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PhD-Dissertation Chapter4.typ
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== Abstract<abstract>Synthetic hydrogels are an attractive platform for cell culture, as theyare water-rich, and they can often be used to encapsulate cells within athree-dimensional matrix under the right chemical conditions. Manyhydrogels form _via_ free radical polymerization in the presence ofinitiating species under UV light. The combination of free radicals andUV light during polymerization can lead to decreased viability andcellular stress. Here, I demonstrate a photoiniferter strategy topolymerize hydrogel networks without exogenous initiators and UV light.We demonstrate formation of soft hydrogels with xanthate-functionalpolymers with visible light. Importantly, polymerization proceedsequally well under nitrogen and in ambient conditions with a range ofmonomers. I suggest this process as a way to diversify monomer choicefor synthetic hydrogel development in the absence of UV-light and freeradical initiators.
== Introduction<introduction>Hydrogels made from synthetic polymer precursors span a wide range ofapplications, from injectable redox-basedhydrogels,@rodriguez-rivera2024 to antifouling coatings,@kolewe2015 tocell culture environments@jansen2022@seidlits2011 and tissueengineering.@parmar2015 Hydrogels are highly useful networks for cellculture applications due to their affinity for water, their stability atphysiological conditions, and their 3D matrix structure that surroundscells.@herrick2013@peyton2007 Most commonly, hydrogels are formed bycrosslinking polymer chains into a network _via_ free radicalpolymerization of vinyl functional monomers, initiated by redox orUV-sensitive species. Limitations of this standard approach includetoxic byproducts@xu2020@temenoff2003@rizzo2023 and high energylight,@masuma2013 both of which can be damaging to embedded cells.Michael-addition reactions of multifunctional polymers are a popularalternative to free radical-initiated network formation, though tuningthe kinetics of Michael-addition reactions can be difficult within thestrict confines of physiological conditions.@jansen2018 I sought tofind an alternative solution to this issue by in situ reversibleaddition-fragmentation chain transfer (RAFT) polymerization. RAFTpolymerizations are traditionally highly oxygen sensitive and polymerizeslowly, so they are rarely used for cell applications.@niu2017 The goalof my work was to explore whether specialized RAFT polymerizationscould be effectively tuned to create soft hydrogels quickly and inoxygen-tolerant conditions.
RAFT polymerizations offer several advantages to free radicalpolymerizations. RAFT polymerizations use far lower radicalconcentrations than typical free radical polymerizations, potentiallyreducing oxidative stress on cells. Owing to their reversibility, theycan be started and stopped at will, enabling the synthesis of complexstructures and sequences through careful reaction design. Additionally,polymers with RAFT endgroups are nontoxic to cells.@pissuwan2010 A finalbenefit of RAFT-crosslinked hydrogels is their ability to produce highlyhomogeneous@wanasinghe2022 crosslinked networks with chain-endfunctionality, enabling the preparation of living hydrogels that can bemodified after fabrication.@thang1999@cortez-lemus2021
A key limitation in the use of RAFT for biocompatible hydrogelfabrication is its high sensitivity to oxygen.@lehnen2023@bhanu1991Oxygen is a well-known radical scavenger; but molecular oxygen isrequired for viable cell culture. Several techniques have been developedto impart oxygen tolerance to RAFT polymerizations such as employingalkyl amine electron donors or photoredox catalysts.@nomeir2019Alternatively, the oxygen can be overwhelmed with excess radicals fromthe initiating species. Here, I have taken inspiration from this lastconcept by exploiting the high radical flux produced by xanthates inphotoiniferter (PI)-RAFT polymerization to rapidly consume environmentaloxygen during polymerization, followed by rapid reoxygenation bydiffusion to maintain cell viability.@lehnen2022
The photoiniferter (PI) concept was recently described by Otsu,@otsu2000and it uses molecules that can simultaneously act as #strong[ini]tiator,trans#strong[fer] agent, and #strong[ter]minator. This means noexogenous initiators are needed, which simplifies reaction set-up andreduces concerns of byproduct cytotoxicity. Given that xanthates enablepolymerization and crosslinking in air,@zhao2022 and thattrithiocarbonate disulfides can mediate PI-RAFT and produce telechelicpolymers,@beres2024@kerr2021 I hypothesized that xanthate disulfideswould provide a facile way to produce telechelic polymers, similar totrithiocarbonate disulfides, and produce gels rapidly in open air,similar to traditional xanthates. To do this, I employed a bisxanthateproposed by Huang _et al._,@huang2024 and translated it to an aqueoussystem to support future cell applications.
