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Title

両性電解質高分子の構造と凍結保護効果の相関および細胞内浸透による凍結保存

Author(s)山﨑, 椋太CitationIssue Date

2024-03

Type

Thesis or Dissertation

Text version

ETD

URL

http://hdl.handle.net/10119/19076

RightsDescription

Supervisor: 松村 和明, 先端科学技術研究科, 博士

Japan Advanced Institute of Science and Technology

Doctoral Dissertation

Cryopreservation by intracellular permeation ofpolyampholytes and correlation between polymer
structure and cryoprotective effect

Ryota Yamasaki

Supervisor: Kazuaki Matsumura

Graduate School of Advanced Science and Technology
Japan Advanced Institute of Science and Technology
Materials Science

March 2024

Abstract

During cryopreservation, cryoprotectants are used to protect cells from cryoinjury. Currently, 10% dimethyl
sulfoxide (DMSO) is commonly used as a cryoprotectant. However, since DMSO is highly cytotoxic andmembrane-permeable, it must be removed promptly after freezing and thawing. Polyampholytes have highcryoprotective effects, but the cryoprotective mechanism is poorly understood. In this thesis, I focused on
elucidating the cryoprotective mechanism of polyampholytes and their application to cryopreservation using
intracellular permeation.
In Chapter 2, in addition to the previously reported poly(2-(dimethyl amino)ethyl methacrylate/methacrylic acid)(pD), I synthesized poly(vinyl acetate/acrylic acid (AA)/2-(dimethyl amino)ethyl acrylate (DMAEA)) (pV) andpoly(methyl vinyl ether/AA/DMAEA) (pM) with alternating cations and anions, and poly(2-acrylamido-2-methyl
propanesulfonic acid/(3-acrylamidopropyl)trimethyl ammonium chloride) (pA) with a higher degree of
dissociation, and correlated their cryoprotective effects and polymer structures. The cell recovery rate of thealternating array pV was the highest and comparable to that of carboxylated -poly-L-lysine (PLL). Cryoprotective
effect of pA was the lowest, and measurements of residual water volume by temperature variable solid state magic
angle spinning NMR showed that freezing of bulk water was significantly higher. In addition, the tendency of
vitrification and trapping of Na ions was observed in the higher temperature range of pV and PLL, which showed
high cryoprotective effect, suggesting that the ability to adjust salt concentration and vitrification during freezingby trapping salt or water is important for the cryoprotective effect of amphoteric electrolyte polymers.
In Chapter 3, we attempted to enhance the cryoprotective effect by utilizing the permeation of the zwitterionicpolymer poly(sulfobtaine) into cells. Furthermore, we synthesized a copolymer of DMSO, a cell-permeable
cryoprotectant, and 2-(methyl sulfinyl)ethyl methacrylate (MSEMA), which has the same methyl sulfinyl group,and confirmed its cryoprotective effect. Intracellular permeation was confirmed by the fluorescently modified
polymer, and the polymer was found to easily counter diffuse by incubating at 37 °C for about 30 minutes after
permeation. Poly(SPB) and poly(SPB/MSEMA10) improved the cell recovery rate by about 20 or 30% throughintracellular permeation. Cell recovery rate after counter diffusion of the polymer at 37 °C was comparable to that
before polymer permeation, and the cell pellets alone can be cryopreserved after polymer permeation, indicatingthat poly(SPB) can improve cryoprotection by intracellular permeation.
This study revealed that vitrification and salt/water trapping are important for cryoprotection of polyampholytes.
This understanding of the cryoprotection mechanism will provide important guidelines for the molecular design ofcryoprotectants in the future. Furthermore, the ability of zwitterionic polymers to reversibly permeate into cells
and enhance their cryoprotective effects offers the potential for cryopreservation of more complex threedimensional cellular structures such as spheroids, and the ability of artificial polymers to It is expected that this will
open up new avenues for development.
[keyword] Cryopreservation, Polyampholytes, solid-state MAS NMR, Intracellular permeationiTable of Contents
Chapter 1 General Introduction

