Biocompatibility of a polymer based on Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+) for neural implants
© Ejserholm et al. 2015
Received: 27 March 2015
Accepted: 4 September 2015
Published: 21 September 2015
The flexibility of implantable neural probes has increased during the last 10 years, starting with stiff materials such as silicone to more flexible materials like polyimide. We have developed a novel polymer based on Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+, consisting of a thiol, two allyls, an epoxy resin and two initiators), which is up to 100 times more flexible than polyimide. Since a flexible neural probe should be more biocompatible than a stiff probe, an OSTE+ probe should be more biocompatible than one composed of a more rigid material. We have investigated the toxicity of OSTE+ as well as of OSTE+ that had been incubated in water for a week (OSTE+H2O) using MTT assays with mouse L929 fibroblasts. We found that OSTE+ showed cytotoxicity, but OSTE+H2O did not. Extracts were analyzed using LC-MS and GC-MS in order to identify leaked chemicals.
Most constituents were found in extracts of OSTE+, whereas only initiators were found in OSTE+H2O extracts. The detected levels of each chemical found in the LC-MS and the GC-MS analysis were below the toxicity level when compared to MTT assays of all the individual chemicals, except for one of the initiators that had an IC50 value close to the detected levels.
Our notion is that the toxicity of OSTE+ was caused by one of the initiators, by impurities in the constituents or by synergistic effects of low doses of leaked chemicals. However, our conclusion is that if OSTE+ is incubated for one week in water, OSTE+ is not cytotoxic and suitable for further in vivo studies.
KeywordsBiocompatibility In Vitro toxicity LC-MS Neural implant Polymer Off-Stoichiometry Thiol-Enes + EpoxyOSTE+
Off-Stoichiometry Thiol-Enes + Epoxy (OSTE+) is a new polymer that was developed to be used in microfluidic devices  instead of PDMS, and we have then further developed it into a potential material for intra-cortical neural probes . The Young´s modulus of OSTE+ can be tuned by shifting the ratio between the constituents and it can easily be patterned using UV-lithography. We have developed a composition of OSTE+ that is as stiff as polyimide at 10 °C, and roughly 100 times more flexible at a physiological temperature, 37 °C (Table 1) . In UV-lithography, OSTE+ is a negative photoresist and can be spin-coated into very thin layers where the thickness of the layer can be controlled by the spin speed. Another advantage OSTE+ affords is that the fabrication protocol is close to that of polyimide and it can therefore easily replace polyimide in current neural probes.
In this study, our goals were to determine the biocompatibility of OSTE+ as well as investigate if there was leaching of constituents from the final polymer. The biocompatibility of various preparations of OSTE+ was tested using in vitro cytotoxicity assays (MTT assays) together with microscopy techniques and the analysis of possible constituents leaching from the materials was performed with liquid chromatography - mass spectroscopy (LC-MS) and gas chromatography – mass spectroscopy (GC-MS). This is an initial study of the cytotoxicity of OSTE+ and the results show that OSTE+, if handled correctly, renders low toxicity to cells, this warrants further studies in compliance with ISO 10993–1 in order to completely deduce the biocompatibility.
The cytotoxicity tests were performed using MTT assays in compliance with ISO standard 10993–5, and all samples were extracted using ISO standard 10993–12.
OSTE+ is produced in a two-step polymerization reaction as previously described . First, a fast UV-initiated radical polymerization step results in a cross-linked network of some of the thiol groups and of all the ene groups in the allyls. Then a thermal anionic polymerization step results in a fully polymerized network with the unreacted thiol groups and the epoxy resin.
In the first polymerization step, UV light was used o activate Lucirin® TPO-L (BASF, Germany) that initiated cross-linking between the thiol, tris[2-(3-mercaptopropionyloxy)ethyl] isocyanurate (90 %, Sigma Aldrich, Germany), and the two allyls, trimethylolpropane diallyl ether (90 %, Sigma Aldrich) and 2,4,6-triallyloxy-1,3,5-triazine (97 %, Sigma Aldrich). In the second polymerization step, 1,5-diazabicyclo[4.3.0]non-5-ene (98 %, DBN, Sigma Aldrich) was used as the initiator for cross-linking between the thiol and the epoxy resin, D.E.N 341 Epoxy Novolac resin (Dow Chemicals, USA).
