Influence of side-chain length on long-term release kinetics from poly(2-oxazoline)-drug conjugate networks

European Polymer Journal 120 (2019) 109217

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Influence of side-chain length on long-term release kinetics from poly(2oxazoline)-drug conjugate networks

T

Jong-Ryul Parka, Joachim F.R. Van Guyseb, Annelore Podevynb, Eleonore C.L. Bollea, ⁎ ⁎ Nathalie Bocka, Erik Lindec, Mathew Celinac, Richard Hoogenboomb, , Tim R. Dargavillea, a

Institute of Health and Biomedical Innovation, Science and Engineering Faculty, Queensland University of Technology, Queensland 4001, Australia Supramolecular Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4, B-9000 Ghent, Belgium c Sandia National Laboratories, Albuquerque, NM 87185-1411, USA b

ARTICLE INFO

ABSTRACT

Keywords: Benazepril Drug conjugation Drug delivery Hydrogel Poly(2-oxazoline)

Four drug-conjugated poly(2-alkyl-2-oxazoline) (PAOx) networks with different hydrophobicity were synthesized via copolymerization of either 2-methyl-, 2-ethyl-, 2-propyl- or 2-butyl-2-oxazoline with the functional monomer, 2-dec-9-enyl-2-oxazoline. The incorporation of a labile ester linkage between the polymer and the drug benazepril allowed for sustained drug release over periods of months with the release rates strongly depending on the hydrophobicity of the polymer pendant groups. Drug loading of 13 ± 2 wt% was used with 10 mol% crosslinking sites simply by tuning the thiol-ene stoichiometry. The networks exhibited negligible cell toxicity but cell repulsion was observed for hydrogels based on poly(2-methyl-2-oxazoline) and poly(2-ethyl-2oxazoline) while those based on poly(2-n-propyl-2-oxazoline) and poly(2-n-butyl-2-oxaoline) showed cell adhesion. These results suggest that PAOx networks have great potential as drug delivery devices for long-lasting drug release applications.

1. Introduction The conjugation of drugs to polymers through biodegradable linkers is a proven approach in drug delivery systems. The advantages over traditional, physically entrapped polymer-based drug delivery systems include improved solubility and metabolic stability, and some control over the tissue distribution, for example by enhanced accumulation in tumors [1–3]. Release kinetics may also be controlled from hours to months by appropriate design of the linkers between the polymer and the drug [4]. So far, many different polymeric carriers have been developed for polymer-drug conjugates, including the well-studied poly (ethylene glycol) (PEG) and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) [5]. However, PEG has limitations such as a limited number of functional groups for the attachment of drug [6], and possible immunogenicity [7–9]. PHPMA also has drawbacks such as challenging synthesis routes and functionalization strategies to yield polymer-drug conjugates [10–12]. Poly(2-alkyl-2-oxazoline)s (PAOx) are a class of synthetic polymers with pseudo-peptide structure, increasingly considered as alternatives to PEG, and to a lesser extent PHPMA, due to potential biocompatibility and versatility of its structure [13–17]. This great structural versatility ⁎

stems from (1) the cationic ring-opening polymerization, which allows excellent control of the chain termini through choice of initiator/terminator, and (2) the wide range of available monomers and postpolymerization modification strategies [13,18,19], introducing control over functionality and hydrophilicity of the polymer side-chains. This versatility has been used to incorporate drugs, imaging agents, and crosslinking points [20–22]. Two recent studies on PAOx-drug conjugates were reported based on the conjugation of the anti-cancer drug doxorubicin using a hydrazide linkage (drug content: 5.3–6.2 wt%) for intravenous administration [23], and conjugation of the Parkinson’s disease drug rotigotine via an ester linkage, named SER-214 (from the company Serina Therapeutics), for sub-cutaneous administration [24,25]. These studies showed that administered drugs could be bound to PAOx macromolecules via cleavable linkers resulting in sustained or pH-controlled drug release profiles. However, both these examples are designed for short term drug delivery and premature clearance of the polymer-drug conjugate prior to release of the drug from the PAOx backbone is possible. One approach for extending drug release and reducing early clearance is to use drug conjugated-networks as drug eluting depots implanted in the body (e.g. subcutaneously) [26–29]. This strategy can

Corresponding authors. E-mail addresses: [email protected] (R. Hoogenboom), [email protected] (T.R. Dargaville).

https://doi.org/10.1016/j.eurpolymj.2019.109217 Received 5 July 2019; Received in revised form 26 August 2019; Accepted 28 August 2019 Available online 29 August 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.