1

1.1 Cryopreservation

1

1.2 Cryoinjury

2

1.3 Cryoprotectants

4

1.3.1 Small molecule CPAs

4

1.3.2 Antifreeze (glycol) Protein (AF(G)P

7

1.3.3 IRI active polymer

9

1.3.4 Nanomaterials

10

1.4 Polyampholytes

11

1.4.1 About polyampholytes

11

1.4.2 Polyampholytes for cryopreservation

14

1.5 Intracellular delivery of cryoprotectants

16

1.5.1 Phospholipid-phase transition18

1.5.2 TRET 1

19

1.5.3 Microinjection

19

1.5.4 Nanoparticles

19

1.5.5 Intracellular delivery of polymers

20ii1.6 Spheroid

21

1.7 Research objects

22

1.8 Reference23

Chapter 2 Relationship between side chain structure of polyampholytes andcryoprotective effect32

2.1 Introduction

32

2..2 Materials & methods

34

2.2.1 Materials

34

2.2.2 Characterization

34

2.2.3 Synthesis of pA

35

2.2.4 Synthesis of pD

35

2.2.5 Synthesis of S-benzyl-O-ethyl dithiocarbonate

36

2.2.6 Synthesis of poly(VA-alt-MA)

37

2.2.7 Modification of poly(VA-alt-MA) and poly(MVE-alt-MA) with 2-(dimethyl
amino)ethanol

38

2.2.8 Modification of -poly-L-lysine with SA

39

2.2.9 Cell culture39

2.2.10 Cryopreservation

40iii

2.2.11 Ice Recrystallization Inhibition (IRI) assay

40

2.2.12 Temperature variable solid-state Magic-Angle-Spinning (MAS) NMR
measurements

41

2.3 Results & discussion

42

2.3.1 Polymer characterization

42

2.3.2 Cryopreservation

49

2.3.3 IRI assay

50

2.3.4 NMR measurements

52

2.4 Conclusion

72

2.5 Reference73

Chapter 3 Enhancement of cryopreservation with intracellularly permeable zwitterionicpolymers

76

3.1 Introduction

76

3.2 Materials & methods

79

3.2.1 Materials

79

3.2.2 Characterization

79

3.2.3 Synthesis of 2-(methylthio)ethyl methacrylate (MTEMA)80

3.2.4 Synthesis of MSEMA

81
iv

3.2.5 Synthesis of zwitterionic polymer

81

3.2.6 Synthesis of fluorescence-modified polymers

83

3.2.7 Cell culture84

3.2.8 Cryopreservation

84

3.2.9 Mitochondrial membrane potential85

3.2.10 Cytotoxicity test

86

3.2.10 Confocal Microscopy

86

3.2.11 Observation of counter diffusion

87

3.2.12 Differential scanning calorimetry87

3.3 Results & Discussion

88

3.3.1 Polymer characterization

88

3.3.2 Cytotoxicity

94

3.3.3 Intracellular permeability

95

3.3.4 Cryoprotective effects

97

3.3.5 Thermal behavior of polymers

99

3.3.6 Freezing and thawing of cell pellets

102

3.4 Conclusion

107

3.5 Reference

109v

Chapter 4 General Conclusion

112

4.1 Conclusion

112

Achievement

116

Acknowledgement

118

vi

List of tables and Figures
Table 2.1 Characteristics of polyampholytes prepared via RAFT polymerization.

46

Table 2.2 Various data for each polymer obtained by temperature-variable solid-state
NMR.

71

Table 3.1 Characterization of zwitterionic polymer.

90

Table 3.2 Thermal properties of zwitterionic polymers as measured by DSC.

100

Figure 1.1 Freezing damage due to freezing speed.

