*#align(center)[#smallcaps[@fast-oxygen-tolerant-raft-polymerization-of-hydrogels[Chapter]]]* = #smallcaps[Fast, oxygen-tolerant RAFT polymerization of hydrogels] #align(center)[Publication in preparation] == Abstract Synthetic hydrogels are an attractive platform for cell culture, as they are water-rich, and they can often be used to encapsulate cells within a three-dimensional matrix under the right chemical conditions. Many hydrogels form _via_ free radical polymerization in the presence of initiating species under UV light. The combination of free radicals and UV light during polymerization can lead to decreased viability and cellular stress. Here, I demonstrate a photoiniferter strategy to polymerize hydrogel networks without exogenous initiators and UV light. We demonstrate formation of soft hydrogels with xanthate-functional polymers with visible light. Importantly, polymerization proceeds equally well under nitrogen and in ambient conditions with a range of monomers. I suggest this process as a way to diversify monomer choice for synthetic hydrogel development in the absence of UV-light and free radical initiators. == Introduction Hydrogels made from synthetic polymer precursors span a wide range of applications, from injectable redox-based hydrogels,@rodriguez-rivera2024 to antifouling coatings,@kolewe2015 to cell culture environments@jansen2022@seidlits2011 and tissue engineering.@parmar2015 Hydrogels are highly useful networks for cell culture applications due to their affinity for water, their stability at physiological conditions, and their 3D matrix structure that surrounds cells.@herrick2013@peyton2007 Most commonly, hydrogels are formed by crosslinking polymer chains into a network _via_ free radical polymerization of vinyl functional monomers, initiated by redox or UV-sensitive species. Limitations of this standard approach include toxic byproducts@xu2020@temenoff2003@rizzo2023 and high energy light,@masuma2013 both of which can be damaging to embedded cells. Michael-addition reactions of multifunctional polymers are a popular alternative to free radical-initiated network formation, though tuning the kinetics of Michael-addition reactions can be difficult within the strict confines of physiological conditions.@jansen2018 I sought to find an alternative solution to this issue by in situ reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT polymerizations are traditionally highly oxygen sensitive and polymerize slowly, so they are rarely used for cell applications.@niu2017 The goal of my work was to explore whether specialized RAFT polymerizations could be effectively tuned to create soft hydrogels quickly and in oxygen-tolerant conditions. RAFT polymerizations offer several advantages to free radical polymerizations. RAFT polymerizations use far lower radical concentrations than typical free radical polymerizations, potentially reducing oxidative stress on cells. Owing to their reversibility, they can be started and stopped at will, enabling the synthesis of complex structures and sequences through careful reaction design. Additionally, polymers with RAFT endgroups are nontoxic to cells.@pissuwan2010 A final benefit of RAFT-crosslinked hydrogels is their ability to produce highly homogeneous@wanasinghe2022 crosslinked networks with chain-end functionality, enabling the preparation of living hydrogels that can be modified after fabrication.@thang1999@cortez-lemus2021 A key limitation in the use of RAFT for biocompatible hydrogel fabrication is its high sensitivity to oxygen.@lehnen2023@bhanu1991 Oxygen is a well-known radical scavenger; but molecular oxygen is required for viable cell culture. Several techniques have been developed to impart oxygen tolerance to RAFT polymerizations such as employing alkyl amine electron donors or photoredox catalysts.@nomeir2019 Alternatively, the oxygen can be overwhelmed with excess radicals from the initiating species. Here, I have taken inspiration from this last concept by exploiting the high radical flux produced by xanthates in photoiniferter (PI)-RAFT polymerization to rapidly consume environmental oxygen during polymerization, followed by rapid reoxygenation by diffusion to maintain cell viability.@lehnen2022 The photoiniferter (PI) concept was recently described by Otsu,@otsu2000 and it uses molecules that can simultaneously act as #strong[ini]tiator, trans#strong[fer] agent, and #strong[ter]minator. This means no exogenous initiators are needed, which simplifies reaction set-up and reduces concerns of byproduct cytotoxicity. Given that xanthates enable polymerization and crosslinking in air,@zhao2022 and that trithiocarbonate disulfides can mediate PI-RAFT and produce telechelic polymers,@beres2024@kerr2021 I hypothesized that xanthate disulfides would provide a facile way to produce telechelic polymers, similar to trithiocarbonate disulfides, and produce gels rapidly in open air, similar to traditional xanthates. To do this, I employed a bisxanthate proposed by Huang _et al._,@huang2024 and translated it to an aqueous system to support future cell applications. == Materials and methods === 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 isodecyl acrylate (iDA, 99%) were each passed over alumina to remove inhibitors before 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 Prakesh and Matthew.