Fatty-acid-derived ester-urethane macromonomers synthesized using bismuth and zinc catalysts

Photocurable materials that can be delivered as liquids and rapidly (within seconds) cured in situ using UV light are gaining increased interest in advanced minimally invasive procedures. The aim of this work was to synthesize and characterize fatty-acid-derived ester-urethane telechelic (methacrylate) macromonomers, suitable for photopolymerization. The commonly used dibutyltin dilaurate catalyst was replaced with bismuth neodecanoate, bismuth tris(2ethylhexanoate), and zinc (II) acetyloacetonate as less-toxic alternative catalysts. Additionally,


Introduction
Advances in diagnostic imaging, as well as surgical technique and instrumentation have synergistically enabled rapid growth of minimally invasive procedures. 1 These procedures have advantages over traditional surgical techniques, such as shortening the treatment time and reducing the risk of complications. These surgical approaches motivate further research into wide range of injectable materials 2,3 that can enable tissue repair, drug delivery, cell therapy, sensing, imaging, etc. Of particular interest are materials that can be injected as a liquid and then crosslinked in situ, 4 under different stimuli, such as light, temperature, or pH. While hydrogel based materials have been most popular, curable elastomeric materials can be well suited for specific tasks, such as heart muscle repair after infarct or hernia repair. 5,6 Towards this aim, we have previously developed injectable and photocurable ester-urethane macromonomers based on fatty acid derivatives obtained from vegetable oils. 7 Polyurethanes are already widely used in medical and pharmaceutical applications due to their favorable safety profiles and mechanical properties, as well as potential for biodegradability. 8 In the case of our injectable macromonomers, once photocured, the obtained elastomeric materials exhibit similar mechanical properties to human soft tissue (i.e. abdominal wall), along with slow enzymatic and hydrolytic degradation. 9 Overall, these materials displayed low toxicity and similar immune response to polylactic acid in a rabbit model, making them promising for minimally invasive surgical procedures. 6 However, in our previous work the ester-urethane macromonomers were synthesized with use of organotin-based catalyst, dibutyltin dilaurate (DBTDL). 7 Overall, tin-based catalysts are commonly used for the synthesis of polyesters and polyurethanes as elastomers and coatings.
They act as Lewis acids 10 and possess high catalytic activity. 11 However, tin-based compounds and organotin compounds, such as DBTDL in particular, exhibit cytotoxicity, 12 are difficult to remove from polymers, and have adverse environmental effects. 13 Therefore, their usage in medical applications is limited 14 and there is growing interest in "greener" organotin-free reaction pathways. 15 Towards this aim, alternative catalytic systems are being explored that replace tin with other metals, such as zinc, titanium, zirconium, manganese, etc. [16][17][18][19][20][21] Metal catalysts based on bismuth 22,23 and zinc 24 are of particular interest, due to their role in the human metabolism and use in pharmaceuticals, cosmetics, etc. Recently, they were tested in the synthesis of biodegradable polyesters, such as poly(ε-caprolactone) or poly(lactide). 25 Overall, the activity of zinc and bismuth catalysts was similar, but lower than that of the organotin catalyst DBTDL.
The aim of this work was to test two bismuth and one zinc-based catalysts as possible alternatives to organotin compounds for the synthesis of injectable and photocurable esterurethane macromonomers with telechelic methacrylic groups. Additionally, we also switched to a "green" solvent, ethyl acetate (EtOAc), 26 instead of dichloromethane (DCM) used in our previous study. We examined the effect of catalyst concentration on the reaction kinetics and chemical structure of the synthesized macromonomers, as well as on the physico-chemical properties and cytotoxicity of final photocured elastomeric materials.