3

Figure 1.2 structure of small molecular weight CPAs

4

Figure 1.3 Structures of AFP. a) Snow mold fungus AFP36) (PDB: 5B5H), b)

Choristoneura fumiferana AFP37) (PDB: 1Z2F), c) Fish Type III AFP38) (PDB: 1UCS).
7
Figure 1.4 Inhibition of ice crystal growth by AF(G)P. When AF(G)P is bound to ice
crystals, ice crystal growth is suppressed by the Kelvin effect.

8

Figure 1.5 Structures of IRI active polymer; a) PVA, b) Facilly amphipathic
glycopolymer48), c) polyproline49).

9Figure 1.6 Structures of nanomaterial type CPAs. a) GO, b) sulphur-doped oxidized
quasi-carbon nitride quantum dots53), c) oxidized quasi-carbon nitride quantum dots53),vii

Zr based MOF (UiO-66-OH)54).

11

Figure 1.7 Structures of zwitterionic polymer. a) Commonly used cationic and anionic,b) Types of zwitterionic polymer.

12

Figure 1.8 Structures of zwitterionic polymers for cryoprotectant. a) PLL-SA66), b)poly(DMAEMA/MAA)67), c) poly(SPB)68), b) poly(CMB)68), poly(DMAEMA/MAA) inalternating periodic arry69).

14

Figure 1.9 Cryoprotection mechanism of PLL-SA by temperature variable solid-state
NMR71).

16

Figure 1.10 Methods of Intracellular delivery trehalose.

17

Figure 2.1 1H NMR spectrum of S-benzyl-O-ethyl dithiocarbonate.

42

Figure 2.2 1H NMR spectrum of pA.

43

Figure 2.3 1H NMR spectrum of pD.

44

Figure 2.4 13C NMR spectrum of pA.

45

Figure 2.5 13C NMR of spectrum poly(VA-alt-MA).

46

Figure 2.6 Confirmation of ring opening by 1H NMR. Red; poly(VA-alt-MA), Blue; pV.
47
Figure 2.7 Confirmation of ring opening by 1H NMR. Red; poly(MVE-alt-MA), Blue;
pM.

48
viii

Figure 2.8. 1H NMR signal assignments for PLL-SA.

48

Figure 2.9 Cell Recovery and of the polymer against L929 cells.

50

Figure 2.10 Polynucleated ice crystals after 30 minutes annealing -6 °C. a) Microscope
image, b) Mean large grain size (MLGS) with 0.3 M NaCl aq.

51

Figure 2.11 1H NMR signal of water. a) pA, b) pD, c) pV, d) pM, e) PLL, f) Comparison
between each polymer.

55

Figure 2.12 Temperature dependent residual water ratio. a) pA, b) pD, c) pV, d) pM, e)
PLL, f) Comparison between each polymer.

56

Figure 2.13 Temperature dependent full-widths-at-half-maximum of the proton signals.
a) pA, b) pD, c) pV, d) pM, e) PLL, f) Comparison between each polymer.

57

Figure 2.14 Water proton T1 relaxation times as functions of temperature. a) pA, b) pD,c) pV, d) pM, e) PLL, f) Comparison between each polymer.

58

Figure 2.15 23Na NMR signal. a) pA, b) pD, c) pV, d) pM, e) PLL, f) Center transition andsatellite transition

62

Figure 2.14 Temperature dependence of Na ion peak shift position. a) pA, b) pD, c) pV,d) pM, e) PLL, f) Comparison between each polymer.

63

Figure 2.15 Temperature dependence of Na ion sharp peak shift intensity. a) pA, b) pD,c) pV, d) pM, e) PLL, f) Comparison between each polymer.

64

Figure 2.16 Temperature dependent full-widths-at-half-maximum of the Na ion signal.
a) pA, b) pD, c) pV, d) pM, e) PLL, f) Comparison between each polymer.
ix

65Figure 2.17 Na ion T1 relaxation times as functions of temperature. a) pA, b) pD, c) pV,d) pM, e) PLL, f) Comparison between each polymer.

