Polymer Self-Assembly Induced Enhancement of Ice Recrystallization Inhibition

Article abstract of DOI:10.1021/jacs.1c01963

Ice binding proteins modulate ice nucleation/growth and have huge (bio)technological potential. There are few synthetic materials that reproduce their function, and rational design is challenging due to the outstanding questions about the mechanisms of ice binding, including whether ice binding is essential to reproduce all their macroscopic properties. Here we report that nanoparticles obtained by polymerization-induced self-assembly (PISA) inhibit ice recrystallization (IRI) despite their constituent polymers having no apparent activity. Poly(ethylene glycol), poly(dimethylacrylamide), and poly(vinylpyrrolidone) coronas were all IRI-active when assembled into nanoparticles. Different core-forming blocks were also screened, revealing the core chemistry had no effect. These observations show ice binding domains are not essential for macroscopic IRI activity and suggest that the size, and crowding, of polymers may increase the IRI activity of "non-active"polymers. It was also discovered that poly(vinylpyrrolidone) particles had ice crystal shaping activity, indicating this polymer can engage ice crystal surfaces, even though on its own it does not show any appreciable ice recrystallization inhibition. Larger (vesicle) nanoparticles are shown to have higher ice recrystallization inhibition activity compared to smaller (sphere) particles, whereas ice nucleation activity was not found for any material. This shows that assembly into larger structures can increase IRI activity and that increasing the "size"of an IRI does not always lead to ice nucleation. This nanoparticle approach offers a platform toward ice-controlling soft materials and insight into how IRI activity scales with molecular size of additives.

Full text of DOI:10.1021/jacs.1c01963

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Article
Polymer Self-Assembly Induced Enhancement of Ice
Recrystallization Inhibition
Panagiotis G. Georgiou, Huba L. Marton, Alexander N. Baker, Thomas R. Congdon, Thomas F. Whale,
and Matthew I. Gibson*
Cite This: J. Am. Chem. Soc. 2021, 143, 7449−7461
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ABSTRACT: Ice binding proteins modulate ice nucleation/
growth and have huge (bio)technological potential. There are
few synthetic materials that reproduce their function, and rational
design is challenging due to the outstanding questions about the
mechanisms of ice binding, including whether ice binding is
essential to reproduce all their macroscopic properties. Here we
report that nanoparticles obtained by polymerization-induced self-
assembly (PISA) inhibit ice recrystallization (IRI) despite their
constituent polymers having no apparent activity. Poly(ethylene
glycol), poly(dimethylacrylamide), and poly(vinylpyrrolidone)
coronas were all IRI-active when assembled into nanoparticles.
Different core-forming blocks were also screened, revealing the
core chemistry had no effect. These observations show ice binding
domains are not essential for macroscopic IRI activity and suggest that the size, and crowding, of polymers may increase the IRI
activity of “non-active” polymers. It was also discovered that poly(vinylpyrrolidone) particles had ice crystal shaping activity,
indicating this polymer can engage ice crystal surfaces, even though on its own it does not show any appreciable ice recrystallization
inhibition. Larger (vesicle) nanoparticles are shown to have higher ice recrystallization inhibition activity compared to smaller
(sphere) particles, whereas ice nucleation activity was not found for any material. This shows that assembly into larger structures can
increase IRI activity and that increasing the “size” of an IRI does not always lead to ice nucleation. This nanoparticle approach offers
a platform toward ice-controlling soft materials and insight into how IRI activity scales with molecular size of additives.
Poly(vinyl alcohol) (PVA) is established as a uniquely
potent polymeric IRI, even though its exact mechanism of
action is still under study.27,28 Graphene oxide has been
reported to interact with ice,29−31 and the self-assembly of
saffranine leads to ice binding aggregates.32,33 Ben and co-
workers have shown that short glycopeptides,34 or even some
surfactants,35 can inhibit ice growth but do not lead to altered
ice crystal morphologies. It has been discovered that thermal
hysteresis activity (a macroscopic property which requires ice
face binding) does not scale with IRI activity in several ice
binding proteins,36,37 and hence there is increasing evidence
that ice binding is not a prerequisite for the macroscopic effect
of IRI. Removing the need for ice binding (which needs
precision placement of the interacting groups) would simplify
INTRODUCTION
■
Ice binding proteins (IBPs)1,2 (and also polysaccharides3,4) are
a diverse class of biological macromolecules that achieve the
molecular recognition of ice crystals in the presence of a large
excess of water. INPs include those classed as antifreeze
proteins (AFPs), which can depress the freezing point of water
(and can also raise the melting point)5 and are ice
recrystallization (growth) inhibitors (IRI). Ice nucleating
proteins (INPs) that promote ice formation are another
distinct class.6−8 There are a huge range of technological
challenges where IBPs (or synthetic mimics) could have
impact, spanning the cryopreservation of donor cells and
tissues,9−13 protecting infrastructure against freeze/thaw
damage,14,15 and improving the texture of frozen foods.16,17
Inspired by IBPs, there has been significant interest in the
development of synthetic materials that can modulate ice
growth and/or materials that can enhance cellular cryopre-
servation.18−22 Synthetic materials have advantages in terms of
scalability, cost, and tunability compared to biological ones, but
there is real need to understand their function and to address
questions around how the size and nanoscale dimensions may
impact activity.23−26
Received: February 19, 2021
Published: May 4, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Synthesis of PEG-corona PISA-derived nanoparticles. (A) Synthesis of PEG45-b-PDAAmn diblock copolymer nanoparticles via aqueous
RAFT-mediated PISA using a commercially available PEG45 macro-CTA ([solids] = 10% w/w, [NaCl] = 0.05 M). (B) Normalized SEC RI
molecular weight distributions. Mn,SEC and ĐM values were calculated from PMMA standards with DMF + 5 mM NH4BF4 as the eluent. (C)
Evolution of Mn (black circles) and ĐM (red circles) values with increasing targeted DPPDAAm. (D) Zeta potential values for PEG45-b-PDAAmn
diblock copolymer nanoparticles. (E) Representative dry-state images of PEG45-b-PDAAmn diblock copolymer nanoparticles, stained with 1 wt %
uranyl acetate (UA) solution. (F) Intensity-weighted size distributions along with average Dh and PD values (the error shows the standard
deviation from five repeat measurements).