Synthesis of telechelic ester-urethane macromonomers
The synthesis of telechelic macromonomers was performed in two steps ( Figure 1) based on our previous work, 7 but with two primary modifications: 1) the reaction temperature was increased from 35 °C to 70 °C and 2) EtOAc was used instead of dichloromethane (DCM). In the first step, 25 ml of EtOAc was introduced into a 250 ml round-bottom flask and degassed with three argon/pump cycles. Next, an appropriate amount of catalyst (2 or 4 mol% calculated relative to amount of polyester polyol) and 6.5 ml (0.052 mmol) of IPDI were added into the flask that was placed in an ice bath. At the same time, 25 g (0.013 mmol) of polyester polyol was dissolved in 25 ml of EtOAc. Next, the dissolved polyol was added dropwise into the ice-cold mixture. When the addition was completed, the flask was transferred to an oil bath and the reaction was continued at 70 °C . Progress of the reaction was monitored by tracking the ratio between FT-IR absorbance at 2262 cm -1 , which corresponds to N=C=O vibration in isocyanate groups of IPDI, and at 1526 cm -1 , which corresponds to N-H bending vibrations of the formed urethane bonds. The first step was considered completed when the ratio stabilized, typically at values between 3 and 5.
In the second step, 6 mg (0.03 mmol) of phenothiazine, a second aliquot of a catalyst (the same mol% as in first step), and 6.6 ml (0.054 mmol) of 2-hydroxyethyl methacrylate (HEMA) were introduced, while protecting the reaction from the light. After all of the isocyanate groups were converted, as determined by FT-IR by the absence of the band at 2262 cm -1 , the flask was removed from the oil bath and cooled down to room temperature. The product was then precipitated into four-fold excess of ice-cold methanol three times and any residual solvent was evaporated under reduced pressure at 50 °C. The obtained product was a transparent, highly viscous, sticky, yellowish liquid. The same procedure was repeated for all catalysts, for both 2 and 4 mol% concentrations. The series of macromonomers synthesized with different catalysts are abbreviated as follows: PrDBTDL, PrBiNDE, PrBiHex, PrZnAc.

Photocuring process
Crosslinked films were prepared according to the following procedure: photoinitiator 2% w/w (Omnirad 819) was mixed with macromonomer (~20 g) dissolved in 25 ml EtOAc. Residual solvent was evaporated under reduced pressure after a homogenous mixture was obtained.
Then, 1-mm-thick films were produced by pouring the final composition onto glass plate and spreading with a steel applicator. The composition was then irradiated with a DYMAX Bluewave LED Prime UVA (USA) light source, with a narrow spectral range and maximum intensity at a wavelength λ max of 385 nm. The intensity of the radiation was adjusted to 20 mW/cm 2 with the help of radiometer, AktiPrint (Technigraf GmbH). Photocrosslinking was carried out in air atmosphere, as well as under argon, in a glove box. The exposure time was 150 seconds for each spot (2.25 cm 2 ) and was carried out stepwise across the entire plate (10 cm x 20 cm).

Characterization methods
Fourier transform infrared spectroscopy (FTIR) was performed by using BRUKER ALPHA Platinum apparatus (Germany) at room temperature in the range of 4000-600 cm -1 , at a resolution of 2 cm -1 and using 32 scans. Liquid (viscous) macromonomers were analyzed in transmission mode, after pouring samples between NaCl plates. Spectra of films after photocrosslinking were obtained using reflection mode and the ATR snap-in with diamond crystal. Spectra were analyzed using EZ OMNIC software.
Nuclear magnetic resonance (NMR) spectra of all obtained macromonomers were recorded The mechanical tensile properties of photocured samples were assessed using an Instron 3366 (UK) testing system with a 500 N load cell, at crosshead speed of 25 mm/min. The crosshead speed was selected from the range of 10-50 mm/min, as typical for tensile testing of soft connective tissue. 27 The samples were of rectangular: 10 mm in width, 60 mm in length, and 0.5 mm thick. The following parameters were determined: tensile strength (s br ), elongation at break (e br ), and modulus of elasticity (E). The modulus was calculated at 2%-3% and 5%-6% elongation, respectively.
Cytotoxicity of materials was tested via extract tests according to ISO10993-5 28 in similar fashion to our previous works. 29,30 Briefly, strips of photocured materials (area: 3 cm 2 , thickness: 0.5 mm) were cut into smaller pieces, placed in a well of a 24-well plate, and incubated in 1 ml 3.

Progress of the reaction based on infrared spectroscopy
Proper choice of non-toxic catalysts is the key to successful synthesis of polymers for biomedical applications. Here, we assessed the potential of two bismuth derivatives (BiNDE and BiHex) and a zinc (ZnAc) compound as alternatives to the organotin catalyst, DBTDL for the synthesis of photocurable macromonomers via two-step reaction (see scheme in Figure 1).
The chemical structures of all four catalysts are presented in Figure 2. Overall, the reaction progress for all of the tested catalysts was similar. As a representative example, the FT-IR spectra used to monitor the progress of the synthesis with the use of BiHex 2 mol% are presented in Figure 3 (the data for the other catalysts are available in the SI, see  Figure 4. A summary of the reaction conditions, times, and yields are presented in Table 1.  These differences in the concentration dependence may be explained by differences in the catalytic mechanism between DBTDL and the remaining non-organotin catalysts (see Figure   5). a) b)