66

Figure 2.18 Change in peak intensity of pD due to temperature change.

70

Figure 3.1 1H NMR spectrum of MTEMA.

88

Figure 3.2 1H NMR spectrum of MTEMA.

89

Figure 3.3 FT-IR spectra of MTEMA and MSEMA.

89

Figure 3.4 1H NMR spectrum of poly(SPB).

91

Figure 3.5 1H NMR spectrum of poly(SPB/MSEMA10).

92

Figure 3.6 1H NMR spectrum of poly(SPB/MSEMA20).

92

Figure 3.7 1H NMR spectrum of poly(SPB/MSEMA30).

93

Figure 3.8 GPC curve of polymers.

93

Figure 3.9 Cytotoxicity of each polymer against L929 cells 1 hour after exposure.

94

Figure 3.10 Fluorescence images of L929 cells. a) Confocal microscope images offluorescein-labelled poly(SPB) and poly(SPB/MSEMA10) in L929 cells. b)
Fluorescence images of L929 cells at 10-minute intervals after incubating at 37 °C
after permeation of fluorescein-labelled poly(SPB). Scale bars: 10 m.
Figure 3.11 Cryoprotective properties of poly(SPB) and poly(SPB/MSEMAn). a)
Comparison of cryoprotective effects with and without polymer permeation. b) Cellx

96

recovery after permeation and counter-diffusion of poly(SPB) andpoly(SPB/MSEMA10).

98

Figure 3.12 Cell growth curve after freeze–thaw cycle.

99

Figure 3.13 DSC trace of polymers.

101

Figure 3.14 Cryoprotective capacity of trehalose- and poly(SPB)-permeabilized cellpellets.

102

Figure 3.15 Cryoprotective capacity of fluorescence-modified poly(SPB)-permeabilized cellpellets.

104

Figure 3.16 Mitochondrial membrane potential before and after freeze-thaw

104

xi

Chapter 1
General introduction
1.1 Cryopreservation
The function and structure of organelles in living cells and tissues may change over time.
Cryopreservation is a technology that enables the preservation of cells, tissues, and organs
by slowing their metabolism at -80 or -196 °C. Therefore, it is a very important
technology for long term preservation of various biological samples and for clinicalapplications1～3). In 1949, Polge et al. reported the cryopreservation of bird semen using
glycerol, the first report on cryopreservation4). Since then, research has been conductedon the cryopreservation of various cells and tissues such as sperm of other animals andplants5-8). In addition, cryopreservation using dimethyl sulfoxide (DMSO) was reported
by Lovelock in 19599). DMSO is currently the commonly used cryoprotectant (CPA) andis typically used at concentrations of around 10%. DMSO has strong hydrogen bonds
with water that lower the freezing point and exhibits excellent cryoprotective effects 10,11).
These small molecules CPAs have membrane permeability and permeate into cells duringfreezing, so an operation to remove CPAs from inside cells is required after thawing 12,13).
Furthermore, DMSO has many problems such as cytotoxicity and induction of stem celldifferentiation, however, it is still used today because there are no CPAs to replace 14-16).
1

Therefore, the development of CPAs to replace DMSO is actively underway. There are
two types of cryopreservation methods: slow freezing and vitrification17). In thevitrification preservation method, cells are directly cryopreserved in liquid nitrogen,which turns intracellular water into an amorphous state and avoids intracellular ice crystal
formation18). On the other hand, in the slow freezing method, cells are slowly frozen to 80 °C, which causes ice crystals to form inside the cells, causing mechanical stress andosmotic shock19, 20). However, the slow freezing method is simpler because it can be carriedout if there is a deep freezer. Therefore, in this study, cells were cryopreserved using theslow freezing method.