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the design process and enable a new range of IRI-active
materials to emerge with potentially easier design rules.
In the absence of ice binding, there is emerging evidence
that materials with hydrophobic patches can display moderate
IRI (but less than AFPs)38 such as self-assembled metal-
lohelices,39 ROMP-derived polymers,40 or nanocellulose.41
Balcerzak et al. synthesized a library of sequentially modified
lysine derivatives showing increased hydrophobicity lead to
more IRI.42 In many cases, IRI activity increases with
molecular weight, observed for proteins43 and polymers,27
and in self-assembly.32,44 However, it is more complicated than
“more polymers on a surface, or larger structures, are always
better”; the incorporation of PVA (IRI active and ice binder)
as the corona of micelles45,46 or grafted to gold nanoparticles47
led to no increase in activity, potentially due to the low surface
density of the polymers. Similarly, dendrimer or gold
nanoparticle conjugated AFPs showed no enhancement on a
per-protein subunit basis but on a molar basis led to large
enhancements in both IRI and thermal hysteresis activity.48,49
Ice nucleating proteins (a distinct class of IBPs) have to be
sufficiently large to match the minimum ice cluster size,50,51
and it is proposed that a key difference in activity between
larger ice nucleating proteins and smaller antifreeze proteins is
their size.23,52,53 Considering this evidence, it is clear that the
impact of macromolecular size, but also crowding, plays a role
in understanding IRI (but also other properties such as thermal
hysteresis) but that there are size limits on what can be
obtained by using linear polymers; increasing the size of
proteins (e.g., linear assemblies of repeat units) is not practical
and will not lead to identical folding or display of surfaces.
Hence, there is a need to discover new platforms where
nanoscale dimensions and composition can be tuned.
Polymerization-induced self-assembly (PISA) is a powerful
approach for in situ development of block copolymer
nanoparticles to generate multivalent nanomaterials at high
concentrations of controlled morphology and size.54−56 During
aqueous PISA, a water-soluble corona-forming block is chain-
extended by using specific water-miscible (dispersion PISA) or
water-immiscible (emulsion PISA) monomers that gradually
form a second, insoluble core block (as the length of the core-
forming block increases), resulting in nanoparticle formation.
Higher-order morphologies such as worm-like micelles and
polymersomes are typically being observed, resulting in higher
packing density for the copolymer chains compared to
spherical (micellar) particles.57,58 The versatility of this
method has allowed the synthesis of nanoparticles for drug
delivery,59−62 cell storage,63−65 and permeable nanoreac-
tors,66−69 and has been extensively reviewed.70−73 PVA-graft-
macroinitiators have been reported to lead to polymer
nanoparticles with enhanced IRI (compared to the already
very potent IRI activity of PVA), potentially due to the high
local PVA density arising from PISA.74 However, the use of
nanoparticle engineering to discover emergent AFP mimetic
materials from components with no intrinsic IRI activity has
not been explored.
providing a previously unreported mechanism to generate
IRI-active materials. By use of complementary ice nucleation
assays, it was found that the size increase did not substantially
increase ice nucleation activity, showing that making IRI-active
materials larger does not lead to ice nucleation in all cases. This
demonstrates that self-assembled nanomaterials and multi-
valent presentation is a crucial tool to understand, and translate
to application, ice growth modifiers.
RESULTS AND DISCUSSION
■
To investigate the hypothesis that aggregation and crowding of
non-ice binding polymers in a nanoparticle format may induce
IRI activity, a PISA strategy was followed to obtain polymer
nanoparticles with tunable sizes and morphologies. Deionized
water gives false positives in the ice growth assays used
(below) as a eutectic phase where ice/water coexistence is
essential, and hence saline solutes (or other solutes) are
required.38 Therefore, to perform PISA, we first chose to use
diacetone acrylamide (DAAm) as the core-forming monomer,
which we have recently discovered to promote saline stability
in PISA formulations (Figure 1A), unlike many other
monomers.74 A series of aqueous dispersion polymerizations
in the presence of [NaCl] = 0.05 M were performed for DAAm
via thermally initiated RAFT polymerization using a PEG-
based macromolecular chain transfer agent (macro-CTA,
PEG45 mCTA, Mn,SEC = 4.1 kg mol−1, ĐM = 1.1) as the
corona-forming polymer. For these experiments the concen-
tration of monomer was held constant at 10% w/w, the
[macro-CTA]:[initiator] ratio was maintained at 1:0.1, and the
[monomer]/[macro-CTA] ratio was varied to target different
core-block degrees of polymerization. The polymerizations
were carried out at 60 °C with 2,2′-azobis(2-methyl-
propionamidine) dihydrochloride (V-50) as the water-soluble
initiator. A gradual turbidity increase was noticed for
polymerization solutions with increasing DPPDAAm, indicating
the onset of particle formation typically observed in PISA.