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address the challenges in long-lasting drug delivery (over 1–2 months with a single device) as the insoluble depot should be resistant to premature clearance. PAOx hydrogels have previously been used to physically entrap and release drugs, but as expected the release kinetics are typified by an initial burst release and complete release within a day [30–33]. The strategy of covalently conjugating drugs via cleavable linkers to PAOx networks ranging in hydrophilicity to target sustained release without burst release characteristics, to the best of our knowledge, has yet to be explored. The drug chosen in this work is an angiotensin-converting enzyme (ACE) inhibitor, benazepril (BNZ). It is an attractive candidate for these drug conjugated networks as it has a low therapeutic dose (meaning that the incorporation of 1–2 month supply into a single device may be feasible), it is relatively inexpensive, and is usually administered on a regular and recurring basis for long-term management of high blood pressure [34–36]. Importantly, it also contains a carboxylic acid group handle suitable for conjugation. Here, we report the effects of hydrophilicity/hydrophobicity of the PAOx networks on the drug release profiles. A homologous series of four different PAOx networks composed of poly(2-methyl-2-oxazoline) (PMeOx), poly(2-ethyl-oxazoline) (PEtOx), poly(2-n-propyl-2-oxazoline) (PPropOx), and poly(2-n-butyl-2-oxazoline) (PButOx) (hydrophobicity increases as the increase of carbon number) with poly(2-(dec9-enyl)-2-oxazoline) (PDecenOx) were synthesized and characterized. BNZ was modified with a thiol group via an ester linkage and subsequently conjugated to the polymers by thiol-ene photo-conjugation. These drug-conjugated precursors were used to prepare PAOx networks by the addition of a dithiol crosslinker followed by thiol-ene photocrosslinking. The release of the drug was monitored using chromatography and the results were compared to the same system except with the drug physically incorporated. The results demonstrated that the versatility of the PAOx networks offers potential for application as drug delivery system.

Fig. 1. Ion mass spectrum (m/z of BNZ-SH: 484.20, scan rage 200–1000 m/z) in positive ionization mode (top) and the 1H NMR spectra (in CDCl3) of benazepril and 2-mercaptoethyl benazepril (bottom).

2. Materials and methods

2.2. Mass spectrometry

Two commercially available monomers, 2-methyl-2-oxazoline (MeOx) (ChemicalPoint, Germany) and 2-ethyl-2-oxazoline (EtOx) (a gift from Polymer Chemistry Innovations, USA), were distilled over barium oxide (BaO) before use. The two monomers, 2-n-propyl-2-oxazoline (PropOx) and 2-n-butyl-2-oxazoline (ButOx), were synthesized by the Witte-Seeliger method [37], while DecenOx was synthesized by the Modified Wenker method [38]. Methyl p-toluenesulfonate (MeOTs) was chosen as the initiator and used after distillation. Acetonitrile (ACN) was purified by using a solvent purification system (J. C. Meyer). Water was purified using a Sartorius arium®pro system. Benazepril hydrochloride (≥98%) was obtained from TCI chemicals. DL-Dithiothreitol (DTT) (≥98%) and 2-mercaptoethanol (≥99%) were obtained from Sigma Aldrich. Irgacure 2959 (I2959; 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) was a gift from BASF.

Mass spectrometry was recorded using gas phase ion-molecule experiments performed on a linear quadrupole ion trap mass spectrometer (LTQ XL, Thermo Fisher Scientific, USA) with a heated electrospray ionization (HESI) source. The sample in methanol was prepared at the concentration of 5 µM and eluted into the source at the rate of 30 µL min−1. Mass analysis was set to scan 200–1000 mass rage in positive ionization mode. 2.3. Size-exclusion chromatography (SEC) Size-exclusion chromatography (SEC) was measured on an Agilent 1260-series HPLC system equipped with an inline degasser, a diode array detector, a refractive index detector and temperature controller set to 50 °C equipped with two PLgel 5 µm mixed D columns and a precolumn in series. The mobile phase was N,N-dimethylacetamide (DMAc) with 50 mM of lithium chloride (LiCl) at a flow rate of 0.5 mL/ min. The average molecular weights and molar mass distribution (Ð) were calculated against poly(methyl methacrylate) (PMMA) standards from PSS (Polymer Standards Service).