== Materials and methods<materials-and-methods>=== Reagents used<reagents-used>Reagents were purchased from Sigma-Aldrich unless otherwise noted.Methyl acrylate (MA, 99%), ethyl acrylate (EA, 99%), n-butyl acrylate(nBA, ≥ 99%), 2-hydroxyethyl acrylate (HEA, 96%), acrylamide (AAm, 99%),N,N-dimethylacrylamide (DMAa, 99%), acrylic acid (AA, 99%), and isodecylacrylate (iDA, 99%) were each passed over alumina to remove inhibitorsbefore use. N-isopropylacrylamide (NIPAM, 97%), carbon disulfide(redistilled, ≥99.9%), iodine (≥99.8%), (DCTB, ≥99.0%), 1,4-dioxane(dioxane, ≥99.0%), deuterated DMSO (99.8%), and sodium thiosulfate (99%)were used as received. Potassium hydroxide (KOH, 99.98%), ethanol(anhydrous, 90%), methanol (99.8%), hexanes (99%), and diethyl ether(anhydrous, 99%) were purchased from ThermoFisher and used as received.Sodium trifluoroacetate (NaTFA) was synthesized as described by Prakeshand Matthew.@suryaprakash2010
=== Instrumentation<instrumentation>Poly(iDA) molecular weight and dispersity were measured by GPC on anAgilent 1260 with a PL gel 5 μm guard column and three, 5 μm analyticalmixed C columns (Agilent). THF was used as the eluent at a flow rate of1 mL/min. The column was standardized with pMMA calibration standardsand toluene was used as a flow marker. All other polymer molecularweights and dispersities were determined on an Agilent Tech 1260Infinity DMF GPC, with a Gel 5 μm guard~column, a PL Gel 5 μm mix D 1°column, a PL~Gel 5 μm Mix C 1° column, and a refractive index detectorusing a 20 μL sampling loop. Analyses were run at 50 °C using DMF with0.01 M LiCl at a flow rate of 1.0 mL/min with toluene as a flow marker(@fig:samplegpc). #super[1]H NMR spectra were recorded using a400 MHz Avance Bruker spectrometer (@fig:bisxannmr - @fig:pea).
#figure(image("Images/C4S1.png", width: 100.0%), placement: bottom, caption: [ GPC traces of polymers studied, synthesized in air or nitrogen. ])<fig:samplegpc>
#figure(image("Images/C4S2.png", width: 100.0%), placement: auto, caption: [ #super[1]H NMR of bis(xan). Spectra recorded at 300 MHz in CDCl#sub[3]. ])<fig:bisxannmr>
#figure(image("Images/C4S3.png", width: 100.0%), placement: bottom, caption: [ #super[1]H NMR of pnBA. Spectra recorded at 300 MHz in CDCl#sub[3]. ])<fig:nba>
#figure(image("Images/C4S4.png", width: 100.0%), placement: auto, caption: [ #super[1]H NMR of pMA. Spectra recorded at 300 MHz in CDCl#sub[3]. ])<fig:ma>
#figure(image("Images/C4S5.png", width: 100.0%), placement: auto, caption: [ #super[1]H NMR of pMA_-b-_EA. Spectra recorded at 300 MHz in CDCl#sub[3]. ])<fig:mabea>
#figure(image("Images/C4S6.png", width: 100.0%), placement: auto, caption: [ #super[1]H NMR of piDA. Spectra recorded at 300 MHz in CDCl#sub[3]. ])<fig:pida>
#figure(image("Images/C4S8.png", width: 100.0%), placement: auto, caption: [ #super[1]H NMR of pEA. Spectra recorded at 300 MHz in CDCl#sub[3]. ])<fig:pea>
=== Polymerization kinetics<polymerization-kinetics>A 50 wt% solution of MA monomer solution (1 eq of bis(xan), 50 eq of MA,and deuterated DMSO queued to 50 wt%) was prepared in a 20 mL vial. Thesolution was added to an NMR tube either sparged with nitrogen or not. A#super[1]H spectra was taken at t = 0 min using a 400 MHz Avance Brukerspectrometer (Bruker Scientific LLC). The tubes were then placed in aphotoreactor under a 405 nm lamp at 4.1 mW/cm#super[\2.] Spectra werecollected at 1, 5, and 10 minutes to monitor monomer conversion.Reactions were stopped when 90+% conversion was reached. Conversion wasdetermined by integrating the vinyl protons and acrylic backbone protons(@fig:nkinetics and @fig:akinetics).