Quantitative monomer conversions (>95%) were achieved in
1
all cases, as determined by H NMR spectroscopic analysis in
methanol-d4 of the crude samples (Figure S5).
SEC analysis of PEG45-b-PDAAmn diblock copolymers in
DMF + 5 mM NH4BF4 revealed the controlled character of the
aqueous RAFT-PISA process (Figure 1B), with symmetric,
monomodal molecular weight distributions shifting linearly
toward higher molecular weight (Mn) values upon increasing
the DP of PDAAm. A small low Mw shoulder of unconsumed
macro-CTA was apparent, due to a small amount of
unfunctionalized PEG. Calculated Mn values agreed well with
theoretically expected values, while dispersity values remained
relatively low for the polymerizations targeting DPs of 12, 25,
50, and 100 (ĐM ≤ 1.6) but was higher for DP 150 (ĐM = 2.1)
throughout (Figure 1C and Table S1). The absence of charges
on the outer surface of the obtained nanoparticles was
confirmed by electrophoretic analysis at neutral pH (measured
zeta potential < 5 mV) (Figure 1D).
DLS analysis of PEG45-b-PDAAmn formulations revealed the
formation of particles with multiple populations and high
polydispersity (PD) values for the lower DPs of PDAAm,
consistent with worm-like micelles or nanoparticles with mixed
morphologies.54,75 Single particle populations with low PD
values were observed for DPPDAAm ≥ 50, indicating the
formation of uniform assemblies (Figure 1F). A single-
exponential decay, smooth autocorrelation function with
optimal signal-to-noise ratio was also recorded (Y-intercept
Considering the above, we herein report the unexpected
enhancement of IRI activity of polymerization-induced self-
assembly derived nanoparticles from constituent components
with no IRI activity of their own. A panel of nanoparticles with
variation in size, corona-forming, and core-forming block
chemistry/functionality were synthesized. In each case IRI
activity was unexpectedly increased relative to the homopol-
ymer, but only in the largest (vesicular) nanoparticles,
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>0.9) for all formulations (Figure S6). Dry-state TEM
supported the DLS findings and showed an evolution in
morphology as the DP of the core-forming PDAAm block
increased from spheres (DPPDAAm = 12) to mixed morphol-
ogies of worms and spheres (DPPDAAm = 25) to mixed worms,
bilayer lamella and vesicles (DPPDAAm = 50) to perforated
vesicles (DPPDAAm = 100), and finally to single-phase vesicles
of uniform size (DPPDAAm = 150) (Figure 1E and Figure S7).
Cryogenic TEM analysis (cryo-TEM) was also performed on
PEG45-b-PDAAM100 vesicles and confirmed a perforation with
uniform pores on the surface of the vesicles.76,77 It has
previously been reported that PISA diblock formulations with
PDAAm as core-forming block have a composition-dependent
thermoresponsive behavior.78−80 Therefore, PEG45-b-
PDAAm100 was also synthesized by using 2,2′-azobis[2-(2-
imidazolin-2-yl)propane] dihydrochloride (VA-044) at lower
temperature, and its morphology was studied via TEM imaging
(see Figure S8). This revealed uniform spherical vesicles being
formed with perforated bilayer surface with fewer and less
uniform pores but indicates that it is not a temperature-
induced effect, but its investigation is beyond the scope of this
work.