2.1. Nuclear magnetic resonance (NMR) and Fourier-transform infrared (FT-IR) spectroscopy 1D 1H NMR spectra were recorded using an Avance 600 MHz Bruker spectrometer using CDCl3 as a solvent. Diffusion-ordered spectroscopy (DOSY) experiments based on 1H NMR spectroscopy were acquired at 299 K on a 400 MHz Bruker UltraShield spectrometer with a Quattro Nucleus Probe (QNP). Polymer samples were prepared in CDCl3 and measurements were performed with gradient intensity linearly sampled from 2% at 0.96 G cm−1 to 95% at 45.7 G cm−1. The number of gradient steps was set to be 32. The NMR spectra were processed with Topspin and Dynamic centre. FT-IR were acquired on a Bruker Alpha-P FT-IR fitted with an ATR accessory.

2.4. Gas chromatography GC was performed on an Agilent Technologies 7890A system equipped with a VWR Carrier-160 hydrogen generator and an Agilent Technologies HP-5 column of 30 m length and 0.32 mm diameter. An FID detector was used and the inlet was set to 250 °C with a split injection ratio of 25:1. Hydrogen was used as carrier gas at a flow rate of 2

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Scheme 1. Synthesis of PMeOx-, PEtOx-, PPropOx- and PButOx-DecenOx copolymers and the preparation of benazepril conjugated PAOx networks via incorporation of BNZ-SH and DTT. Table 1 Properties of the synthesized copolymers of PMeOx, PEtOx, PPropOx or PButOx with DecenOx. Name

M1

M2

M2/[M1 + M2]a

Mn,theob (kDa)

Mn,SECc (kDa)

Ða

Recovered mass (%)

PMeOx-DecenOx PEtOx-DecenOx PPropOx-DecenOx PButOx-DecenOx

MeOx EtOx PropOx ButOx

DecenOx DecenOx DecenOx DecenOx

14.6 13.7 14.2 14.3

20.65 22.85 25.35 27.79

25.80 28.90 23.80 8.80

1.59 1.13 1.25 1.22

89 79 57 66

a b c

Obtained from the 1H NMR spectra. The theoretical molecular weight (Mn) for 100% conversion. Number-average molecular weight (Mn) and polydispersity index (Ð) by SEC.

2 mL min−1. The oven temperature was increased with 20 °C min−1 from 50 °C to 120 °C, followed by a heating ramp of 50 °C min−1 from 120 °C to 300 °C.

Hydrogels were snap frozen in liquid nitrogen and lyophilized then mounted onto aluminium stubs using adhesive carbon tabs and subsequently coated in 5 nm platinum (Pt) using a Leica ACE EM600 Pt coater. Images were aquired on a Zeiss Field Emission SEM (Carl Zeiss Microscopy, Germany) at an accelerating voltage of 3–5 kV.

Chromatography (HPLC) (Agilent 1100 Series) equipped with InertSustain® 5µ C18, 250 × 4.6 mm, 5 µm particle size HPLC column. The mobile phase was 40% acetonitrile in water with 0.1% trifluoroacetic acid (TFA; ≥99%) with a flow of 1 mL/min and an injection volume of 100 µL. Released BNZ was detected at 224 nm. The cumulative data for the observed drug release yields were fitted with a MATLAB based 3D FEM diffusion model using Fickian behavior with a single diffusivity. The geometry for the disc-shaped specimens was 1 mm height and a diameter of 3 mm. Similar diffusion models have recently been applied to water desorption from thermoset materials [39].

2.6. Swelling ratio

3. Synthetic procedures

Network swelling ratios were measured using a gravimetric method. The networks were washed 3× with ethanol and immersed in DI water for 24 h to remove impurities. Then the networks were dried under reduced pressure at room temperature until constant weights were obtained. The dried networks were immersed in fresh DI water and the weights of swollen networks were measured periodically after the excess water on the surface was removed using tissue paper. The swelling ratio is defined as:

3.1. Polymerizations

2.5. Scanning electron microscopy (SEM)

Swelling ratio =

Ws

After the successful synthesis of the three monomers (nPropOx, nButOx, and DecenOx) shown in Fig. S1, all polymerizations were carried out by cationic ring opening polymerization (CROP). The polymerization mixtures containing [M1]:[DecenOx]:MeOTs = 170:30:1 (with M1 = MeOx, EtOx, nPropOx or nButOx) and a monomer concentration of 4 M in acetonitrile were prepared in a dry nitrogen atmosphere in a glovebox with a water and oxygen concentration ≤0.5 ppm. These polymerization mixtures were reacted at 70 °C for 2–3 days until a conversion of ± 98% was reached. After cooling the mixtures to ambient temperature the polymerizations were terminated with dry piperidine (1.5 eq. relative to MeOTs) and stirred overnight. The polymers were precipitated in cold diethyl ether and dried in a vacuum oven at 50 °C for 2 days. 1H NMR (500 MHz, CDCl3) (Fig. S2): PMeOx-DecenOx: δ = 5.73 (m, 31H, CHCH2), 4.89 (m, 62H, CHCH2), 3.73–3.12 (m, 800H, NCH2CH2), 2.31 (m, 60H, CHCH]CH2), 2.14–1.90 (s, m, 570H, CH3 and (C]O)CH2), 1.5 (m, 60H, CH2), 1.37–1.10 (m, 300H, CH2). PEtOx-DecenOx: 5.78 (m, 30H, CHCH2), 4.93 (m, 60H, CHCH2), 3.77–3.21 (m, 800H, NCH2CH2), 2.62–2.12 (m, 400H, (C]O)CH2), 2.08–1.20 (m, 420H, CH2), 1.10 (m, 510H, CH3). PPropOx-DecenOx: 5.78 (m, 30H, CHCH2), 4.93 (m, 60H, CHCH2), 3.81–3.12 (m, 800H, NCH2CH2), 2.51 (m, 60H, CHCH]CH2), 2.42–2.08 (m, 400H, (C]O)CH2), 1.80–1.11 (m, 700H, CH2), 0.92 (m, 510H, CH3) PButOx-DecenOx: 5.72 (m, 30H,

Wd Wd

where Ws = weight of the networks after swelling in H2O and Wd = weight of dry networks. 2.7. Drug release PAOx networks with physically entrapped BNZ or covalently linked BNZ were incubated in phosphate-buffered saline (PBS, pH 7.4), whereas conjugated BNZ networks were incubated in PBS as well as in sodium hydroxide solution (NaOH, pH 10) at the concentration of 1 g/L at 37 °C. For all release studies 1 mL of buffer was taken periodically and the solution was refilled with 1 mL of fresh buffer. The amount of released drug was quantified by High Performance Liquid 3

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3.2. Synthesis of 2-mercaptoethyl benazepril (BNZ-SH) Thionyl chloride (0.3 mL, 1.42 mmol, 2 eq.) was added dropwise to a 2 mL flask containing benazepril (300 mg, 0.71 mmol, 1.0 eq.) in 1 mL of chloroform at 0 °C and was, then, allowed to warm to ambient temperature overnight. The resulting product, benazepril acid chloride, was isolated by removing residual thionyl chloride and solvent under vacuum, and dissolved in 1 mL chloroform. The solution containing benazepril acid chloride was added dropwise to a 5 mL flask containing 2-mercaptoethanol (222 mg, 2.84 mmol, 4 eq.) in 1 mL of chloroform. After stirring overnight under argon atmosphere, 30 mL of chloroform was added and the product was extracted with water (2 × 30 mL) and saturated sodium bicarbonate solution (30 mL). The organic phase was dried over magnesium sulfate (MgSO4) and the solvent was evaporated under reduced pressure. The product, 2-mercaptoethyl benazepril (BNZ-SH), was obtained to give 67% yield after purification by column chromatography (ethyl acetate: dichloromethane = 5:5) 1H NMR (600 MHz, CDCl3) (Fig. 1): 7.5–6.9 (m, 9H, CH), 4.62–4.26 (d, 4H, CH2(C]O)O), 4.02 (m, 2H, CH2CH2SH), 3.30 (m, 1H, CH2), 3.13 (m, 2H, CH2SH), 2.65 (m, 4H, CH2), 2.45 (m, 3H CH2), 1.39 (m, 1H, SH), 1.06 (m, 3H, CH3); ESI-MS = m/z 483.2 (MH+). 3.3. Drug conjugation The PAOx copolymers (10 mg, 1 eq.) and BNZ-SH (0.30 eq. relative to the alkene of the PAOx) were dissolved in ethanol (85 µL), followed by addition of I2959 (5 µL of a 2 w/v % in ethanol) to achieve 10 wt% polymer solutions. The solutions were irradiated with UV light (either 1.7 W/cm2 365 nm (Omnicure S1000) or 370–380 nm using 3 × 3 W LEDs (Avonec)) at room temperature for 480 s (Scheme 1). The obtained poly(2-oxazoline)-benazepril (PAOx-BNZ) conjugates were used for the next step without further purification. The reactivity of BNZ-SH towards PAOx was studied by a colorimetric thiol-quantification assay using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Ellman’s reagent; Thermo Scientific). Therefore, 50 µL polymer solutions were prepared in a similar manner as the experiments described above and 3 µL samples of these mixtures were taken at different UV irradiation times followed by addition of 30 µL Ellman’s buffer (10 mM DTNB in DMSO) and incubation for 10 min. The sample solutions were diluted with 2 mL dilution buffer consisting of 100 mM Tris·HCl and measured by UV/Vis spectrophotometry.