DBTDL catalyst acts as a Lewis acid that can interact with N=C=O groups-in this case
coming from IPDI-and increase their electrophilicity (Figure 5a). Further, DBTDL is a Lewis acid with pKa 11 and therefore this catalyst can be expected to be more active at higher concentrations, resulting in reduced reaction times. In contrast, according to literature, 11

Chemical structure of macromonomers by IR spectroscopy
The FT-IR measurements also permitted us to confirm that the chemical structures of the obtained macromonomers were consistent with the assumed reaction mechanism and with our previous work where DBTDL was used as catalytic system. 7 Overall, all of the obtained spectra were similar, indicating that the macromonomers obtained using the new catalysts had the same chemical structures. A representative FT-IR spectrum of PrBiHex_2 macromonomer, as compared to the starting material, polyester polyol Priplast-1838, is presented in Figure 6.
Spectra for the other macromonomers are available in the SI, see Figure S9 and S10. The

Chemical structure of macromonomers by NMR spectroscopy
The chemical structure of the obtained macromonomers was also confirmed by 1 H-NMR and 13 C-NMR spectroscopy. The analysis of NMR spectra also confirmed that all of the obtained macromonomers had similar structures, consistent with our expectations based on our prior work. 7 As a representative example, Figure 7 presents 1 H-and 13 C-NMR spectra of macromonomer obtained using BiHex at 2 mol% per step (PrBiHex_2). The remaining 1 H-NMR spectra for the materials synthesized with other catalysts are included in the SI (see Figure   S11 and S12).

Molecular mass and viscosity of macromonomers
The results of GPC analysis are presented in Figure 8 and Table S1 in the SI. For all of the macromonomers, an increase of molecular mass and decrease of dispersion was observed, as compared to the starting polyester polyol, Priplast 1838. For reactions conducted with 2 mol% of catalyst per step, the highest M w (~13000 g/mol) was obtained with BiHex as catalyst.
Meanwhile, for the case of reactions with 4 mol% of catalyst per step, the highest M w , also approx. 13000 g/mol, was obtained with use of ZnAc. The Ð of all obtained macromonomers was similar, approx. 1.75. Likewise, for all macromonomers, the shapes of the chromatograms are similar, with only small shifts observed. Importantly, the obtained macromonomers do not differ significantly in M w from those obtained in our previous work, 7 despite the changes in reaction conditions (catalysts and solvent).

Figure 8 GPC results for (a) 2 and (b) 4mol% of catalysts. Closed symbols correspond to Mw and open to Ð (Mw/Mn). c), d) Chromatograms of materials obtained with use of 2 and 4mol%, respectively
The results of dynamic viscosity measurements are presented in Table S1 and Figure 9. For all of the macromonomers a marked increase in viscosity was observed, as compared to Priplast, indicating successful synthesis. As expected, the data largely correlated with the M w results (see Figure S13 in SI).
For use in minimally invasive procedures, the rheological properties of an injectable materials play a key role. 35 They must be viscous enough to be locally retained, but not so much that the material cannot be properly delivered. We conclude that the high viscosity of the obtained macromonomers should ensure that they are well retained at the desired site during the duration of the photocuring process. At the same time, with viscosities <1000 Pa s, the macromonomers show injectability with the use of 16 G needle ( Figure 10 and short video in Supplementary   Information). While details of a clinical delivery device (syringe, catheter, etc.) are beyond the scope of this work, we conclude that these materials should be well-suited for minimally invasive surgical procedures that use relatively large gauges of instruments, including laparoscopic and catheter procedures, such as those that may be used for cardiac repair 36 . Figure 10 Injectability of PrBiHex_2 through a 16 G needle.

Chemical structure of photocured elastomeric networks
In order to confirm photocrosslinking functionality, all of the obtained macromonomers were photocured in air or under argon, following the addition of 2% w/w of photoinitiator (Omnirad 819). The chemical structures of cured films were confirmed by ATR FT-IR spectroscopy.
Overall, the spectra of all of the cured elastomeric materials were similar. Figure 11 shows representative IR spectra of films obtained after photocuring PrBiHex_2 macromonomer in air or under argon atmosphere.