All ingredients were weighed according to a stoichiometric ratio of monomers of 1.5 : 0.47 : 0.53 : 0.5 for thiol, diallyl, triallyl and the epoxy resin, respectively. TPO-L and DBN were used at 0.2 %. Finally, 3 % acetone (Sigma Aldrich) was added to the mixture. All ingredients were mixed and degassed under vacuum for 5 min. The mixture was then poured into a pre-fabricated PDMS mold. The mold containing the mixture was then covered with a polycarbonate film and exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. The polycarbonate film was removed after the exposure and the OSTE+ pieces were hard-baked (to finalize the thiol-epoxy polymerization) in an oven at 65 °C overnight. The ratio of the different constituents results in an OSTE+ with a glass transition temperature of 39 °C .
Polyimide was selected as a reference material since it is a material used in neural probes today [8, 9, 11, 13, 20, 22]. Polyimide (Durimide 7505, FujiFilm, Belgium) was poured into a PDMS mold, followed by a 2 h soft-bake (to remove the solvents) at 85 °C. The mold with polyimide was exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. The polyimide was then hard-baked (cured) in an oven at 200 °C for 4 h. Finally, the polyimide pieces were rinsed in MilliQ water.
A sheet of HDPE (Bay Plastics LTD, England) was cut into pieces using a milling machine. The pieces were cleaned with acetone (Sigma Aldrich) in an ultrasonic bath followed by a rinse in ethanol (Solveco, Sweden) and MilliQ water.
Extractions of polymer samples were obtained according to ISO standard 10993–12. The extraction was performed in cleaned borosilicate glass vials and water (LC-MS Ultra Chromasolv, Sigma Aldrich, for the chemical analysis and MilliQ water for the cytotoxicity analysis) was used as the extraction medium. The ratio of the surface area of the OSTE+, OSTE+H2O, polyimide or HDPE samples and the water was 3 cm2/ml. The samples were washed in MilliQ water before extraction. The vials were gently shaken at 37 °C for 72 h. When the extraction solution was used in cytotoxicity tests, the polymer samples were first sterilized using an autoclave at 121 °C and the extractions were performed with sterile water.
To obtain standards for the LC-MS analysis, an exact amount of each chemical used for the production of OSTE+ was dissolved in methanol (Sigma Aldrich) overnight (except acetone and D.E.N 431). The following day the solutions were diluted to a concentration of 500 μg/ml using 50 % MilliQ water and 50 % methanol. This was followed by a second dilution step to 50 μg/ml using MilliQ water. Acetone was not tested since it is not detectable in the LC-MS system used; D.E.N 431 was not tested since it is not water-soluble. To simulate the UV-lithography process of the fabrication of OSTE+, all standards were exposed to UV-light using a Karl Suss MA4 mask aligner for 40 min. All standards, extractions (described above) of OSTE+, extractions of OSTE+H2O as well as blank samples, were analyzed using LC-MS.
A LC-MS system (QStar XL, Sciex, together with an Agilent 1100 LC system) was used with an Acquity CHS C18 1.7 μm 2.1x50 mm column and a sample volume of 5 μl. The mass spectrometer was configured to scan 120–1000 Da at 2 scans/s. Acetonitrile (LC-MS, Chromasolv, Sigma Aldrich) and MilliQ water were used at a flow rate of 250 μl/min. The concentration of acetonitrile in the mobile phase was: starting at 5 % for 1 min, ramp to 95 % over 3 min, hold for 2 min, ramp down to 5 % in 0.1 min.
To obtain standards for the GC-MS analysis, an exact amount of all constituents (except acetone) was dissolved in toluene (Sigma Aldrich and VWR). To simulate the UV-lithography process of the fabrication of OSTE+, all samples were exposed to UV-light for 40 min using a Karl Suss MA4 mask aligner. 400 μl of the OSTE+ extractions or OSTE+H2O extractions (described above) was pipetted to a new vial containing 1 ml of toluene. The toluene extraction samples were mixed using a vortex for 40 h, followed by centrifugation for 1 min. To ensure that all chemicals in the extracts were transferred into the organic phase, 2 samples of each extraction were made acidic by adding 50 μl of 0.1 M HCl (VWR) and 2 samples of each extraction were made basic by adding 50 μl of 0.1 M NaOH (VWR) just before the addition of toluene to each extraction. The extracts spiked with HCl are referred to as OSTE+/HCl and OSTE+H2O/HCl throughout the paper and the extracts spiked with NaOH are referred to as OSTE+/NaOH and OSTE+H2O/NaOH throughout the paper. All samples and extracts together with a sample of pure toluene were analyzed using GC-MS. Acetone was not tested since it is used as a cleaning step in the GC-MS analysis.