1.2 Cryoinjury
During cell freeze-thaw cycles, cells are subjected to various chemical and physicaldamages, including osmotic shock, exposure to toxic CPAs such as DMSO, and theformation of intracellular ice crystals15,19-21). In the cooling process, the extracellularsolution first begins to freeze. At this time, since the ice crystals do not contain solutes,
the osmotic pressure increases due to the concentration of the extracellular solution,
causing cell dehydration. If the cooling rate is made faster, the outflow of cell water
gradually decreases, causing intracellular ice to form. According to Mazur's two-factor
hypothesis, during cryopreservation, cell states are classified into three types depending2

on the freezing rate21) (Figure 1.1). When the cooling rate is appropriate, intracellulardehydration increases the solute concentration and inhibits intracellular freezing.
However, if the cooling rate is too fast, the dehydration necessary to achieve osmotic
equilibrium does not occur, causing intracellular freezing. If the cooling rate is too slow,
irreversible damage will occur due to solute concentration by excessive dehydration andcell membrane adhesions. Furthermore, in the thawing process after cryopreservation,water may flow into dehydrated cells beyond the cell membrane, causing swelling andlysis of the cells. Additionally, ice recrystallization is induced during thawing, which cancause mechanical damage to cell membranes by growing small ice crystals into larger ice
crystals22, 23).

Figure 1.1 Freezing damage due to freezing speed.
31.3 Cryoprotectants
1.3.1 Small molecule CPAs
Small molecule CPAs can be divided into two types, membrane permeable and nonmembrane permeable, depending on whether they permeate into cells or not (Figure 1.2).

Figure 1.2 structure of small molecular weight CPAs.

DMSO, the most common CPA, is a membrane permeable CPA. DMSO exhibits freezing
point depression at certain concentrations9). In addition, molecular dynamics simulations4

researched between DMSO and water revealed that the proportion of unfrozen water
during freezing increases due to hydrogen bonding between DMSO and water 10).
Additionally, because of investigating the hydrogen bonding ability of DMSO and acetone
with water using Raman spectroscopy, it was revealed that the hydrogen bond between
DMSO and water is stronger than that of acetone and is very similar to the hydrogen bond
between water and water. As a result, DMSO is not removed from the hydrogen bond
network during freezing and freezes in an amorphous state11). However, as mentioned
above, DMSO is cytotoxic and may induce differentiation and induction14-16). For example,
it has been reported that differentiation occurs in embryonic stem cells even at very low
concentrations of DMSO of 0.125%. In addition to differentiation, patients transplantedwith cells cryopreserved with DMSO have been reported to have significantly increased
DMSO-induced side effects. Other side effects such as nausea, hypotension, andarrhythmia have been reported in patients receiving infusions of hematopoietic stem cells
cryopreserved in DMSO24-26). DMSO is also used to cryopreserve humaninducedpluripotent stem cells (hiPS), which are attracting attention in the field of cell therapy
and regenerative medicine because they differentiate into all cell types. However, the useof DMSO should still be avoided in the cryopreservation of reprogrammed cells, as itincreases mRNA levels of the de novo DNA methyltransferase DNMT3A in mouse5embryoid bodies and is accompanied by hyper- or hypomethylation of many gene loci27).
Furthermore, in order to avoid the side effects of DMSO as raised so far, DMSO needs tobe removed when actually used in clinical applications, etc. However, this process ofremoving DMSO results in fewer cells being recovered. Therefore, DMSO-freecryopreservation methods and alternative CPAs are being actively investigated.
Glycerol is non mutagenic and less toxic than DMSO. It also acts through salt buffering,binding with metal ions, and dehydration of cells, and suppresses intracellular freezing,
thereby alleviating cell freezing stress28-30). However, glycerol also needs to be removed
from the cells after thawing, and some cells may be lost in the process31).
Sugars are mainly used as non-membrane permeable CPAs. Among them, trehalose isnaturally found in high concentrations in the bodies of some yeasts, nematodes,
tardigrades, etc., and these microorganisms are known to be resistant to extremely low
temperatures and dryness32-34). Two hypotheses have been proposed regarding the
cryoprotective mechanism of trehalose35). First, trehalose forms hydrogen bonds withbiopolymers or promotes hydration of biopolymers, allowing cells to retain theirfunctional structure. Second, it halts metabolic activity by forming a glass like matrix withlow molecular mobility. However, trehalose has a very weak function as an extracellular
CPA, and only when trehalose is present inside and outside cells can it exert a high6

cryoprotective effect36). Common methods for introducing trehalose into cells include
electroporation and expressing trehalose transporter 1 (TRET1) in cells 37, 38). However,these methods have problems such as membrane damage due to electric shock and theneed for genetic modification.