#figure(image("Images/C4S9.png", width: 100.0%), placement: auto, caption: [ #super[1]H NMR of bis(xan) and MA evolution over time under 405 nm light in nitrogen. ])<fig:nkinetics>
#figure(image("Images/C4S10.png", width: 100.0%), placement: auto, caption: [ #super[1]H NMR of bis(xan) and MA evolution over time under 405 nm light in air. ])<fig:akinetics>
=== Synthesis of O,O-diethyl 1,2-disulfanedicarbothioate (bis(xan))<synthesis-of-oo-diethyl-12-disulfanedicarbothioate-bisxan>KOH (5 g) was added to anhydrous ethanol (200 mL) in a 500 mL roundbottom flask and stirred until dissolved. The flask was then cooled to 0°C on ice. Carbon disulfide (5 mL) was added to the solution dropwise.Immediately, the solution turned bright yellow. Subsequently, the roundbottom flask was stirred for three hours at room temperature. Thesolution was precipitated in diethyl ether (1 L) to yield potassiumethyl xanthogenate as a slightly off white solid. The solid wascollected by vacuum filtration and further dried under 0.001 mbar vacuumovernight. Once dry, 10 g of the pure solid was added to a methanol (50mL) in a 100 mL round bottom flask and dissolved. Iodine (2.5 g) wasadded portionwise, and the solution was stirred for three hours at roomtemperature. The solution was precipitated in ice cold DI water (2 L),yielding O,O-diethyl 1,2-disulfanedicarbothioate as a bright yellowsolid. The solid was washed with saturated sodium thiosulfate solutionand DI water, then dried in a vacuum dessicator to yield the finalproduct.
=== Photopolymerization<photopolymerization>Polymerizations from methyl acrylate (MA), ethyl acrylate (EA), n-butylacrylate (nBA), isodecyl acrylate (iDA), acrylic acid (AA),hydroxyethylacrylate (HEA), acrylamide (AAm), N,N-dimethylacrlamide(DMAa), and N-isopropylacrylamdie (NIPAM) monomers were conducted insimilar fashions. Briefly, a 50 wt% solution of monomer was prepared ina 20 mL vial (1 eq bis(xanthate), 50 eq of monomer, and 50 wt% dioxane).The vial was then sealed with a rubber septum and either left under airor sparged with nitrogen for 15 minutes. Next, the vial was placed intoa photoreactor under a 405 nm lamp either in a ventilated (MA, EA, nBA,iDa, AA, and HEA) or in a saled chamber (AAm, DMAa, and NIPAM) at 4.1mW/cm#super[2] for 30 min. A small aliquot of the resultant polymersolution was reserved to measure monomer conversion, and the remainderwas diluted with dioxane. Polymers containing nBA or iDA wereprecipitated in cold methanol, and all other polymers were precipitatedin cold hexanes.
=== Hydrogel preparation<hydrogel-preparation>Bis(xan) (1 eq) and hydroxyethyl acrylate (100 eq) were vortexed in a 20mL vial until the iniferter dissolved. Then 1 wt% trimethylolpropanetriacrylate and 50wt% DI water were added resulting in precipitation ofthe bis(xan). Under agitation, 100 µL of the well-mixed, cloudy mixturewas quickly pipetted into a 24 well plate. The hydrogel solution wasthen placed under a 405 nm LED lamp for ten minutes. The circular, cleargels were then removed from the wells and placed into 100 mL 1X PBS pH7.4 buffer for 48 hours to fully swell, exchanging the PBS buffer after24 hours.