@suryaprakash2010 === Instrumentation Poly(iDA) molecular weight and dispersity were measured by GPC on an Agilent 1260 with a PL gel 5 μm guard column and three, 5 μm analytical mixed C columns (Agilent). THF was used as the eluent at a flow rate of 1 mL/min. The column was standardized with pMMA calibration standards and toluene was used as a flow marker. All other polymer molecular weights and dispersities were determined on an Agilent Tech 1260 Infinity 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 detector using a 20 μL sampling loop. Analyses were run at 50 °C using DMF with 0.01 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 a 400 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. ] ) #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]. ] ) #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]. ] ) #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]. ] ) #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]. ] ) #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]. ] ) #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]. ] ) === 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. The solution 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 Bruker spectrometer (Bruker Scientific LLC). The tubes were then placed in a photoreactor under a 405 nm lamp at 4.1 mW/cm#super[\2.] Spectra were collected at 1, 5, and 10 minutes to monitor monomer conversion. Reactions were stopped when 90+% conversion was reached. Conversion was determined 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. ] ) #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. ] ) === Synthesis of O,O-diethyl 1,2-disulfanedicarbothioate (bis(xan)) KOH (5 g) was added to anhydrous ethanol (200 mL) in a 500 mL round bottom 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 round bottom flask was stirred for three hours at room temperature. The solution was precipitated in diethyl ether (1 L) to yield potassium ethyl xanthogenate as a slightly off white solid. The solid was collected by vacuum filtration and further dried under 0.001 mbar vacuum overnight. Once dry, 10 g of the pure solid was added to a methanol (50 mL) in a 100 mL round bottom flask and dissolved. Iodine (2.5 g) was added portionwise, and the solution was stirred for three hours at room temperature. The solution was precipitated in ice cold DI water (2 L), yielding O,O-diethyl 1,2-disulfanedicarbothioate as a bright yellow solid. The solid was washed with saturated sodium thiosulfate solution and DI water, then dried in a vacuum dessicator to yield the final product. === Photopolymerization Polymerizations from methyl acrylate (MA), ethyl acrylate (EA), n-butyl acrylate (nBA), isodecyl acrylate (iDA), acrylic acid (AA), hydroxyethylacrylate (HEA), acrylamide (AAm), N,N-dimethylacrlamide (DMAa), and N-isopropylacrylamdie (NIPAM) monomers were conducted in similar fashions. Briefly, a 50 wt% solution of monomer was prepared in a 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 air or sparged with nitrogen for 15 minutes. Next, the vial was placed into a 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 4.1 mW/cm#super[2] for 30 min. A small aliquot of the resultant polymer solution was reserved to measure monomer conversion, and the remainder was diluted with dioxane. Polymers containing nBA or iDA were precipitated in cold methanol, and all other polymers were precipitated in cold hexanes. === Hydrogel preparation Bis(xan) (1 eq) and hydroxyethyl acrylate (100 eq) were vortexed in a 20 mL vial until the iniferter dissolved. Then 1 wt% trimethylolpropane triacrylate and 50wt% DI water were added resulting in precipitation of the bis(xan). Under agitation, 100 µL of the well-mixed, cloudy mixture was quickly pipetted into a 24 well plate. The hydrogel solution was then placed under a 405 nm LED lamp for ten minutes. The circular, clear gels were then removed from the wells and placed into 100 mL 1X PBS pH 7.4 buffer for 48 hours to fully swell, exchanging the PBS buffer after 24 hours. === Rheological characterization of hydrogels After they reached equilibrium swelling, the storage and loss moduli were measured by oscillatory shear rheology using a Kinexus Pro parallel plate rheometer (Netzsch, Selb, Bayern, Germany) with a 20 mm diameter platens. Each platen was affixed with 60-grit sandpaper to prevent slippage. 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 trap filled with DI water was used to maintain gel hydration. === MALDI-ToF To characterize the mass distribution of polymers, I adapted a MALDI-ToF technique described in Beres _et al._@beres2024 Briefly, we dissolved (DCTB) as the matrix (40 mg/mL), sodium trifluoroacetate (NaTFA, 1 mg/mL) as a cationizing agent, and the polymer sample (10 mg/mL) in spectroscopy grade acetonitrile. 