All analyses were performed on a gas chromatograph (Agilent 6890) equipped with an injector in split mode at 250 °C, equipped with an auto sampler (sample volume of 5 μl) and a mass spectrometry detector (Agilent 5973-N). The column used in the system was a HP-5 ms fused silica capillary column (5 % phenyl-methylpolysiloxane) of 30 m × 0.25 mm with a phase thickness of 0.25 μm. The temperature program of the column was: 50 °C, hold for 1 min, ramp to 300 °C over 10 min, hold for 15 min. The carrier gas used was helium at a flow rate of 1.2 ml/min. The mass spectrometer was run in either scan mode (30–600 Da with 2.6 scan/s) or in selected ion monitoring (SIM) mode, using mass to charge ratios (m/z) of 70, 81, 82, 99, 123, 125, 147, 187, 197, 254, 321 together with a dwell time of 20 ms.
Scanning electron microscopy analysis of OSTE+
Four OSTE+ pieces were cut into two halves. One half was put into MilliQ water for 7 days and then each pair was studied by scanning electron microscopy (SEM, SU 8010, Hitachi, Japan) to see if 7 days of incubation in water affected the surface of OSTE+.
Four different materials were tested in the MTT assay: OSTE+, OSTE+H2O, polyimide and HDPE. Polyimide was selected since it is regarded as biocompatible and there are several studies that can be used for comparison. HDPE is a biocompatible material that sometimes is used as a negative control in different cell viability assays [22, 25, 26].
The mouse fibroblast cell line L929 was purchased from American Type Culture Collection (ATCC, LGC standards AB, Borås, Sweden) and was grown as a monolayer culture in complete cell culture medium, i.e. RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine, at 37 °C in 5 % CO2 in air. The cells were sub-cultured twice every week at a density of 12 500 cells/cm2.
Polymer extractions for cell culture
The extracts were obtained as described above and kept at 4 °C for a maximum time of 24 h before use in the cytotoxicity assay with L929 cells. The extracts were diluted 1:2 in 2x concentrated RPMI-1640 medium supplemented with 20 % FBS, 200 U/ml penicillin, 200 μg/ml streptomycin and 2 mmol/l L-glutamine, i.e. a final extract concentration of 50 % in 1x concentrated RPMI-1640 containing 10 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine, before addition to cell cultures Further dilutions of the 50 % concentrated polymer extracts were done in 1x concentrated RPMI-1640 medium supplemented with 10 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mmol/l L-glutamine.
MTT assay – extract testing
L929 cells were seeded in complete cell culture medium in 96-well plates (2000 cells/well in 180 μl medium) and were then incubated at 37 °C in 5 % CO2 in humidified air for 24 h. The medium was then removed and changed to medium containing the different extracts at concentrations ranging from 6.25–50 % medium containing 2.5 % dimethyl sulfoxide (DMSO: positive control) or control medium (complete cell culture medium with 0 % extract). The cells were then kept at 37 °C in 5 % CO2 in humidified air for 72 h before addition of MTT to a final concentration of 0.5 mg/ml. The 96-well plates were incubated with MTT at 37 °C for 1 h before removal of the medium, and the cells with blue formazan crystals were then dissolved in DMSO. The following spectrometry reading at 540 nm was done using a SPECTRAmax M2 instrument (Molecular Devices, Sunnyvale, CA, USA) and analyzed using SoftMax® Pro v. 4.6 (Molecular Devices). A number of 3–6 biological replicates (i.e. the testing of independent extracts of OSTE+, OSTE+H2O, polyimide and HDPE) were used for all the treatments of the L929 cells and a number of 6 technical replicates (i.e. number of n) from each extract were used for each biological replicate. After correcting all absorbance values for background, the percent of control was calculated as absorbance units in the presence of test compound as percentage of that in control. The results of the MTT assay are resumed to reflect the cell number, thus, 50 % percent MTT reduction of control implies that there were 50 % less cells at that treatment concentration .
MTT assay - individual chemicals
Each chemical used in the fabrication (except acetone), 1 or 0.1 mg, was added to a sterile flask and 1000 μl sterile water was added. Further dilutions were made using sterile water. D.E.N 431 was not soluble in water and after shaking the sample and letting it stand for a while, only the water solution was used in the test. L929 cells were seeded in complete medium in 96-well plates (2000–3000 cells/well in 180 μl medium) and the plates were incubated in a CO2 incubator for 24 h before addition of the chemicals. Indicated concentrations of the chemicals were added to the wells and the cells were incubated for 72 h at 37 °C in 5 % CO2 in humidified air. The dose response was evaluated using MTT as described above.