Next, IRI activity was assessed by the “splat” assay. This
assay involves seeding a large number of small ice crystals,
which are then allowed to grow at −8 °C.27,81 IRI-active
materials will slow ice growth, resulting in smaller crystals
relative to the negative control (saline), and the results are
plotted as the mean grain size (MGS). To conduct these
assays, it is essential to have some salt to prevent false negatives
(any additive in pure water in this assay leads to ice growth
inhibition).38,81 To ensure colloidal stability, 0.05 M NaCl was
used here (as opposed to the more common 0.137 M), which
was validated to not produce false positive/negatives in the IRI
assays, but this lower concentration must be considered when
comparing to other reported activities as it is non-identical for
each saline concentration (see Figure S9). Figure 2A shows
dose−response curves for the PEG-based particles, compared
to the PEG macroinitiator, with concentrations normalized to
PEG concentration (as the coronal component) to enable
comparison of multivalent effects. Remarkably, all the
formulations showed some IRI activity. Considering PEG is
widely used as a negative control (as it does not bind ice), the
induction of observable IRI was unexpected. It is important to
note that any material of sufficiently high concentration can
inhibit ice growth, and this is not an on/off property.38 Larger
particles (Dh = 350 and 730 nm corresponding to DPPDAAm of
100 and 150, respectively) showed higher activity than the
smaller (Dh = 42 nm for DPPDAAm = 12), demonstrating that
size and potential packing of the corona (due to less curvature
for large particles compared to small) are crucial to IRI activity,
although this cannot be proved at this stage. The largest
particles inhibited all growth at a PEG concentration of 5 mg
mL−1, placing them in the medium activity range of antifreeze
protein mimetics (less active than PVA or antifreeze
proteins).38,43 Cryomicrographs of annealed ice wafers (Figure
2B) visualize this unexpected activity and clearly demonstrate
that a self-assembly strategy to induce IRI activity into soft
materials is possible and that particle size is a tool to control
the magnitude of activity.38 In the case of PEG45-b-PDAAm100
vesicles with perforated topologies, their IRI activity was
compared with a formulation synthesized at lower temperature
and showed no significant difference in IRI activities (Figure
S10). To further explore the dynamics of this system, PEG45-b-
Figure 2. Assessment of ice recrystallization inhibition activity for
PEG-based nanoparticles. (A) IRI activity summary of PEG45-b-
PDAAmn nanoparticles corrected to [PEG]. Error bars are SD from
a minimum of three repeats (key: S, spherical micelles; W, worm-like
micelles; V, vesicles). The percent mean grain size (MGS) was
reported relative to saline control ([NaCl] = 0.05 M). (B) Example
cryomicrographs of ice wafers from the “splat” assay of PEG45-b-
PDAAmn nanoparticles, [PEG] = 10 mg mL−1.
PDAAM100 block copolymer vesicles were annealed for
different time points (t = 30 and 60 min) showing complete
inhibition even after this time (Figure S13). Colloidal stability
for PEG45-b-PDAAm150 vesicles upon multiple freeze−thaw
cycles was assessed by DLS analysis and showed the particles
were recovered with no significant changes in their hydro-
dynamic diameter (Dh) (Figure S11). The IRI activity as a
function of total solid (polymer) concentration is also plotted
in Figure S12. This shows that even when the core-polymer
mass is included (which is not in contact with ice/water), the
nanoparticles are still more active than a PEG (corona) alone
on a mass basis. The larger particles were also still more active,
and hence the activity is not an artifact of concentration
calculations and the nanoparticle size is a key criteria. To
further demonstrate the IRI activity, an alternative assay was
conducted in 45 wt % sucrose (“sucrose sandwich”) which has
a lower ice fraction.43 Figure S14 shows nucleated ice crystals,
which after 30 min have grown significantly (recrystallized) in
the presence of PEG45 macro-CTA, while Figure S15 shows
that a solution containing 10 mg mL−1 of PEG45-b-PDAAm150
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Figure 3. Synthesis, characterization, and ice recrystallization inhibition activity of PEG-coronal nanoparticles using PiBuOMAm- and PDAAm-
based cores. (A) PEG45-b-PiBuOMAm100, (B) PEG113-b-PDAAm100 (i), and PEG113-b-PDAAm400 vesicles (ii) synthesized at [solids] = 10% w/w
via aqueous RAFT-mediated PISA in the presence of [NaCl] = 0.05 M with commercially available PEG45 and PEG113 macro-RAFT agents. Insets
show intensity-weighted size distributions along with average Dh and PD values (the error shows the standard deviation from five repeat
measurements) and representative dry-state TEM images stained with 1 wt % uranyl acetate (UA) solution. (C) IRI activity summary corrected to
[PEG].
nanoparticles displays inhibition of ice recrystallization,
demonstrating activity in a range of different formulations.
To evaluate whether the observations made above were only
due to the PEG corona (as the component with most solvent
interactions), a higher Mw PEG macro-CTA (PEG113 mCTA)
was employed, and the composition of the core-forming block
was separately examined. Few PISA core-forming monomers
are reported to be saline-tolerant.82 We employed an
acrylamide-based monomer that can undergo PISA, N-
(isobutoxymethyl)acrylamide (iBuOMAm), which was se-
lected based on in silico computation/experimental study by
O’Reilly and co-workers.83 iBuoMAm was polymerized via
aqueous RAFT in the presence of [NaCl] = 0.05 M by using
PEG45 mCTA at 10% w/w solids content targeting a
DPPiBuoMAm of 100. The prepared PEG45-b-PiBuOMAm100
diblock copolymer possessed bimodal molecular weight
distribution due to unconsumed PEG mCTA with relatively
spherical nanoparticles and uniform size (Figure S12D). Cryo-
TEM imaging of PEG45-b-PiBuOMAm100 nanoparticles in
solution confirmed the formation of perforated spherical
nanoparticles (Figure S16E). A higher Mw PEG macroinitiator
(PEG113 mCTAto probe effect of coronal chain length) was
subsequently used for the polymerization of DAAm to yield
PEG113-b-PDAAm100 and PEG113-b-PDAAm400 (to enable
comparison of the same morphology with PEG45-b-
PDAAM100 vesicles) diblock copolymer nanoparticles. TEM
and DLS analyses showed the formation of uniform spherical
nanoparticles and unilamellar vesicles for DPPDAAm = 100 and
400, respectively (Figure 3B, Figures S17 and S18), with
monomodal size distributions, mean hydrodynamic diameters
(Dh) of 96.6 1.8 nm (DPPDAAm = 100) and 280.8 8.2 nm
(DPPDAAm = 400), and low polydispersities (PD = 0.20 0.04
and 0.08 0.04). These particles were tested for IRI and were
found to show nearly identical activity to those that had
PDAAm cores (above). Additionally, PEG113-b-PDAAm400
vesicles showed higher IRI activities compared to PEG45-b-
PDAAm100 vesicles. This experiment proves that the nano-
particle size and morphologies were crucial for IRI activity, but
not the nature of the core-forming block (other than its role in
ensuring saline stability), primarily because of its inaccessibility
by the solvent molecules (Figure 3C).