Fig. 2. Representative DOSY NMR spectra for (a) a mixture of PMeOx and BNZSH and (b) the PMeOx-drug conjugate. Vertical axis represents the diffusion coefficient and horizontal axis indicates chemical shifts.

3.4. PAOx networks Dithiothreitol (DTT) (0.5 eq. to alkene moiety of the PAOx-BNZ conjugates or PAOx-DecenOx copolymers) was added to a solution containing 10 mg PAOx-BNZ conjugate and 5 µL I2959 (2 w/v %) in 85 µL ethanol. The solutions (100 µL) were pipetted onto glass slides pre-treated with Sigmacote (Sigma Aldrich) and compressed with coverslips suspended on 1 mm spacers to form discs, followed by UV light irradiation (365 nm) for 600 s. The PAOx networks were washed with ethanol and distilled water three times and dried under reduced pressure overnight. 3.5. Cell toxicity/adhesion Human primary fibroblasts were isolated according to previously published protocols [40–42] and cultured in Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10% foetal calf serum, 50 U/ mL penicillin, 50 µg/mL of streptomycin and 2 mM L-glutamine (all Invitrogen), up to passage 5. Ethical approval was obtained from the Queensland University of Technology Research Ethics Committee (1300000063) and Uniting Healthcare/St Andrew's Hospital Ethics Committee (0346). The PAOx networks were sterilized in 70% ethanol for 3 h and washed in PBS (3×) and immersed in DMEM overnight. The number of cells seeded onto the samples was 3 × 104 in 10 µL of culture

Fig. 3. Consumption of BNZ-SH with the four copolymers as determined by Ellman’s assay. The stock solution containing 10 wt% polymer BNZ-SH and 0.1% (w/v) I2959 in ethanol were irradiated with 365 nm light source and samples taken periodically. Mean ± standard error (n = 3).

CHCH2), 4.88 (m, 60H, CHCH2), 3.70–3.10 (m, 800H, NCH2CH2), 2.43–2.06 (m, 400H, (C]O)CH2), 1.96 (m, 60H, CHCH = CH2), 1.68–1.05 (m, 1060H, CH2), 0.85 (m, 510H, CH3).

4

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Fig. 4. The normalized RI SEC traces of PAOx-BNZ conjugates with 10 wt% polymer in ethanol with BNZ-SH (0.3 eq. of alkene groups) and 0.1% I2959 before UV irradiation and after 6, 12, and 18 min. (a) PMeOx-DecenOx (b) PEtOx-DecenOx (c) PPropOx-DecenOx, and (d) PButOx-DecenOx. Eluent = DMAc.

revealing peaks at 3.4 ppm characteristic for the polymer backbone methylene groups as well as the typical signals for the terminal double bond of the DecenOx at 5.7 ppm and 4.9 ppm (Fig. S2). All copolymers showed similar values of the alkene content, namely 14.1 ± 0.5 mol%, which is close to the targeted degree of modification. The SEC data (Table 1) showed that each copolymer, with the exception to PButOxDecenOx, had a molar mass (Mn) in good agreement with the monomerto-initiator feed ratios ([M]/[I] = 200), despite the use of a relative PMMA calibration. The molar mass of the PButOx-DecenOx measured by SEC was less than half that expected from the feed ratios due to poor solvation of the hydrophobic polymer by DMAc, resulting in an underestimation of the molar mass. Similar observations have been reported for other hydrophobic PAOx, for example poly(2-nonyl-2-oxazoline) [45]. It is noted that the PMeOx-DecenOx copolymer exhibited relatively broad elution peaks (Ð = 1.59), which is commonly observed when targeting higher degrees of polymerization with the MeOx monomer, which can be attributed to more extensive chain transfer reactions [43]. The other copolymers showed narrower peaks in SEC indicating a lower dispersity, Ð ≤ 1.25.