Figure 11 ATR FT-IR spectra of cured films of PrBiHex 2mol%
A difference in absorbance of the band at 1643 cm -1 , corresponding to stretching vibration of C=C, was observed. The carbon-carbon double bonds present in the macromonomer from the attached HEMA are converted into single carbon-carbon bonds during photopolymerization process. Thus, the higher absorbance of this band after photocuring in air indicates that oxygen inhibition had occurred. However, the gel fractions (see Table S2, in the SI) of all photocured elastomers were similar, approx. 93%-curing atmosphere did not affect gel fraction results.

Mechanical analyses
A summary of the mechanical properties of all of the obtained materials is presented in Figure   12, while representative stress-strain curves can be found in the Supplementary Information

Cell viability
Finally, to confirm suitability for biomedical applications, we assessed cytotoxicity based on ISO 10993-5. Cell viability assay results for L929 mouse fibroblasts exposed to extracts of the tested materials for 24 hours are presented in Figure 13 (representative phase contrast photomicrographs are available in the Supplementary Information Figure S18-19). As anticipated, the use of tin-free catalysts resulted in higher cell viability for all tested conditions (catalyst concentration and atmosphere), with a trend towards higher viability in the case of materials synthesized with ZnAc catalyst. As an organotin catalyst, DBTDL is known to be highly toxic and-equally importantly-is very difficult to remove from polymers during purification. 43,44,45 Based on the literature, DBTDL can be estimated to have an IC50 of approx.
3 µg/mL, 43 which is an order of magnitude more toxic than various zinc and bismuth salts. 46,47 However, the comparisons are not perfect due to differences in experimental details. We estimate that the upper bound for residual catalyst content in our samples as prepared for the cell culture tests is in the range of 100-150 µg. Thus, given the similar gel fractions for all samples, the differences in cytotoxicity are likely the result of toxicity of residual DBTDL (see also Figure S15). In order to assess the potential for oxygen inhibition 48 , we photocured materials in both inert (argon) atmosphere as well as in open air, as may be expected to occur during a surgical procedure. We did not observe any effect of the atmosphere present during photocuring, indicating that the effect of oxygen inhibition is relatively modest. Likewise, FTIR analysis of all of the materials confirms the complete purification of residual HEMA ( Figure   S14), which has a reported IC50 of approx. 1.3 mg/mL 49 . We conclude that all of the obtained materials using non-organotin catalysts can be considered well-suited for photocuring in situ without specialized oxygen-free conditions. At the same time, this study and the cell culture experiments were intended to serve as a proof-of-concept and screening; additional animal studies will be needed to address in vivo outcomes such as inflammatory response and fibrosis.
However, previous data obtained for materials obtained with use of DABCO and DBTDL catalysts in a rabbit model were very encouraging 6 and we anticipate that similar-or betterresults may be obtained for materials synthesized with use of Zn or Bi catalysts.

Conclusion
We report here the synthesis of macromonomers containing ester-urethane linkage using three different non-organotin catalysts (BiNDE, BiHex, and ZnAc). FT-IR and NMR studies indicated that all of the tested catalysts resulted in the same structures for the ester-urethane macromonomers. Further, after photocuring, the elastomeric networks obtained from macromonomers synthesized with zinc and bismuth catalysts had suitable mechanical properties for soft tissue regenerative medicine and lower cytotoxicity, as compared to DBTDL-regardless of photocuring atmosphere. Collectively, our results indicate that it is possible to reduce the overall health and environmental safety impact of this macromonomer synthesis reaction by using less toxic catalysts and "green" solvent (ethyl acetate). This is very beneficial not only from the point of view of potential biomedical applications, but also from the safety of the process and overall life cycle of the materials. After considering of all measured parameters as well as the reaction times, we conclude that 2 mol% of BiHex catalysts may offer the best compromise between reaction time, mechanical properties, and cell viability.

Acknowledgements
This work has been supported by research project OPUS17 from the Polish National Science Center (Narodowe Centrum Nauki) "Hybrid and elastomeric polymer networks: synthesis, structure and properties", UMO-2019/33/B/ST5/01445. The authors thank Karol Fijałkowski, PhD (Faculty of Biotechnology and Animal Husbandry, ZUT) for access to the multi-functional plate reader.

Conflict of interests
M. El Fray is co-inventor of patents that are licensed to PolTiss Sp. z o.o. P. Sobolewski has performed paid consulting for PolTiss Sp. z o.o. which is commercializing photocrosslinkable biomaterials.