low dispersity value, as determined by SEC analysis (Mn,SEC
=
25.1 kg mol−1, ĐM = 1.6) (Figure S16). Dynamic light
scattering (DLS) analysis revealed the formation of nano-
particles with a monomodal size distribution and mean
hydrodynamic diameter (Dh) of 357.9
1.3 nm and low
polydispersity (PD = 0.24 0.01) (Figure 3A). Dry-state
transmission electron microscopy (TEM) imaging confirmed
the development of uniform PEG45-b-PiBuOMAm100 porous
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Figure 4. Summary of PISA formulation using non-PEG-based coronas. (A) Synthesis of PVP40-b-PDAAmn diblock copolymer nanoparticles via
aqueous RAFT/MADIX-mediated PISA. PVP40 macro-CTA was synthesized by photoinitiated RAFT polymerization ([solids] = 20% w/w, [NaCl]
= 0.05 M). (B) Representative dry-state TEM images of PVP40-b-PDAAmn diblock copolymer nanoparticles, stained with 1 wt % uranyl acetate
(UA) solution. (C) Normalized intensity-weighted size distributions obtained by DLS. (D) Zeta potential values for PVP40-b-PDAAmn diblock
copolymer nanoparticles. (E) Synthesis of PDMAC40-b-PDAAm200 diblock copolymer vesicles via aqueous RAFT-mediated PISA using a
PDMAC40 macro-CTA ([solids] = 20% w/w, [NaCl] = 0.05 M). (F) (i) Representative dry state and (ii) cryo-TEM images; (iii) intensity-
weighted size distribution along with average Dh and PD values (the error shows the standard deviation from five repeat measurements). Inset:
autocorrelation function.
With the above observation of PEG activity and con-
firmation that the nature of core was not crucial, we envisioned
that introducing a different corona composition, as it is the
component that is in direct contact with the aqueous/ice
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Figure 5. Ice recrystallization inhibition (IRI) activity of PVP and PDMAC-based nanoparticles. (A) PVP40-b-PDAAmn nanoparticles corrected to
total PVP concentration in particle format. (B) PVP40-b-PDAAmn nanoparticles at [PVP] = 10 mg.mL−1. (C) PDMAC40-b-PDAAm200 diblock
copolymer vesicles and PDMAC40 corrected to [PDMAC]. (D) Comparison of IRI activity for diblock copolymer vesicles of PVP40-b-PDAAm200
,
PDMAC40-b-PDAAm200, and PEG45-b-PDAAm150. Error bars are SD from a minimum of three repeats. The percent mean grain size (MGS) was
reported relative to saline control. [PVP] was recalibrated based on chain extended PVP40 present particle format after estimating amount of
unconsumed PVP40 macro-CTA based on Table S3 data.
1
phases, would result in different IRI activities. PEG is not
known to present IRI, so as it was important to compare it to
other PISA-derived particles with a linear non-ionic hydro-
philic corona-forming polymer, with similar repeat unit
dimensions (to avoid comb-type PEG-(meth)acrylates which
might affect packing density). Here, poly(vinylpyrrolidone)
(PVP) was employed for the first time as a steric stabilizing
block for performing PISA by using DAAm as the core-forming
monomer (Figure 4). Initially, N-vinylpyrrolidone (NVP) was
polymerized by using xanthate chain transfer of 2-(ethoxy-
carbonothioyl)sulfanyl propanoate (EXEP) (see Supporting
Information for full synthetic procedure, Figures S3 and S4)
via photoinitiated RAFT/MADIX to ensure maximum end-
group fidelity (PVP40, Mn,SEC RI = 4600 g mol−1, ĐM = 1.3)
(Figure S19).84,85 Then, a series of aqueous dispersion
polymerizations in the presence of [NaCl] = 0.05 M were
performed for DAAm via RAFT by using the synthesized
PVP40 macromolecular chain transfer agent. The concentration
of monomer was held constant at 20% w/w, and the
[monomer]/[macro-CTA] ratio was varied to target DPPDAAm
= 25, 50, 100, and 200. A gradual turbidity increase was
after 6 h, as determined by H NMR spectroscopic analysis in
methanol-d4 of the crude samples (Figure S20 and Table S2).