1.3.2 Antifreeze (glyco) Protein （AF(G)P）
Because the Antarctic and Arctic regions have extremely low temperatures and arecovered with ice, organisms that live there have developed several survival strategies suchas antifreeze peptides and extracellular polysaccharides39). AF(G)P is one of these, andhas been discovered in various organisms such as diatoms, insects, and fish 40-42) (Figure
1.3).

Figure 1.3 Structures of AFP. a) Snow mold fungus AFP36) (PDB: 5B5H), b)

Choristoneura fumiferana AFP37) (PDB: 1Z2F), c) Fish Type III AFP38) (PDB: 1UCS).

7

AF(G)P is thought to exert cryoprotective effects by lowering the freezing point throughthermal hysteresis (TH) and inhibiting ice crystal formation through ice recrystallization
inhibition (IRI). TH is caused by the interaction between AF(G)P and the ice crystal
surface due to the difference between the freezing and melting points of the solution43, 44).
Although the structure, TH, and IRI of AF(G)P differ among insects, fish, and plants, themechanism of inhibition of ice crystal growth is common. First, the ice-binding surface(IBS) of AF(G)P recognizes ice crystals, and then AF(G)P and ice crystals bind 45). Ice
crystal growth is then inhibited by non-IBS46). Because non-IBS is a hydrophobic andcharged structure, it effectively increases the curvature of the interface and lowers the
local freezing point according to the Kelvin effect, suppressing the crystallization of theincoming water (Figure 1.4).

Figure 1.4 Inhibition of ice crystal growth by AF(G)P. When AF(G)P is bound to ice
crystals, ice crystal growth is suppressed by the Kelvin effect.
8

Although AF(G)P is a naturally occurring CPA, it is not always suitable forcryopreservation due to drawbacks such as high cost, potential immunogenic and toxic
effects, and needle-shaped ice crystals47-49).

1.3.3 IRI active polymer
Many of the polymers used as CPAs focus on the IRI of AF(G)P (Figure 1.5). A typical
CPA with IRI activity is polyvinyl alcohol (PVA)50).

Figure 1.5 Structures of IRI active polymer; a) PVA, b) Facilly amphipathic
glycopolymer52), c) polyproline53).

Because the atomic distance between hydroxy groups on the molecule (2.92 Å) is close
to the prismatic plane of ice crystals (2.74 Å), PVA has higher IRI activity thancommercial polymers such as poly(ethylene glycol) (PEG) and hydroxy ethyl starch(HES) used for CPAs51). However, when used alone, PVA does not have a very highcryoprotective effect, and other CPAs must be added. Other CPAs that mimic the three9

dimensional structure of AFGP are also being developed. In natural AF(G)P, isolatedhydrophobic and hydrophilic groups are also known to be important factors in inhibiting
ice crystal growth, and Graham et al. designed a glycopolymer to increase the solubility
and IRI activity of the polymer52). In addition, since polyproline does not have an amide
NH and cannot form intramolecular hydrogen bonds, it has amphipathic properties liketype 1 AFP, which contains 70% alanine. Graham et al. have shown that polyproline hashigh IRI activity and improves the recovery of cell monolayers after cryopreservationcompared to DMSO53). Furthermore, Qin et al. achieved cryopreservation of oocytes
using polyproline54). However, these CPAs with IRI activity still have the problem ofrequiring the addition of DMSO as CPA.