5 μL each of matrix and sample and 1 μL of salt were mixed, and 1 μL of this mixture was deposited onto a ground steel target. Data was acquired on an Ultraflex III MALDI-TOF/TOF mass spectrometer equipped with a smartbeam laser (Bruker) operating in linear or reflectron positive ion mode. External calibration was performed using mixtures of commercial PEGs to overlap with the m/z range of interest. Spectra were generated by averaging 5,000-10,000 shots from non-overlapping positions. Data was analyzed using FlexAnalysis v3.4 and PolyTools v1.31 (Bruker). == Results and discussion === Kinetics and livingness Our goal was to develop a new approach to synthesize polymers and hydrogels rapidly, without toxic radicals and side products, in the presence of oxygen and water. If successful, this would represent a fundamentally new strategy to form polymers and hydrogels in the presence of living cells. To accomplish this, I employed xanthate-mediated PI-RAFT polymerizations because of their fast kinetics.@bowman2023@li2017@li2018a Inspired by recent work on bis(trithiocarbonates)@beres2024@kerr2021 and xanthates, I selected a xanthogen disulfide (bis(xan)) as the PI. Polymerization reactions of bis(xan) follow a traditional RAFT mechanism. 405 nm light puts bis(xan) into an excited state where it can fragment through β-scission into two thiyl radicals. These radicals can then react with vinyl monomers through a single unit monomer insertion (SUMI) pathway (@fig:pischeme).@beres2024 The addition of a monomer into bis(xan) changes its chemistry, resulting in better initiating radicals upon excitation and fragmentation relative to thiyl radicals. When the xanthates are again excited by light, a secondary carbon radical is formed that propagates rapidly. From there, the growing 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. ] ) We assessed the compatibility of bis(xan) to create polymers with a suite of acrylic monomers (@xanthatepolym) under nitrogen atmosphere in dioxane. Water insoluble monomers tested were alkyl esters, including methyl acrylate (MA), ethyl acrylate (EA), n-butyl acrylate (nBA), and isodecyl acrylate (iDA). Water soluble monomers tested included acrylates (and hydroxyethylacrylate (HEA)) and acrylamides (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 ) All polymerizations save for AAm yielded a viscous liquid after 30 minutes. For the alkyl esters, smaller pendent alkyl groups corresponded to 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 smaller peaks in the chromatogram (@fig:samplegpc). For the acrylamides, only monomers with substituted amides polymerized. AAm did not polymerize, whereas pDMAA and pNIPAAm formed readily with pDMAA showing lower dispersities than pNIPAAm (@fig:samplegpc). The amide protons are likely responsible for the large dispersity and failed polymerizations due to aminolysis reactions of bis(xan).@thomas2004 Regardless, the polymers that did form represent a wide range pendent functionality, demonstrating broad utility and applicability of bis(xan). To determine the air tolerance and suitability towards cell encapsulation of these polymerizations, the same monomers were polymerized with bis(xan), this time without nitrogen purging to remove air. Mirroring the Nitrogen condition, all polymerizations except AAm yielded a viscous liquid after 30 minutes. Further, similar molecular weights and dispersities were observed when polymerizations were conducted in air and nitrogen (@xanthatepolym). To further investigate the influence of air on these reactions, we analyzed polymerization kinetics. Ideally, polymerizations would approach full conversion within ten minutes to be useful for encapsulating cells. To this end, a MA polymerization under air or nitrogen was monitored _via_ NMR. Polymerizations were conducted in air or under 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 air displayed nearly identical kinetics, each reaching 97% conversion within 10 min. The polymerization is remarkably fast compared to prior publications 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. ] ) A brief study was then conducted to determine the impact of heat on the polymerization reactions was conducted with MA monomers. I compared reactions at 38°C and 60°C in air (@pmatemp). In comparison to pMA synthesized at 38°C, pMA synthesized at 60 °C was more narrowly dispersed. This is most likely due to the change in the RAFT equilibrium at elevated temperatures.