SEC analysis of PVP40-b-PDAAmn diblock copolymers
revealed a rather uncontrolled character of chain extensions
of PVP40 macro-CTA with high dispersity values (ĐM > 2.0) as
would be expected for this macroinitiator (Figure S21).86 PVP
is known to undergo undesirable side reactions in aqueous
media, leading to poor RAFT control and incomplete
monomer conversions,87,88 and it can only be readily
copolymerized with a limited set of other lesser activated
monomers (LAM), such as vinyl acetate or acrylics,89,90 but no
LAM dispersion PISA monomer is known, necessitating this
approach. It is crucial to note that a relatively high proportion
of PVP stabilizer chains remained unreacted at the end of the
polymerization, leading to bimodal molecular weight distribu-
tions and is discussed in terms of the IRI activity below. Peak
deconvolution was also performed on SEC curves for resulting
block copolymer nanoparticles (Figures S23−S26), and the
ratios of the areas between chain extended and unconsumed
PVP macro-CTA are summarized in Table S3.
Dry-state TEM analysis showed an evolution in morphology
upon increasing the DP of the core-forming block from spheres
(DPPDAAm = 25 and 50) to mixed morphologies of spheres and
vesicles (DPPDAAm = 100) to pure single-phase vesicles of
uniform size (Figure 4B). For DPPDAAm = 100, the appearance
noticed for polymerization solutions with increasing DPPDAAm
,
indicating the onset of particle formation. Quantitative
monomer conversions (>97%) were achieved in all cases
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Figure 6. Ice binding and ice nucleation analysis of nanoparticles. (A) Modified “sucrose sandwich” ice shaping assay images for 1 mg mL−1 of
PEG45-b-PDAAm100 nanoparticles in 45 wt % sucrose solution; (B) 1 mg mL−1 of PVP40 and (C) 1 mg mL−1 of PVP40-b-PDAAm200. (D)
Assessment of ice nucleation activity in microliter droplets showing immersion mode fraction frozen curves as a function of temperature for
droplets containing 1 mg mL−1 PEG45-b-PDAAm150, PDMAC40-b-PDAAm200, and PVP40-b-PDAAm200 along with their corresponding macro-
CTAs (E) and PVP40-b-PDAAmn (n = 25, 50, 100, 200) nanoparticle series. Example temperature errors bars are included on selected data points
on panels D and E.
of mixed spheres and vesicles can be attributed to the high
dispersity values with a large proportion of PVP stabilizer
chains being chain-extended to different degrees. Cryo-TEM
analysis further confirmed the resulting morphologies (Figure
S27). DLS analysis supported TEM findings and revealed the
stable formation of particles with single populations and low
polydispersity (PD) values (Figure 4C and Figure S28). The
absence of charges on the outer surface of the obtained
nanoparticles was also confirmed by electrophoretic analysis at
neutral pH (measured zeta potential < 5 mV) (Figure 4D).
A stabilizing block of poly(N-dimethylacrylamide)
(PDMAC) was also employed to perform PISA by using
DAAm as the core-forming monomer (Figure 4E). DMAC was
initially polymerized in dioxane at 70 °C by using 2-
(((butylthio)carbonothiolyl)thio)propanoic acid as the chain
transfer agent (see Supporting Information for full synthetic
solids in the presence of [NaCl] = 0.05 M, targeting a final
DPPDAAm of 200. This system has been extensively studied in
the past, and previous synthetic procedures were followed to
achieve vesicular morphologies.79,80,91−93 Quantitative mono-
mer conversion (>98%) was achieved after 6 h, as determined
by 1H NMR spectroscopic analysis in methanol-d4 of the crude
sample (Figure S31). The prepared PDMAC40-b-PDAAm200
diblock copolymer possessed monomodal molecular weight
with relatively low dispersity value, as determined by SEC
analysis in DMF + 5 mM NH4BF4 (Mn,SEC = 31600 g mol−1,
ĐM = 1.5) (Figure S32). Dynamic light scattering (DLS)
analysis revealed the formation of nanoparticles with a
monomodal size distribution and mean hydrodynamic
diameter (Dh) of 308.8
5.5 nm and low polydispersity
(PD = 0.12 0.02) (Figure 4Fiii). Dry-state and cryo-TEM
imaging finally confirmed the development of single-phase
unilamellar vesicles (Figure 4Fi,ii).
Figure 5 shows the IRI activity of the non-PEG-based
corona formulations. For PVP, the same particle size
dependence on activity as seen for PEG was observed with
larger particles (vesicles) being more active than smaller ones
procedure, Figures S5 and S6). The resulting homopolymer
1
was analyzed by H NMR and SEC (PDMAC40, Mn,SEC RI
=
4800 g mol−1, ĐM = 1.1) (Figure S30). This water-soluble
polymer precursor was chain-extended with DAAm via RAFT
aqueous dispersion polymerization at 60 °C and 20% w/w
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(micelles), and both were more active than the macro-CTA
alone. It is crucial to note that due to inefficient macro-
initiation (see above), the PVP particles as synthesized
included a population of free polymer chains. The particle
IRI activity (shown in Figure 5) was determined after removal
of unreacted PVP40 macro-CTA (by centrifugation, Figures
S21 and S22). The presented PVP concentration was
determined from deconvolution of the SEC traces (Figures
S23−S26 and Table S3). IRI activity before removing excess
PVP40 is also shown in Figure S29 for comparison. PDMAC-
based particles also showed enhancement relative to the
homopolymer alone, with the magnitude similar to the PVP-
based particles (Figure 5C). Comparing vesicular particles
whereby the PDAAm core-block DP was similar (Figure 5D)
shows that all presented similar activity, and hence the
formation of self-assembled structures rather than the chemical
nature of the corona-forming block is the key requirement. All
the data are summarized (independent of chemical composi-
tion) as MGS versus diameter in Figure S33, confirming that
particle size correlated to activity. We should highlight this
does prove a mechanism but does show that nanoparticle
engineering is a tool to control the macroscopic IRI activity.