Phase contrast and fluorescence microscopy
L929 cells were seeded in 12-well plates containing sterile glass coverslips, whereupon they were incubated at 37 °C in 5 % CO2 in humidified air for 24 h. The growth medium was then removed and changed to growth medium without extract or containing the different polymer extracts (at a concentration of 50 %, obtained as described above). The cells were then incubated for 72 h before they were photographed with an Olympus CKX41 inverted phase contrast microscope (Olympus Corporation, Tokyo, Japan) equipped with an Olympus SC30 camera (Olympus Corporation) then analyzed using the software cellSense Standard version 1.4 (Olympus Corporation). The cells were then fixed in 3.7 % paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS). The fixed cells were stained with Alexa Fluor 647-conjugated phalloidin in PBS containing 1 % bovine serum albumin and 1 % Tween-20 for 1 h and in PBS containing 1 μg/ml bisbenzimide for 1.5 min to visualize the cytoskeleton and nuclei, respectively. The glass coverslips were mounted on glass slides using Mowiol 4–88 and left in a refrigerator overnight. The samples were imaged with a LSM510 confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a Hamamatsu R6357 photomultiplier (Hamamatsu Photonics K.K., Hamamatsu, Japan). A 405 nm diode-pumped solid-state laser was used to excite the bisbenzimide and a band-pass filter of 420–480 nm was used for the emission. The Alexa Fluor 647-conjugated phalloidin was excited with a 633 nm HeNe ion laser and a 650 nm long-pass filter was used for the emission. The cells were visualized by z-stacks composed of single optical planes in high magnification and the images were analyzed with the software ZEN 2012 (black edition) version 8.0 (Carl Zeiss Microscopy GmbH).
Chemical analysis using LC-MS
Standards of all constituents (except acetone and D.E.N 431), extracts of OSTE+, extracts of OSTE+H2O and blank samples were analyzed using LC-MS.
Corresponding mass to charge ratio, LOD and detected level for each constituent found using LC-MS
Detected level in OSTE+ extracts (μg/ml)
Detected level in OSTE+H2O extracts (μg/ml)
163, 325–327, 488-491
121, 147, 317
Chemical analysis using GC-MS
LOD of GC-MS (SIM mode)
LOD of GC-MS
All extracts of OSTE+, OSTE+H2O, OSTE+/HCl, OSTE+/NaOH, OSTE+H2O/HCl, OSTE+H2O/NaOH and all blank samples were run through the GC-MS both in SCAN mode and in SIM mode. No detectable traces of the constituents were found in any of the extracts.
IC50 values obtained from the dose response curves shown in Fig. 4b and levels detected in the LC-MS analysis modified to represent extracts diluted to 50 %
50 % OSTE+ extracts (μg/ml)
50 % OSTE+H2O extracts (μg/ml)
OSTE+ is a new polymer that is more flexible then other polymers used for neural probes. This should allow probes constructed in OSTE+ to follow the micro motions of the brain, hence increasing the biocompatibility of the neural probe . Our group has previously shown that it is possible to make implantable neural probes in OSTE+ . However there is one important question: is OSTE+ biocompatible? In our study we decided to give an answer to this question by using standardized biocompatibility toxicity tests used by the industry. Cytotoxicity was investigated using MTT assays with L929 cells in compliance with ISO standard 10993–5. The MTT assay was used to investigate the cytotoxicity of extracts of four different materials (OSTE+, OSTE+H2O, polyimide and HDPE) and of the individual chemicals that were used in the OSTE+ fabrication process. The morphology of cells treated with extracts was investigated using phase contrast and confocal microscopy. GC-MC and LC-MS were used to analyze chemicals found in extracts of OSTE+ and extracts of OSTE+H2O. SEM was used to investigate the surface structure of OSTE+ and OSTE+H2O.
The results of the cytotoxicity test (Fig. 4a) imply that OSTE+ used directly after production may be toxic if not washed properly before implantation. The reduction of MTT is assumed to be proportional to cell number and thus at the highest concentration (extract diluted 50 %), the OSTE+ extract significantly reduced the cell number thus implying an effect on cell proliferation. Using confocal microscopy and phase contrast microscopy we could not find any morphological changes imposed by any of the treatments (Fig. 4c). We cannot, however, exclude the possibility that higher magnification microscopy or the use of other cellular staining probes than phalloidin staining of actin may indicate morphological changes. To be able to draw conclusions about the exact mechanism of toxicity, more elaborate investigations have to be made. On the other hand, that may not be warranted since the toxicity disappeared when the OSTE+ samples were pre-incubated in water as an extra washing step.