Table 2 Benazepril content in PAOx networks. Networks

BNZ content (wt%)

PMeOx-DecenOx PEtOx-DecenOx PPropOx-DecenOx PButOx-DecenOx

15.7 14.5 13.4 12.4

medium. Following an incubation period of 1 h to allow for cell attachment, 1 mL of fibroblast growth medium was added to each well. Following cell culture for 3 days, samples were washed in PBS supplemented with calcium and magnesium and fixed in 4% paraformaldehyde (PFA) overnight. Cells were then permeabilized with 0.2% Triton X for 5 min at room temperature, blocked in 1% bovine serum albumin for 30 min and fluorescently labelled by placing them in a working solution containing blocking solution and 0.8 U/mL TRITC–conjugated phalloidin (phalloidin tetramethylrhodamine B isothiocyanate, Sigma Aldrich, 200 U/mL) and 5 µg/mL 4′6-diamidino–2–phenylindole (DAPI, Life Technologies, USA) for 30 min.

4.2. Benazepril conjugation

4. Results and discussion

To conjugate the ACE inhibitor benazepril to PAOx, the drug was functionalized via esterification with 2-mercaptoethanol to incorporate a thiol functional group. The synthetic route to 2-mercaptoethyl benazepril (BNZ-SH) started with conversion of the carboxylic acid group of benazepril to an acid chloride with thionyl chloride followed by reaction with mercaptoethanol (Scheme S1). Ion mass analysis (Fig. 1 top) showed the correct molar mass of BNZ-SH (m/z = 483.2 + H) and the successful synthesis was further confirmed by 1H NMR spectroscopy (Fig. 1 bottom), which revealed the absence of the acid group at 9.2 ppm and 10 ppm and presence of the thiol proton (1.4 ppm, ‘c’) and the adjacent methylene groups at 4.0 ppm and 3.1 ppm (‘a’ and ‘b’). The benazepril conjugation to the PAOx copolymers was carried out with

4.1. Polymerizations A series of four PAOx copolymers based on MeOx, EtOx, nPropOx or nButOx (M1) with DecenOx were synthesized with a monomer to initiator ratio of [M1]:[DecenOx]:[MeOTs] ratio of 170:30:1 by cationic ring-opening polymerization (CROP) to high conversions (> 98% by GC) and terminated with piperidine (Scheme 1). Conventional heating at relatively low temperature (70 °C) was used for the polymerizations instead of the standard microwave heating method [43] as it allows for easier scale-up and can lead to fewer side-reactions for PAOx [44]. The resulting polymers were characterized by 1H NMR spectroscopy 5

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Fig. 5. SEM images of the lyophilized PAOx hydrogels.

Fig. 7. Release of benazepril physically loaded into hydrogels (data points) in PBS (pH 7.4) at 37 °C, fitted with a Fickian single diffusivity 3D FEM diffusion model (solid lines).

of the 1H NMR spectroscopic peaks attributed to the alkene group at 4.9 ppm and 5.7 ppm. Further confirmation of the benazepril conjugation was obtained from FT-IR showing the presence of the ester linking group at 1730 cm−1 (Fig. S3) and a DOSY experiment. For the DOSY experiment the diffusion coefficients of the signals corresponding to BNZ-SH and the PAOx backbone were equal (Fig. 2b and Fig. S4), which was not the case for a mixture of free PAOx and free BNZ-SH (Fig. 2a) evidencing the success of the thiol-ene photo-conjugation reaction. In addition, a colorimetric thiol-quantification assay showed no difference in the rates or conversion (> 90% in all cases) of photo-conjugation between the four copolymers (Fig. 3). The photoconjugation of BNZ-SH to the different PAOx copolymers was very fast as it was nearly completed within three minutes. Conjugation of the drug to the copolymer should result in an overall increase in molar mass. Size exclusion chromatography (SEC; Fig. 4) does indeed show an increase in molar mass with photo-conjugation time although the magnitude of the increase as reflected by the SEC did

Fig. 6. Swelling ratio of (a) PAOx networks without drug and (b) PAOx-BNZ conjugated networks at 37 °C, expressed as means with standard error (n = 3).