The key property investigated here, IRI, is a macroscopic
observation which could be caused by a range of molecular-
level mechanisms from single, or multiple, ice face binding or
disruption of water transfer at the ice/water interface without
actual binding.35,41,94 IRI is also known to increase with
molecular weight for many materials, and hence the effective
very high Mw of a nanoparticle could provide a simple reason
for the observations here. It is important to again highlight that
IRI is not a binary on/off property but that sufficient
concentrations of any material can induce it.38 To investigate
whether the particles were affecting ice crystal morphology,
which may indicate ice binding (as opposed to a non-specific
“size” related effect), crystals were grown in sucrose solution
(which leads to segregated ice crystals whose morphology can
be viewed). Figure 6A shows ice crystals grown with a PEG-
coronal nanoparticle formulation (PEG45-b-PDAAm100) did
not induce significant faceting (Figure S34). This fits a
hypothesis that the PEG-coronal nanoparticle activity enhance-
ment is not due to ice binding. In contrast, PVP alone (Figure
6B and Figure S35) showed some weak faceting of the crystals
and formation of dendritic morphologies. However, the PVP-
coronal nanoparticles (PVP40-b-PDAAm200) resulted in ex-
tensive dendritic ice crystals (Figure 6C and Figure S36). This
would imply that PVP, while itself only having weak (or no)
IRI activity,27 when assembled in the correct format can
engage ice or affect the morphology of ice produced. The
increased faceting could be due to the nanoparticles spanning
between multiple ice faces due to their large size. This is an
exciting observation as if ice crystal morphology can be probed
or modulated by these easy-to-make synthetic soft materials, it
opens doors to fundamental studies on the ice interface,
without needing protein engineering as well as potential
applications. It also suggests that the enhanced activity seen for
PEG/PDMAC coronas (without ice engagement) still apply
when using coronas with some affinity for ice.
nucleus in supercooled water: known as heterogeneous ice
nucleation. A feature of ice nucleating proteins, INPs (as
discussed in the Introduction), is that assembly of small
domains into larger ones is essential for activity, as macro-
molecules must be of a similar scale to the classical nucleation
theory derived critical ice cluster size to nucleate ice at a given
temperature.50,51 The ice critical cluster size increases rapidly
as temperature approaches the melting point. For example, the
critical ice cluster has a diameter of about 20 nm at −5 °C.51
This means large aggregates of (for example) bacterial ice
nucleating proteins are required to induce ice nucleation close
to 0 °C. There is theoretical evidence that molecular size is a
key criteria for ice nucleation,23 and it has been observed
experimentally that some AFPs can also nucleate ice, with the
size of the AFP being an essential descriptor.52,53 Given that
some of the polymer particles synthesized bind ice, all show
IRI and are large compared to ice critical nuclei; it might be
thought that they would be capable of nucleating ice at warm
temperatures as seen for higher molecular weight antifreeze
proteins. To test this, microliter ice nucleation assays were
undertaken by using a bespoke apparatus described in the
Supporting Information. This instrument freezes around 50
droplets per experiment to establish the spread of likely
nucleation temperatures for a given heterogeneous nucleator.
The ice nucleation temperatures of 1 mg mL−1 nanoparticle
and polymer solution are reported in Figure 6D,E. It was found
that the PEG- and PDMAC-based formulations nucleated ice
at average temperatures of −25.8 and −26.8 °C, respectively,
only slightly warmer than the average freezing temperature of
pure water on the instrument, −28.1 °C. PVP and the various
PVP-based nanoparticles tested nucleated at average temper-
atures between −23.6 and −24.5 °C. Notably, the fraction
frozen curves for PVP and PVP40-b-PDAAm200 were very
similar. While the PEG- and PDMAC-derived nanoparticles do
nucleate ice at slightly warmer temperatures than their
precursor polymers, the difference is not large. Figure 6E
shows a slight increase in nucleation temperature with
increasing particle size for PVP-derived nanoparticles;
however, the enhancement is not large. The nucleation data
show that aggregation of polymers into particles does not
substantially enhance ice nucleation effectiveness. This
supports the theory that correct long- and short-range
structure, as well as sufficient size, is required for a nucleant
to be highly effective. These combined observations support
emerging evidence that the observation macroscopic properties
of ice binding proteins are not always caused by the same
underlying molecular mechanisms36−38 and that a particular
property can be dialled into a material. There is an opportunity
to achieve desirable levels of ice growth inhibition (for a
specific application) by deploying nanoparticle formulations
such as those presented here, even if their overall activity is
substantially weaker than IBPs, and indeed their mechanism is
non-specific.
