PhD-Dissertation / Chapter4.typ

*#align(center)[#smallcaps[@fast-oxygen-tolerant-raft-polymerization-of-hydrogels[Chapter]]]*
= #smallcaps[Fast, oxygen-tolerant RAFT polymerization of hydrogels]
<fast-oxygen-tolerant-raft-polymerization-of-hydrogels>
#align(center)[Publication in preparation]

== Abstract
<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
<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
<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 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
<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.
  ]
)
<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. 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.
  ]
)
<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 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
<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
<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
<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
<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
<results-and-discussion>
=== Kinetics and livingness
<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.
  ]
)
<fig:pischeme>

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
)<xanthatepolym>

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.
  ]
)
<fig:xanthatekinetics>

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
  )
<pmatemp>

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
  )
<xanthategpc>

=== End group fidelity of polymers
<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.
  ]
)
<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 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
<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.
  ]
)
<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 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
<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.]