1.3.4 Nanomaterials
Some use nanomaterials as ice crystal growth inhibitors (Figure 1.6). For example,
graphene oxide (GO) exhibits a structure similar to natural AFP55). Due to thehoneycomb-like structure of GO, the ice lattice matches the hydrogen bonds formed
between the hydroxyl groups on the top surface of GO and the water and ice molecules,
which greatly suppresses the growth of ice crystals56). Other carbon-based nanomaterials
being studied include oxide quasi-carbon nitride quantum dots57). This is also thought tohave a high affinity with ice because the interatomic distance on the surface is close to the10hexagonal ice prism surface, increasing IRI activity. In addition, the IRI activity of Zrbased metal-organic frameworks (MOFs) can be adjusted by adjusting the amount ofhydrogen-donating groups on the (1,1,1) face of the MOF58). However, the cryoprotective
effect of these nanomaterials is not very high.

Figure 1.6 Structures of nanomaterial type CPAs. a) GO, b) sulphur-doped oxidized
quasi-carbon nitride quantum dots57), c) oxidized quasi-carbon nitride quantum dots57),
Zr based MOF (UiO-66-OH)58).

1.4 Polyampholytes
1.4.1 About polyampholytes
Zwitterionic polymers are polymers that were first reported by Alfrey et al. in 1950. The
entire polymer is composed of a mixture of charged regions, and due to the mixed charge

11state of cations and anions, it has been associated with proteins 59, 60). Commonly usedfunctional groups include protonated amino, quaternary ammonium, and pyridine as
cationic groups, and carboxylates, sulfonates, and phosphates as anionic groups 61-65)(Figure 1.7 a)). Zwitterionic polymers can be divided into two types: betaine types andpolyampholyte types (Figure 1.7 b)).

Figure 1.7 Structures of zwitterionic polymer. a) Commonly used cationic and anionic, b)
Types of zwitterionic polymer.
12Betaine type of polymers refers to a polymer that has both cation and anion regions in
one side chain. There are equal numbers of positive and negative charges on the polymer,
so the net charge is zero, and the electrostatic charge within the molecule upon interaction,
it assumes a spherical folded conformation61, 66, 67). In addition, betaine polymer chains are
salt responsive and swell due to external inorganic salts, which affects the mechanicalproperties of hydrogels, etc.68). In the polyampholyte type, the polymer is composed of a
mixture of several charged monomers. Unlike betaine type of polymers, the charge can beeasily adjusted by changing the ratio of monomers, so it has the advantage of allowing one
charge to be dominant. Additionally, polymers with environmentally responsive
properties can be designed based on the composition and structure of the polymer. Thesezwitterionic polymers have excellent hydrophilicity because they have abundant anions
and cations in the polymer chain. Furthermore, the zwitterionic polymer selectively binds
to oppositely charged species through electrostatic interactions, so it has pH
responsiveness. Therefore, there is growing interest in applying polymer brushes tobiomedical applications such as antifouling coatings and drug delivery systems (DDS) 61,
.

69)

13

1.4.2 Polyampholytes for cryopreservation
In addition to antifouling coatings and DDS, zwitterionic polymers are also being
studied as CPAs (Figure 1.8). However, intracellular freezing and membrane adhesion
due to dehydration rates and other factors are more important in actual cryoinjury. In fact,
the cryoprotective effect of polymers with high IRI activity alone is low. It has also beenreported that AFPs with similar IRI activity have different cryoprotective effects.