=== Rheological characterization of hydrogels<rheological-characterization-of-hydrogels>After they reached equilibrium swelling, the storage and loss moduliwere measured by oscillatory shear rheology using a Kinexus Pro parallelplate rheometer (Netzsch, Selb, Bayern, Germany) with a 20 mm diameterplatens. Each platen was affixed with 60-grit sandpaper to preventslippage. Measurements were conducted at room temperature at 0.1%strain, 0.1 and 1 Hz, and a constant 0.01 N normal force. A solvent trapfilled with DI water was used to maintain gel hydration.
=== MALDI-ToF<maldi-tof>To characterize the mass distribution of polymers, I adapted aMALDI-ToF technique described in Beres _et al._@beres2024 Briefly, wedissolved (DCTB) as the matrix (40 mg/mL), sodium trifluoroacetate(NaTFA, 1 mg/mL) as a cationizing agent, and the polymer sample (10mg/mL) in spectroscopy grade acetonitrile. 5 μL each of matrix andsample and 1 μL of salt were mixed, and 1 μL of this mixture wasdeposited onto a ground steel target. Data was acquired on an UltraflexIII MALDI-TOF/TOF mass spectrometer equipped with a smartbeam laser(Bruker) operating in linear or reflectron positive ion mode. Externalcalibration was performed using mixtures of commercial PEGs to overlapwith the m/z range of interest. Spectra were generated by averaging5,000-10,000 shots from non-overlapping positions. Data was analyzedusing FlexAnalysis v3.4 and PolyTools v1.31 (Bruker).
== Results and discussion<results-and-discussion>=== Kinetics and livingness<kinetics-and-livingness>Our goal was to develop a new approach to synthesize polymers andhydrogels rapidly, without toxic radicals and side products, in thepresence of oxygen and water. If successful, this would represent afundamentally new strategy to form polymers and hydrogels in thepresence of living cells. To accomplish this, I employedxanthate-mediated PI-RAFT polymerizations because of their fastkinetics.@bowman2023@li2017@li2018a Inspired by recent work onbis(trithiocarbonates)@beres2024@kerr2021 and xanthates, I selected axanthogen disulfide (bis(xan)) as the PI.
Polymerization reactions of bis(xan) follow a traditional RAFTmechanism. 405 nm light puts bis(xan) into an excited state where it canfragment through β-scission into two thiyl radicals. These radicals canthen react with vinyl monomers through a single unit monomer insertion(SUMI) pathway (@fig:pischeme).@beres2024 The addition of amonomer into bis(xan) changes its chemistry, resulting in betterinitiating radicals upon excitation and fragmentation relative to thiylradicals. When the xanthates are again excited by light, a secondarycarbon radical is formed that propagates rapidly. From there, thegrowing polymer enters RAFT equilibrium.
#figure(image("Images/C4F1.png", width: 100.0%), placement: auto, caption: [ Schematic of photoiniferter polymerization of vinyl monomers. PI polymerization proceeds through a SUMI step to yield a better initiating R group capable of initiating the propagation step, followed by conventional RAFT kinetics. ])<fig:pischeme>
We assessed the compatibility of bis(xan) to create polymers with asuite of acrylic monomers (@xanthatepolym) under nitrogenatmosphere in dioxane. Water insoluble monomers tested were alkylesters, including methyl acrylate (MA), ethyl acrylate (EA), n-butylacrylate (nBA), and isodecyl acrylate (iDA). Water soluble monomerstested included acrylates (and hydroxyethylacrylate (HEA)) andacrylamides (acrylamide (AAm), (DMAa), and N-isopropylacrylamdie(NIPAM)).
