization.

Milling Experiments
Milling experiments were performed with hydrogels of the composition pEtOx 100 -pDc = Ox 50 -pTMBO 6 (FITC) 2.8 and pEtOx 100 -pDc = Ox 50 -pTMBO 30 -FITC 0.1 .Both networks could not be successfully ground in a ball mill in the dry state, but only as aqueous suspensions (100 mg/15 mL), yielding particles with dimensions in the micrometer range (Figure 2a).The ground particles tended to re-aggregate and, upon water removal under reduced pressure, correspondingly yielded films instead of powders.Even by the application of ultrasound for several hours, these films could not be re-suspended in water to yield microparticles.Hence, the micrometer range of the microparticle size distribution was only stable in aqueous media for a limited time.Even though the tendency of the particles to re-aggregate in aqueous media was lowered, the application of ultrasound was required for the re-establishment of the microparticle size distribution after storage in the range of hours or longer.Currently on-going investigations aim at the addition of (biocompatible) tensides to the mixture during grinding.The first experiments with sodium dodecyl sulfate (SDS), which has been approved as an excipient in mixtures of hydrogel/SDS/water = 100 mg/100 mg/15 mL, have revealed a shift of microparticle size distribution to the lower micrometer range (Figure 2b), while the storage stability, however, was not in the day range yet, and the suspensions still needed to be ultrasonicated prior to application.Nonetheless, the proof-of-concept that smaller particles need surfactants for stabilization could be established.

Surface Functionalization
The UV-induced crosslinking of poly(2-oxazoline)-based copolymers with oligovalent thiols and the functionalization of decenyl side-chains of poly(2-oxazoline)s by thiol-ene reactions have been described in the literature [31][32][33].In this study, the concept was transferred to the functionalization of ground gel particles in suspension by thiol-ene reactions, benefiting from the fact that they are insensitive to water [39].The cyclic pentapeptide, c(RGDfC), was chosen, because of its enhanced cancer cell recognition, due to the RGD motif geometrically constrained in the pentacycles bearing a bulky D-amino acid (D-phenylalanine in this case) [15,18,19] and the presence of the thiol-bearing L-cysteine for thiol-ene reactions.After UV-irradiating a mixture of the peptide and the freshly ground, FITC-functionalized hydrogel, the suspension was used for cell tests (see below).In order to prove that the RGD functionalization was successful and that it had occurred preferentially at the microgel surface, a cross-section was cut with a razor blade from particles with diameters in the 150 µm range and subjected to SEM-EDX measurements that revealed the elemental mapping of carbon, oxygen and sulfur throughout the inner and the outer domains (Figure 3).While carbon and oxygen atoms constituting the polymer network were evenly distributed on the surface of the microgel particle, as well as its interior, sulfur atoms were predominantly detected on the surface of the particle.This finding strongly suggests that the sulfur-containing thiol groups are attached to the microgel surface and supports the claim of the surface functionalization with the RGD motif.

Cell Tests
The adhesion of freshly ultrasonicated suspensions of ground microgels (pEtOx 100 -pDc = Ox 50 -pTMBO 30 -FITC 0.1 ) with or without RGD-surface functionalization were tested against endothelial EA.hy cells and human pancreatic cancer BON cells [38][39][40][41].BON cells were chosen, because of their pronounced surface expression of highly active α v β 5 -integrins, according to fluorescence-activated cell sorting measurements [42].The α v β 5 -integrin, like its α v β 3 congener, has been reported to show an interaction of supreme strength with the RGD motif in peptide pentacycles [18,19].In order to investigate the adherence of microparticles to these types of cells, the cells were seeded on glass cover slips and incubated with the hydrogel-containing media.Subsequently, the cover slips were gently rinsed in PBS to remove non-adhered hydrogel particles.Samples were then evaluated by transmission light and green fluorescence microscopy.
Due to the covalent attachment of FITC, fluorescence microscopy enabled the clear recognition of the microgel particles (Figure 4).In all experiments, only adhesion, but no cellular uptake, of the hydrogels was observed.In n = 5 independent experiments, the time-dependent adherence of RGD-functionalized vs. RGD-free hydrogels to BON cells after 24 h was analyzed for n ≥ 350 cells/type of hydrogel, revealing an approximately 4.3-fold higher adhesion of RGD-bearing hydrogels compared to the RGD-free ones.Only in RGD-functionalized hydrogels were multiple attachments of several hydrogel-particles to BON cells observed, while RDG-free hydrogels showed only single and random attachments to individual BON cells.Additionally, control experiments were performed with non-transformed low α v β 5 -expressing endothelial EA.hy cells: significant adhesion of the particles to the cells could not be observed, neither as a function of time nor as a function of the RGD-functionalization (Figure 4, top).These observations supported the enhanced attachment of RGD-functionalized gels to α v β 5 -expressing tumor cells.The adhesion of larger particles to the cells is likely to be caused by mechanical interlocking and is not indicative of adhesion due to specific microgel-cell interactions.