CONCLUSIONS
■
We report that biomimetic ice recrystallization inhibition
activity can be introduced into polymer nanoparticles from
components which themselves have no ice binding or
associated activity. Saline-tolerant polymerization induced
self-assembly (PISA) was exploited to obtain nanoparticles of
controlled size and morphology by using a library of different
corona-forming polymers [poly(ethylene glycol), poly(vinyl-
pyrrolidone) (for the first time via dispersion PISA), and
With the above observations that the PISA particle format
led to increased IRI activity and ice shaping, the ability of the
particles to nucleate ice was next studied. On cooling, pure
water can persist as liquid to temperatures as low as −40 °C.
This phenomenon is known as supercooling, and some
substances such as INPs are capable of inducing a stable ice
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Authors
poly(dimethylacrylamide)] and core-forming blocks [poly-
(diacetone acrylamide) and poly((isobutoxymethyl)-
acrylamide)]. In all cases, ice recrystallization inhibition was
observed despite the constituent homopolymers (i.e., corona-
forming blocks) having no activity themselves, suggesting that
size and confinement of the coronal blocks can enhance this
macroscopic property. Larger particles (vesicles) were more
active than smaller ones (micelles/spheres and intermediate
morphologies), confirming that the size and nanoarchitecture
of the particles were crucial for inducing activity. The activity
enhancement relative to linear polymers was seen both in
terms of the total coronal mass (as the solvent-contacting
component) or as the total mass (including the core), showing
this was not simply due to the increased mass applied in a
nanoparticle formulation. The core-forming blocks were also
varied, and this had minimal or no impact on the observable
ice recrystallization inhibition activity, confirming this was a
general effect. In the case of poly(vinylpyrrolidone) coronal
particles, there was observation of ice shaping, suggesting these
particles may engage specific ice faces, which was not seen with
the polymers alone. This additional binding did not lead to
significantly higher IRI activity though, suggesting IRI was
dominated by their size and packing density for each
morphology. Ice nucleation was also studied, but no activity
was seen. This was a crucial observation as it has been reported
that larger assemblies of antifreeze proteins can lead to ice
nucleation activity but that the assembly of these polymers
does not lead to ice nucleation. This may suggest a unique
mechanism, but the huge structural differences between the
particles and AFPs make a real comparison difficult. The data
here also supports emerging evidence that the magnitude of
each macroscopic property associated with ice binding proteins
mimics does not scale equally between all materials. The exact
molecular mechanism for this increased activity is not clear and
may be simply related to the overall size and molecular weight
of the particles, which exceeds what is accessible with linear
polymers. However, the PVP data supports that increased ice
binding can occur in these assemblies. These observations
show that coronal confinement of polymers can induce the
macroscopic effect of ice recrystallization inhibition but that
the same is not true for ice-nucleation activity, providing
evidence that simply enlarging IRI-active components does not
guarantee ice nucleation. While the overall magnitude of
activity here is less than ice binding proteins, there is huge
technological potential in the development of freeze-stable
colloids, exploiting this size-driven enhancement to translate to
applications, as well as a tool to study the fundamentals of ice-
binding/modifying materials.
Panagiotis G. Georgiou − Department of Chemistry,
University of Warwick, CV4 7AL Coventry, U.K.;
orcid.org/0000-0001-8968-1057
Huba L. Marton − Department of Chemistry, University of
Warwick, CV4 7AL Coventry, U.K.; orcid.org/0000-
0001-7547-6001
Alexander N. Baker − Department of Chemistry, University of
Warwick, CV4 7AL Coventry, U.K.; orcid.org/0000-
0001-6019-3412
Thomas R. Congdon − Department of Chemistry, University
of Warwick, CV4 7AL Coventry, U.K.
Thomas F. Whale − Department of Chemistry, University of
Warwick, CV4 7AL Coventry, U.K.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c01963
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
M.I.G. thanks the ERC for a Consolidator Grant (866056) and
the Royal Society for an Industry Fellowship (191037) joint
with Cytivia. This project has received funding from the
European Union’s Horizon 2020 research and innovation
programme under the Marie Skłodowska-Curie Grant Agree-
ment No. 814236. BBSRC-funded MIBTP program (BB/
M01116X/1) and Cytivia are thanked for supporting H.M.
T.F.W. thanks the Leverhulme Trust and the University of
Warwick for supporting an Early Career Fellowship (ECF-
2018-127). T.R.C. thanks the BBSRC Emerging Innovations
Grant ref BB/S506783/1. The Warwick Polymer Research
Technology Platform is acknowledged for SEC analysis, and
the Warwick Electron Microscopy Research Technology
Platform is acknowledged for TEM. We also acknowledge
the University of Warwick Advanced Bioimaging Research
Technology Platform supported by BBSRC ALERT14 Award
BB/M01228X/1 and Dr. S. Bakker for cryo-TEM. The
BBSRC-funded MIBTP program (BB/M01116X/1) and
Iceni Diagnostics Ltd. are thanked for a studentship for A.N.B.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c01963.
■
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AUTHOR INFORMATION
Corresponding Author
Matthew I. Gibson − Department of Chemistry and Warwick
Medical School, University of Warwick, CV4 7AL Coventry,
U.K.; orcid.org/0000-0002-8297-1278;
Email: m.i.gibson@warwick.ac.uk
■
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