#figure( placement: auto, align(center)[#table( columns: 8, align: (center,right,right,right,right,right,right,right,), table.header([Polymer], [Temp (°C)], [Atm], [Target DP], [Measured DP], [Mn (g/mol)], [Đ], [Conv (%)],), [PMA], [38], [Air], [50], [57], [5773], [1.46], [96], [PMA], [38], [N2], [50], [45], [4562], [1.49], [97], [PEA], [38], [Air], [50], [49], [4969], [1.63], [95], [PEA], [38], [N2], [50], [41], [4180], [1.59], [97], [PnBA], [38], [Air], [50], [38], [3847], [1.6], [96], [PnBA], [38], [N2], [50], [41], [4106], [1.41], [98], [PHEA], [38], [Air], [50], [139], [13993], [1.81], [95], [PHEA], [38], [N2], [50], [154], [15411], [1.66], [96], [PiDA], [38], [Air], [50], [73], [7361], [2.26], [90], [PiDA], [38], [N2], [50], [63], [6364], [2.25], [92], [PDMAa], [60], [Air], [50], [44], [4457], [1.32], [N/A], [PNIPAM], [60], [Air], [50], [66], [6613], [1.4], [N/A], )] , caption: [Characterization of xanthate polymerizations for a range of monomers. Polymerization conditions (temperature, atmosphere, targeted DP) and characterization (measured DP, molecular weight (M#sub[n]), dispersity (Đ), and conversion are detailed.] , kind: table)<xanthatepolym>
All polymerizations save for AAm yielded a viscous liquid after 30minutes. For the alkyl esters, smaller pendent alkyl groups correspondedto better controlled polymerizations than their larger counterparts(pEA, pnBA, piDA, @xanthatepolym). The largest pendent alkyl group polymer,piDA, had a large dispersity (Đ \> 2) and several unidentified smallerpeaks in the chromatogram (@fig:samplegpc). For the acrylamides, onlymonomers with substituted amides polymerized. AAm did not polymerize,whereas pDMAA and pNIPAAm formed readily with pDMAA showing lowerdispersities than pNIPAAm (@fig:samplegpc). The amide protons are likelyresponsible for the large dispersity and failed polymerizations due toaminolysis reactions of bis(xan).@thomas2004 Regardless, the polymersthat did form represent a wide range pendent functionality,demonstrating broad utility and applicability of bis(xan).
To determine the air tolerance and suitability towards cellencapsulation of these polymerizations, the same monomers werepolymerized with bis(xan), this time without nitrogen purging to removeair. Mirroring the Nitrogen condition, all polymerizations except AAmyielded a viscous liquid after 30 minutes. Further, similar molecularweights and dispersities were observed when polymerizations wereconducted in air and nitrogen (@xanthatepolym).
To further investigate the influence of air on these reactions, weanalyzed polymerization kinetics. Ideally, polymerizations wouldapproach full conversion within ten minutes to be useful forencapsulating cells. To this end, a MA polymerization under air ornitrogen was monitored _via_ NMR. Polymerizations were conducted in air orunder nitrogen with 405 nm light at 38 °C with spectra collected at 0,1, 5, and 10 minutes (@fig:xanthatekinetics a). Polymerization under nitrogen or airdisplayed nearly identical kinetics, each reaching 97% conversion within10 min. The polymerization is remarkably fast compared to priorpublications using 365 nm light.@huang2024
#figure(image("Images/C4F2.png", width: 100.0%), placement: auto, caption: [ Bis(xanthates) mediate ultra-fast polymerizations of acrylates and produce living polymers. (a) Conversion over time of xanthate-mediated PI polymerization of 50 wt% MA under air and nitrogen atmosphere. (b) GPC chromatogram showing degree of polymerization of pMA and pMA_-b-_EA synthesized in air and under nitrogen. ])<fig:xanthatekinetics>
A brief study was then conducted to determine the impact of heat on thepolymerization reactions was conducted with MA monomers. I comparedreactions at 38°C and 60°C in air (@pmatemp). In comparison to pMAsynthesized at 38°C, pMA synthesized at 60 °C was more narrowlydispersed. This is most likely due to the change in the RAFT equilibriumat elevated temperatures.@nwoko2025 Although not useful for cellapplications, temperature could be another useful handle for controllingthis polymerization.