10 wt% polymer solution, BNZ-SH (0.3 equivalents relative to DecenOx units), and I2959 in ethanol by irradiating with 365 nm light. The drug conjugation by thiol-ene photo-conjugation was inferred by a decrease 6

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suggests there is some phase separation in these cases. For the most hydrophobic copolymer gel synthesized from ButOx monomer there is additional topographical undulations on the scale of 10–100 μm perhaps due to a volume change during gel synthesis. The apparent lack of interconnectivity of the pores and small size does not lead to additional water uptake of the hydrophobic networks. Instead, the relationship between swelling of the networks in water and polymer structure correlated well with the hydrophilicity of the side-chain (Fig. 6a). Here, the PMeOx and PEtOx copolymer networks can be considered hydrogels, but the more hydrophobic PPropOx and PButOx copolymer networks are essentially glassy resins. Conjugation of the drug leads to less swollen networks (Fig. 6b) due to the increased hydrophobicity upon incorporating of BNZ [46,47]. The swelling ratio should influence the drug release rate in two ways. Firstly high swelling will result in high diffusion rates [48], and secondly, higher water content will result in increased rates of hydrolysis of the linking ester group (and vice versa in both cases for lower water content). 4.4. In vitro drug release In vitro drug release studies were initially carried out from networks with the BNZ physically entrapped to study the influence of network swelling as mentioned above as well as the drug-polymer interactions. As expected the release kinetics (Fig. 7) mirrored the swelling ratios of the networks (Fig. 6). The rate of release from the most hydrophilic network, PMeOx-DecenOx, was similar to that observed previously for PAOx networks physically loaded with drugs [30–32], but the more hydrophobic networks had more sustained release that was has been observed previously for this class of polymers. Interestingly, not all of the added material appears to be accessible for delivery as apparent from the lower than expected final release level (on the order of 50 µg/g instead of the added 100 µg/g). A possible reason is some loss of drug due to adherence to the glass slides during curing or to plasticware during the drug release measurements. Nonetheless, the cumulative drug release data agreed reasonably well with typical Fickian diffusion based on the good agreement with the 3D FEM diffusion model for single diffusivity (bold lines Fig. 7). The PPropOx-DecenOx network showed more scatter (the two data points close to the 1 day mark) but timing and the leveling-off release quantity still followed overall Fickian diffusion behavior for the relevant geometry. PPropOx is known to have thermoresponsive properties close to the temperature used here [49] and we can tentatively state that there may be possible hydrophobic interactions between the drug and n-propyl side chain accounting for the increased scatter. Conjugating the BNZ to the polymer dramatically changed the release kinetics compared to the physically entrapped BNZ. At pH 7.4 very little drug was released from the two BNZ-conjugated hydrophilic networks (PMeOx-DecenOx and PEtOx-DecenOx) and almost no drug was released from the two hydrophobic BNZ-conjugated networks (PPropOx-DecenOx and PButOx-DecenOx) (Fig. 8a) even after 35 days due to the stability of the ester at this pH. At pH 10 (Fig. 8b) the drug release was more rapid than at pH 7.4 as the ester linkage between drug and polymer is more prone to cleavage in a basic environment, thereby facilitating the faster drug release [50]. The rate of drug release at pH 10 showed pronounced differences depending on the pendant group. PMeOx-DecenOx released the drug the fastest (≈70 mg BNZ per 1 g PAOx network per month, ≈50% total accumulated release), with the release from the other networks in order of swelling degree (i.e. PEtOx-DecenOx > PPropOx-DecenOx > PButOx-DecenOx). Diffusivity could in theory be determined for a drug delivery system where the drug generation first depends on hydrolytic cleavage and hence a ‘scission based’ release from the matrix material. However, in this case a 3D FEM model will have to be based on a competitive reactive diffusion model linking chemistry (drug generation rate) with the

Fig. 8. Release profiles from PAOx-BNZ conjugated networks expressed as drug amount per gram of gel in: (a) PBS (pH 7.4) and (b) NaOH (pH 10), at 37 °C.