The chemical analysis (LC-MS) showed that the water-soluble chemicals DBN, diallyl, triallyl and TPO-L were found in the extracts made from OSTE+. But only DBN and TPO-L were detected in the OSTE+H2O extracts, which is expected since they are only used as initiators in the process and are not immobilized in the final polymer network. Because the detected levels of most constituents were close to the respective LOD, some may still have leached out into the OSTE+H2O extracts. The IC50 value found for DBN (Table 4) is close to the detected levels of DBN in extracts of OSTE+ and in extracts of OSTE+H2O, and as seen in Fig. 4b the steep curve for DBN could explain the difference in toxicity between the extracts OSTE+ and the extracts of OSTE+H2O. Comparing the IC50 values for diallyl and triallyl in the OSTE+ extracts with the detected levels (Table 4) the IC50 values are six times greater for diallyl and 60 times greater for triallyl. Hence, our conclusion is that neither diallyl nor triallyl are responsible for the toxicity found in the OSTE+ extracts. The lack of results from the extracts analyzed using the GC-MS is probably because most of the constituents are water soluble and thus were not extracted into the toluene phase from the water phase. Since acetone was used in the fabrication process, it is a possible candidate for toxicity. However, acetone was used as a 3 % concentration in the fabrication mixture which was then degassed, exposed to UV-light and baked at 65 °C over night. Since acetone is a highly volatile substance, the concentration was likely lowered substantially. Remaining acetone may of course have leaked into the extract but a hypothetical calculation of concentration in the extract with no evaporation during the production process would give at hand a maximum concentration in the medium of 0.2 % which is below a concentration that has shown toxicity in cells [30, 31]. Thus, we do not think acetone contributed to toxicity of OSTE+. The unidentified peaks in the LC-MS spectra (Fig. 2) support the existence of impurities, but since we were unable to identify the peaks their role in the toxicity cannot be determined. Neither have we tested possible synergistic effects of the low doses of the leaked compounds. Since we did not autoclave the samples used in the chemical testing, there could be a small variation between the extracts used in the MTT assays and in the chemical testing. However, the variation should be insignificant since the state of the material does not change when increasing the temperature above the temperature used in the hard-baking step.
Since oxygen will inhibit the UV-initiated polymerization step and PDMS has a high oxygen permeability [32, 33], the use of a spin-casting technique instead of PDMS molds should allow for a better polymerization of OSTE+ and decrease the amount of unreacted constituents in the final polymer. The surface analysis, using SEM (Fig. 3) shows that there was a small difference between OSTE+ and OSTE+H2O samples. The nano-sized cracks were somewhat wider in the OSTE+H2O sample. In our previous work we did not use acetone in the mixture and we did not see any cracks when using that formula . We therefore hypothesize that these cracks appeared during a fabrication step where acetone evaporated. Since acetone was added in order to make the mixture less viscous and easier to use in a mold, the use of spin casting should allow for a fabrication protocol without the use of acetone, which hopefully will prevent the formation of nano-sized cracks.
OSTE+ is a very promising material to use for neural probes because of its superior flexibility compared to other polymers used in neural probes today. The toxicity of OSTE+ extracts was presumably derived from DBN, unknown impurities in the constituents or from low dose synergetic effects of the constituents. However, by pre-incubating the final polymer in water for 7 days, OSTE+ was shown to be nontoxic to cells. The use of high-purity chemicals in the fabrication process, removal of acetone from the fabrication, the addition of a cleaning step in water during fabrication and the use of a silicon wafer as the substrate instead of PDMS should minimize the toxicity rendering implants made from OSTE+ nontoxic. In order to ensure the biocompatibility of OSTE+ for neural implants both short and long term in vivo tests have to be conducted.
This project was sponsored by two grants from the Swedish Research Council (project number 80337401 and project number 60012701; 60012701 is a Linnaeus grant) and one grant from The Knut and Alice Wallenberg Foundation (project number: KAW 2004.0119). We would also like to thank Professor emeritus Jan Åke Jönsson and Margareta Sandahl at the Centre for Analysis and Synthesis, Department of Chemistry at Lund University, for all the help with the GC-MS analysis.
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