#figure( placement: auto, align(center)[#table( columns: 8, align: (center,right,right,right,right,right,right,right,), table.header([Polymer], [Temp (°C)], [Atm], [Target DP], [Measured DP], [Mn
(g/mol)], [Đ], [Conv (%)],), table.hline(), [PMA], [38], [Air], [50], [57], [5773], [1.46], [96], [PMA], [60], [Air], [50], [84], [8496], [1.25], [N/A], )] , caption: [Impact of temperature on xanthate polymerization of pMA.] , kind: table )<pmatemp>
To further characterize the air tolerance, I conducted further analysison the degree of polymerization (DP) _via_ GPC. Polymerizations of MA inair and nitrogen with a targeted DP of 25 were conducted using amonomer:bis(xan) ratio of 25:1. The resultant polymers yielded DP of 32and 35 monomers per chain for air and nitrogen respectively (@fig:xanthatekinetics b, @xanthategpc). I then attempted tore-initiate polymerization by redissolving the pMA poymers and adding EA(1 eq. bis(xan): 1 eq. EA). Successful chain extension was demonstratedfor both samples _via_ the shift to earlier elution times (@fig:xanthatekinetics b).Interestingly, the final DP measured by GPC were nearly twice as high asthe targeted DP in both air and under nitrogen. This behavior isconsistent with prior reports studying bis(trithiocarbonates),@beres2024though the reason for this doubling of DP is not clear. Despite thehigher than predicted DP, I was able to produce living polymerscapable of further reaction in open air and in nitrogen.
#figure( placement: auto, align(center)[#table( columns: 8, align: (left,left,left,left,left,left,left,left,), table.header([Polymer], [Temp (°C)], [Atm], [Target DP], [Measured DP], [Mn (g/mol)], [Đ], [Conv (%)],), [PMA], [38], [Air], [25], [32], [3278], [1.45], [94], [PMA], [38], [N2], [25], [35], [3578], [1.4], [95], [PMA_-b-_EA], [38], [Air], [64], [115], [11559], [1.32], [94], [PMA_-b-_EA], [38], [N2], [70], [114], [14414], [1.32], [95], )] , caption: [GPC characterization of polymerizations of MA and block polymers built from living PMA.] , kind: table )<xanthategpc>
=== End group fidelity of polymers<end-group-fidelity-of-polymers>To confirm the telechelic nature of polymers resulting from synthesisvia bis(xan), I analyzed the structure of pMA _via_ MALDI-ToF, GPC, andNMR (@fig:xanthateendgroup). For these reactions, pMA was synthesized at a target DPof 25 (25:1 MA:bis(xan)), either in air or nitrogen, and characterizedby MALDI-ToF (@fig:xanthateendgroup a,b). MALDI-ToF showed thatpMA polymerized through a photoiniferter approach with bis(xan) is wellcontrolled, with polymerizations done in air and in nitrogen having adispersity of 1.11 (@fig:maldin and @fig:maldia). The MALDI-ToFspectra also showed air and nitrogen synthesized polymers to have verysimilar molecular weights (Mn = 4584 g/mol in nitrogen, M#sub[n] = 4821 inair). Additionally, MALDI-ToF analysis detected the sodiated forms ofboth polymers, consistent with pMA bearing the $alpha$ and $omega$functionality expected in the telechelic polymer (@fig:pischeme).