Conclusions and Outlook
Aiming at the development of novel drug delivery systems for chemotherapeutics, poly(2-oxazoline)-based hydrogels were surface-functionalized with a pentacyclic RGD motif for integrin recognition at cancer cell surfaces.The swelling degrees in water, ethanol and dichloromethane of the copoly(2-oxazoline)-based networks composed of aliphatic 2-oxazolines can be adjusted by the variation of the monomers, namely EtOx, NonOx and Dc = Ox.This allows for one-pot/one-step syntheses of amphi-and lipo-gels that could be loaded with compounds in situ or post-synthetically.The incorporation of Dc = Ox into the networks enabled the surface functionalization of (ground) gels by photo-initiated thiol-ene reactions with the cyclic peptide, c(RGDfC).
Microparticles bearing covalently surface-bound RGD peptide sequences were found to adhere to BON cancer cells to significant degrees, while hardly any adherence to non-cancerous human endothelial cells could be observed.This preferential adhesion makes these microgels potential candidates for the delivery of otherwise non-selective chemotherapeutics to cancer cells.Notably, multiple adhesions to adjacent cancer cells could be observed for RGD-functionalized particles.
Current investigations aim at optimizing the grinding procedure, in particular, by stabilizing the particle size distribution in a suspension (additives for the stabilization of the particle size distribution), and the introduction of stimuli-responsive chemical linkers for the attachment of chemotherapeutics.While poly(2-oxazoline)s are stable towards hydrolysis under physiological conditions [43], acid-labile linkers are one key strategy for the introduction of stimuli-responsive bonds.Hence, drug release will be triggered only at the targeted cells.

Figure 3 .
Figure 3. Scanning electron microscopy image of the cross-section of a poly(2-oxazoline)-based gel after grinding and subsequent surface functionalization by (a) UV-induced thiol-ene reactions; (b) as well as the SEM-EDX images of the cross-section showing the distribution of carbon atoms; (c) oxygen atoms; and (d) sulfur atoms, respectively.The center plain visible in the images was the interior of the particle prior to cross-sectioning, while all other plains and edges represent the surface.

Figure 4 .
Figure 4. Superimposed transmission and fluorescence photographs of EA.hy endothelial (top) and BON cancer cells (bottom) that were incubated with pEtOx 100 -pDc = Ox 50 -pTMBO 30 -FITC 0.1 hydrogels for 3 h (left) and 24 h (right), respectively.Significant adhesion of the hydrogels to cells was only observed for the combination RGD-functionalized particles/BON cancer cells.

Table 1 .
The swelling degrees of pEtOx 150 -pTMBO x networks in water, ethanol, and dichloromethane.The swelling degrees according to (m swollen − m dry )/m dry were determined in duplicate [SD1 and SD2, respectively; the average is represented by the arithmetic mean value (MV) and the standard deviation, σ].

Table 2 .
The swelling degrees of pEtOx 100 -pDc = Ox 50 -pTMBO x networks in water, ethanol and dichloromethane.The swelling degrees according to (m swollen − m dry )/m dry were determined in duplicate [SD1 and SD2, respectively; the average is represented by the arithmetic mean value (MV) and the standard deviation, σ].

Table 3 .
The swelling degrees of pEtOx 50 -pNonOx 50 -pDc = Ox 50 -pTMBO x networks in water, ethanol and dichloromethane.The swelling degrees according to (m swollen − m dry )/m dry were determined in duplicate [SD1 and SD2, respectively; the average is represented by the arithmetic mean value (MV) and the standard deviation, σ].

Table 4 .
The swelling degrees of pNonOx 100 -pDc = Ox 50 -pTMBO x networks in water, ethanol and dichloromethane.The swelling degrees according to (m swollen − m dry )/m dry were determined in duplicate [SD1 and SD2, respectively; the average is represented by the arithmetic mean value (MV) and the standard deviation, σ].

Table 5 .
The swelling degrees of pNonOx 150 -pTMBO x networks in water, ethanol and dichloromethane.The swelling degrees according to (m swollen -m dry )/m dry were determined in duplicate [SD1 and SD2, respectively; the average is represented by the arithmetic mean value (MV) and the standard deviation, σ].