Developing Antifreeze Protein Mimics for
Cryopreservation
Thomas R. Congdon, Dr Matthew I. Gibson
t.r.congdon@warwick.ac.uk
www.warwick.ac.uk/go/gibsongroup
Control of Ice Crystal Size and Shape
Poly(vinyl alcohol) as an Antifreeze Agent
200 µm
PVA has been shown to be the most potent synthetic recrystallisation
inhibitor, but does not shape the ice or greatly affect the freezing
point.
200 µm
(3)
(2)
(1)
Antifreeze proteins display strong ice recrystallisation inhibition (IRI) - PVA is non-toxic and readily available, and has been recently shown to
slowing the rate of ice crystal growth (1), but causes damaging needle- greatly enhance the cryopreservation of human red blood cells.
like crystals to form, due to Dynamic Ice Shaping (2). This is due to
Deller, R.C.; M. & Gibson, M.I. et. al. Nat. Comms., 2014, 5 (3244)
adsorption of proteins onto the ice crystal surface (3), which also
Deller, R.C.; Congdon, T.; Gibson, M.I., et al Biomat. Sci., 2013, 1, 478
induces thermal hysteresis, lowering the freezing point of water.
100
(4)
The specific properties/mechanisms that make PVA such an efficient
ice recrystallisation inhibitor are currently unknown. In order to
examine the effect of molecular weight, dispersity, and the effect of
copolymers a MADIX methodology was employed (4).
DP 10
DP 20
DP 50
DP 150
DP 250
DP 350
Normalised RI Intensity
1.0
1000
10000
(6)
MW/Da
(7)
(8)
Monomers compatible with
this MADIX agent. Vinyl
Acetate (6), Isopropenyl
Acetate (7), N-vinyl
Pyrrolidone (8).
100000
(5)
100
50
50
0
0
25
50
75
Mol % Acetate Groups on PVA 350
0
0.0
0.5
1.0
-1
Concentration/ mg.ml in PBS
0.5
0.0
DP150
DP250
DP350
% MLGS compared to PBS
DP10
DP20
DP50
DP80
(9)
PVA oligomers display activity similar
to longer chain PVA, with activity
‘switching on’ between 10 and 20
repeat units (9). The inclusion of
acetate groups stops IRI activity above
25 mol% (10), whereas pyrrolidone
…groups lessen activity (11).
% MLGS comapred to PBS
% MLGS compared to PBS
Ice Recrystallisation Inhibition (IRI) Activity of PVA
Using Controlled Polymerisation to Probe
Structure/Activity Relationships
(10)
100
50
0
0
50
100
Mol % Pyrrolidone Groups on PVA 50
(11)
Congdon, T.C.; Notman, R., Gibson, M.I., Biomac, 2013, 14, (1578)
Preparation and IRI Activity of PVA Stars
IRI Activity of PVA(b)PVP Block Copolymers
The ‘living’ polymerization of vinyl pyrrolidone using MADIX allows
for the addition of blocks of another monomer to be added (14).
% MLGS comapred to PBS
100
PVA Star, DP(arm) 16
PVA 20
PVA 50
50
0
0
1
2
3
(13) Concentration/ mg.ml-1
4
These stars displayed
no greater increase in
IRI compared to
similarly sized linear
PVA, suggesting that
there is no direct
adsorption to a crystal
face (13).
Background References
Gibson, M.I., Polym. Chem., 2010, 1, 1141
O’Reilly, R.K, et. al., Macromol., 2011, 4, 886
Stenzel, M. H. et. al., Macromol. Chem. Phys., 2003, 9, 1160
% MLGS compared to PBS
(12)
Three-arm stars were prepared using a novel trifunctional MADIX
agent, which afforded stars that even at high conversion had a low
polydispersity (Đ 1.17) and no trace of a linear component (12).
PVA 10
PVA 20
PVP.PVA 20.10
PVP.PVA 20.20
PVP.PVA 20.25
100
50
0
0
1
-1
PVA Concentration/ mg.ml
(15)
Acknowledgements
Dr Matthew I. Gibson • RSC Materials Division Travel Grant
Dr Rebecca Notman • With thanks to Birmingham Science
• Dr Robert Deller
• Alaina Emmanuella
• Daniel Mitchel
2
City Advanced Materials 2
•The MIG group
3
(14)
When IRI activity is plotted
against the amount of PVA in each
polymer (15), it becomes clear
that the PVP block is not affecting
activity and, in the case of
PVP.PVA 20.10, the IRI activity is
slightly better compared to homo
PVA 10.