#figure(image("Images/C4F3.png", width: 100.0%), placement: auto, caption: [ Structural characterization of pMA synthesized with bis(xanthates) in air and nitrogen. (a) MALDI-ToF spectra of pMA synthesized in nitrogen (Mn = 4310 g/mol). (b) MALDI-ToF spectra of pMA synthesized in air (Mn = 4396.2 g/mol). (c) GPC (DMF) chromatogram of pMA polymers with a targeted DP of 25 (d) #super[1]H NMR spectra showing characteristic xanthate peaks. ])<fig:xanthateendgroup>
#figure(image("Images/C4S11.png", width: 100.0%), placement: auto, caption: [ Linear MALDI-ToF of pMA synthesized under nitrogen. ])<fig:maldin>
#figure(image("Images/C4S12.png", width: 100.0%), placement: auto, caption: [ Linear MALDI-ToF of pMA synthesized under air. ])<fig:maldia>
GPC confirmed the polymers had nearly identical molecular weights anddispersities (@fig:xanthateendgroup c). #super[1]H NMR resultsrevealed CH#sub[3]-#strong[CH#sub[2]]-O peaks characteristic of chain end O-ethylxanthates, attached to pMA synthesized in air and under nitrogen (@fig:xanthateendgroup d). Further, these results unambiguously showed endgroup fidelity _via_ a downward shift in the xanthate functional groups inpMA relative to their ppm in the bis(xan) precursor (4.6 ppm@fig:xanthateendgroup d, 4.7 ppm @fig:bisxannmr).
=== Rapid hydrogel fabrication<rapid-hydrogel-fabrication>We then set about testing their utility in forming hydrogels. The goalfor hydrogel fabrication is to enable a hydrogel system useful fortissue engineering, namely cell culture. Hydrogels for cell cultureshould reach the critical gel point within ten minutes without agitationso that cells do not settle to the bottom of the solution. Gelationcannot be too fast, as proper mixing must occur to limitheterogeneity.@jansen2018 I hypothesized that a fast gelation ratecould be attained in water with one of the water soluble polymerssynthesized _via_ bis(xan).
#figure(image("Images/C4F4.png", width: 100.0%), placement: auto, caption: [ Structural characterization of pMA synthesized with bis(xanthates) in air and nitrogen. (a) MALDI-ToF spectra of pMA synthesized in nitrogen (Mn = 4310 g/mol). (b) MALDI-ToF spectra of pMA synthesized in air (Mn = 4396.2 g/mol). (c) GPC (DMF) chromatogram of pMA polymers with a targeted DP of 25 (d) #super[1]H NMR spectra showing characteristic xanthate peaks. ])<fig:xanthategel>
To test gelation rate, pHEA was selected as a model hydrophilic polymer.First, HEA was polymerized in water _via_ bis(xan). After ten minutes of405 nm light irradiation, a trifunctional vinyl crosslinkertrimethylolpropane triacrylate was added to be 1 wt% of the solution.The sample was again irradiated and within 1.5 min, a crosslinked gelformed. Following the successful fabrication of gels in water, severalgels were prepared by the same protocol and left to swell in PBS for 48hrs. The resultant gels were colorless and optically transparent (@fig:xanthategel a). To determine the properties of the resultanthydrogels, modulus was then characterized using parallel plate rheology.The $G'$ and $G''$ values of the gels were determined, resulting in a $G'$ of72 ± 24 Pa and $G''$ of 4.5 ± 0.9 Pa at 1 Hz (@fig:xanthategel b).$G'$ and $G''$ were observed to be frequency independent, indicating thatthe samples had reached the critical gel point defined by theWinter-Chambon criterion.@winter1986 The small standard deviation ofthese modulus values indicates the hydrogel formed had consistentnetwork properties. Therefore, using bis(xan), I was able to rapidlyfabricate soft hydrogels in water with consistent modulus values.
== Conclusion<conclusion>In this study, I demonstrated bis(xan)-mediated PI polymerizations tobe very fast and not impacted by the presence of oxygen. The polymersproduced could be chain extended with additional monomers. Finally,hydrogels were fabricated using pre-synthesized polymer and atri-functional crosslinker in ~1.5 min. This approach provides a facileroute to quick hydrogel fabrication using light in the visible spectrumat physiological temperature without radical initiators and theirdecomposition products. Overall, my approach enables an optimalcombination of rapid polymerization/gelation kinetics, and water and airtolerance while producing well defined telechelic polymers with minimalcomponents. These properties make this system suitable for preparingcustom water-soluble acrylic and acrylamide polymers used to formhydrogels to culture cells _via_ crosslinking.
#emph[MALDI-ToF spectra of pMA were collected and analyzed by Dr. CedricBobst.]