@nwoko2025 Although not useful for cell applications, temperature could be another useful handle for controlling this 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 ) To further characterize the air tolerance, I conducted further analysis on the degree of polymerization (DP) _via_ GPC. Polymerizations of MA in air and nitrogen with a targeted DP of 25 were conducted using a monomer:bis(xan) ratio of 25:1. The resultant polymers yielded DP of 32 and 35 monomers per chain for air and nitrogen respectively (@fig:xanthatekinetics b, @xanthategpc). I then attempted to re-initiate polymerization by redissolving the pMA poymers and adding EA (1 eq. bis(xan): 1 eq. EA). Successful chain extension was demonstrated for both samples _via_ the shift to earlier elution times (@fig:xanthatekinetics b). Interestingly, the final DP measured by GPC were nearly twice as high as the targeted DP in both air and under nitrogen. This behavior is consistent with prior reports studying bis(trithiocarbonates),@beres2024 though the reason for this doubling of DP is not clear. Despite the higher than predicted DP, I was able to produce living polymers capable 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 ) === End group fidelity of polymers To confirm the telechelic nature of polymers resulting from synthesis via bis(xan), I analyzed the structure of pMA _via_ MALDI-ToF, GPC, and NMR (@fig:xanthateendgroup). For these reactions, pMA was synthesized at a target DP of 25 (25:1 MA:bis(xan)), either in air or nitrogen, and characterized by MALDI-ToF (@fig:xanthateendgroup a,b). MALDI-ToF showed that pMA polymerized through a photoiniferter approach with bis(xan) is well controlled, with polymerizations done in air and in nitrogen having a dispersity of 1.11 (@fig:maldin and @fig:maldia). The MALDI-ToF spectra also showed air and nitrogen synthesized polymers to have very similar molecular weights (Mn = 4584 g/mol in nitrogen, M#sub[n] = 4821 in air). Additionally, MALDI-ToF analysis detected the sodiated forms of both 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. ] ) #figure(image("Images/C4S11.png", width: 100.0%), placement: auto, caption: [ Linear MALDI-ToF of pMA synthesized under nitrogen. ] ) #figure(image("Images/C4S12.png", width: 100.0%), placement: auto, caption: [ Linear MALDI-ToF of pMA synthesized under air. ] ) GPC confirmed the polymers had nearly identical molecular weights and dispersities (@fig:xanthateendgroup c). #super[1]H NMR results revealed CH#sub[3]-#strong[CH#sub[2]]-O peaks characteristic of chain end O-ethyl xanthates, attached to pMA synthesized in air and under nitrogen (@fig:xanthateendgroup d). Further, these results unambiguously showed end group fidelity _via_ a downward shift in the xanthate functional groups in pMA relative to their ppm in the bis(xan) precursor (4.6 ppm @fig:xanthateendgroup d, 4.7 ppm @fig:bisxannmr). === Rapid hydrogel fabrication We then set about testing their utility in forming hydrogels. The goal for hydrogel fabrication is to enable a hydrogel system useful for tissue engineering, namely cell culture. Hydrogels for cell culture should reach the critical gel point within ten minutes without agitation so that cells do not settle to the bottom of the solution. Gelation cannot be too fast, as proper mixing must occur to limit heterogeneity.@jansen2018 I hypothesized that a fast gelation rate could be attained in water with one of the water soluble polymers synthesized _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. ] ) To test gelation rate, pHEA was selected as a model hydrophilic polymer. First, HEA was polymerized in water _via_ bis(xan). After ten minutes of 405 nm light irradiation, a trifunctional vinyl crosslinker trimethylolpropane triacrylate was added to be 1 wt% of the solution. The sample was again irradiated and within 1.5 min, a crosslinked gel formed. Following the successful fabrication of gels in water, several gels were prepared by the same protocol and left to swell in PBS for 48 hrs. The resultant gels were colorless and optically transparent (@fig:xanthategel a). To determine the properties of the resultant hydrogels, modulus was then characterized using parallel plate rheology. The $G'$ and $G''$ values of the gels were determined, resulting in a $G'$ of 72 ± 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 that the samples had reached the critical gel point defined by the Winter-Chambon criterion.@winter1986 The small standard deviation of these modulus values indicates the hydrogel formed had consistent network properties. Therefore, using bis(xan), I was able to rapidly fabricate soft hydrogels in water with consistent modulus values. == Conclusion In this study, I demonstrated bis(xan)-mediated PI polymerizations to be very fast and not impacted by the presence of oxygen. The polymers produced could be chain extended with additional monomers. Finally, hydrogels were fabricated using pre-synthesized polymer and a tri-functional crosslinker in ~1.5 min. This approach provides a facile route to quick hydrogel fabrication using light in the visible spectrum at physiological temperature without radical initiators and their decomposition products. Overall, my approach enables an optimal combination of rapid polymerization/gelation kinetics, and water and air tolerance while producing well defined telechelic polymers with minimal components. These properties make this system suitable for preparing custom water-soluble acrylic and acrylamide polymers used to form hydrogels to culture cells _via_ crosslinking. #emph[MALDI-ToF spectra of pMA were collected and analyzed by Dr. Cedric Bobst.]