vary depending on the copolymer. It should be noted that the LED UV source used for this part of the work was of lower intensity than that used above and hence resulted in longer irradiation times. In theory the molar mass of the copolymers should increase due to the benazepril conjugation (11–15 wt% theoretical increase) and for PEtOx-DecenOx, PPropOx-DecenOx and PButOx-DecenOx the observed molar mass increased with 14.9%, 18.2%, and 19%, respectively, matching theory quite well. However, only a 2.6% increase in molar mass was observed for PMeOx-DecenOx upon conjugation. We can speculate that PMeOxDecenOx has a higher hydrodynamic volume than the other polymers and therefore the conjugation of the drug results in a smaller percentage increase in the hydrodynamic volume, and hence a smaller change in retention time by SEC. Importantly though, there was a consistent increase in molar mass for all copolymers after conjugation. It is noted that there was no dramatic increase in high molar mass fraction, implying the absence of side-reactions when the feed amount of BNZ-SH was 30 mol% of alkene groups of PAOx. However, it was found that with a BNZ-SH feed equivalent of > 50 mol% of alkene groups there was a large increase of molar mass (data not shown), probably due to radical polymer-polymer coupling [38]. The increased loading of BNZ also led to a decrease in the polymer solubility (especially in water and ethanol) leading to difficulties in making networks. The change in solubility is not surprising considering the poor water solubility of BNZ. For this reason not more than 30 mol% of alkene groups were functionalized with BNZ resulting in networks with BNZ content between 8.9 wt% and 10.4 wt% (Table 2). 4.3. PAOx networks The synthesis of PAOx-BNZ conjugate networks was achieved by adding the dithiol, DTT, to the PAOx-BNZ conjugate and subsequent irradiation with 365 nm light using our previously optimized conditions for similar PAOx copolymers [38]. SEM of the lyophilized gels (Fig. 5) show the existence of small pores, approximately 1–2 μm, in the networks synthesized using the EtOx, PropOx and ButOx monomers. This 7

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J.-R. Park, et al.

Fig. 9. Cell attachment images of PMeOx-DecenOx, PEtOx-DecenOx (white dotted line was drawn to make a distinction between the cells surrounding the gel and the cells on the gel), cells on PPropOx-DecenOx, and PButOx-DecenOx after a 3 day culture period. Scale bar denotes 500 µm for A and B and 100 µm for C and D.

(physical or covalent). These results suggest that drug conjugated PAOx networks could be used as drug delivery devices for short or long term drug release applications, although further studies are needed to enhance the release at pH 7.4 by modification of the linker or through esterase mediated hydrolysis.

diffusion physics for yield behavior over time [51] and was not attempted in this study. Ultimately, we are interested in release of the drug in a subcutaneous environment where some enzymatic load may be present and although this is difficult to model, the use of high pH in this study acts as a proxy for possible esterases, which may be present in vivo. Alternatively, the chemical environment of the ester linkage can be modified to make it more labile at pH 7.4.

Acknowledgements T.D. is supported by the ARC Future Fellowship scheme (FT150100408). Some of the data reported in this paper were obtained at the Central Analytical Research Facility operated by the Institute for Future Environments (QUT). Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DENA0003525. Note: This paper describes objective technical results and analyses. Any subjective views or opinions that might be expressed in the paper do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

4.5. Cell attachment PAOx surfaces and hydrogels are considered to be protein and cell repellent [20,52,53], however, caution is needed as it greatly depends on the hydrophilicity of the polymer as was demonstrated by Dworak et al. who developed thermoresponsive PAOx coatings for switchable cell adhesion [54]. To determine cell attachment and toxicity of the materials studied here human dermal fibroblasts were seeded onto each network. Our previous work has shown that PMeOx-DecenOx networks are cell resistant [20] so it was no surprise that no cells adhered to PMeOx-DecenOx and PEtOx-DecenOx networks. Because there was no cell attachment, there were no cells to image. Instead the well plates with the hydrogels removed are shown in Fig. 9A and B to demonstrate that cells were viable and exhibited typical spindle-like morphology but did not adhere to the networks, which also indicates that the samples were not cytotoxic. In contrast to the hydrophilic networks, cells did attach to the PPropOx-DecenOx and PButOx-DecenOx networks and grew to confluency in the 3 day culture period (Fig. 9C and D). This result suggests strong protein adsorption and consequent cell attachment on these more hydrophobic networks.

Data availability All data generated or analyzed during this study are included in this published article (and its Supplementary information files). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.109217.

5. Conclusions

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PAOx networks based on a range of 2-oxazoline monomers were used to deliver the drug benazepril by either physical entrapment or by conjugation to the polymer backbone including a labile ester group. A wide range of release profiles could be achieved (from hours to months) simply by changing the monomer or the drug incorporation technique

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