Figure 1.8 Structures of zwitterionic polymers for cryoprotectant. a) PLL-SA66), b)poly(DMAEMA/MAA)71), c) poly(SPB)72), b) poly(CMB)72), poly(DMAEMA/MAA) inalternating periodic arry73).
14

Matsumura et al. synthesized carboxylated poly-L-lysine (PLL-SA), an polyampholyte,
using -poly-L-lysine (PLL) and succinic anhydride (SA), and demonstrated its
cryoprotective effect70). PLL-SA shows a high cryoprotective effect when the carboxylgroup substitution rate is 50-76%. Rajan et al. also synthesized poly(DMAEMA/MAA)
from methacrylic acid (MAA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA)
and showed a high survival rate in a 1:1 copolymer71). This polymer, like PLL-SA, was also
found to have lower cytotoxicity compared to DMSO. Rajan et al. also reported
cryoprotective effects for poly(SPB) with sulfobetain (SPB) and poly(CMB) withcalboxybetain (CMB)72). These polyampholyte types CPAs are promising as new CPAs
because the polymers can be cryopreserved on only polymer solution and are lesscytotoxic than DMSO. There are many other studies on poly(DMAEMA/MAA) as CPAs,
such as Zhao et al. reported on cell survival when the net charge of the polyampholyte isbiased negatively, and Bailey et al. alternating periodic polymer73, 74). In PLL-SA, it is
known that it is important to trap polymer water and salt and form a polymer matrix byusing temperature-variable solid-state NMR75) (Figure 1.9). However, for other polymers,
although interaction with cell membranes is known to be important, the clear mechanismof cryoprotection is not known72). As a result, the advantages of methacrylate polymers
such as poly(DMAEMA/MAA), such as free molecular design, are not exploited.
15

Therefore, in this study, by synthesizing polyampholytes with different side chain
structures and similarly using temperature-variable solid-state NMR, I will correlate thepolymer structure and cryoprotective effect and use this as a steppingstone to optimal
molecular design of CPAs. In addition, as the present series of studies focused only on the
cryoprotective function of the polymer, mouse fibroblasts were used as a model for theactual cryopreservation.

Figure 1.9 Cryoprotection mechanism of PLL-SA by temperature variable solid-state
NMR75).

1.5

Intracellular delivery of cryoprotectants

As already mentioned, trehalose, a membrane-impermeable CPAs, acts as water byforming hydrogen bonds with biomacromolecules and promoting hydration of
biomacromolecules so that cellular components can maintain their functional
conformation. It is also thought to obtain a cryoprotective effect by temporarilysuspending metabolic activity by forming a glass like matrix with extremely low molecular16

mobility. However, if trehalose exists only outside the membrane, the cryoprotective
effect is not high, and it is thought that trehalose needs to be present inside and outside
the cell. Therefore, research is underway to transport trehalose into cells to enhance thecryoprotective effect (Figure 1.10). However, although there are various options fortransporting trehalose, they reduce membrane stability and require genetic modificationof cells, which limits their use in clinical applications and reduces their cryoprotective
effects.

Figure 1.10 Methods of Intracellular delivery trehalose.

It has been reported that the cryoprotective effect of polymeric CPAs can be enhanced
by transporting PEG with a low degree of polymerization into cells76). In other reports
using polymers with the same DMSO structure for cryopreservation, poly(methyl glycidyl
sulfoxide) with a PEG structure in the main chain was as effective as DMSO in
cryoprotection, whereas when methacrylate polymers were used, the cryoprotective effectmethacrylate polymers showed lower cryoprotection77 78). The difference between thesetwo polymers is that the polymer with a PEG main chain permeates into cells, whereas
17the methacrylate polymer does not permeate into cells. However, these polymers haveproblems such as long incubation times and endocytosis. It has also been proposed thatcytoplasmic macromolecular condensation reduces cytoplasmic free water, providing cellswith a rapid thermodynamically driven defense against rapid fluctuations in osmotic
strength, temperature, and pressure79). Therefore, it is thought that the efficiency of
cryoprotection can be increased by transporting the polymer into cells. Here, I willsummarize intracellular transport of trehalose and polymers.

1.5.1 Phospholipid-phase transition
The first method to introduce trehalose into cells was to utilize the phase transition ofphospholipids. By inducing the phase transition of phospholipids by heat, osmoticpressure, and electrical stress, the permeability of the cell membrane is increased and thetransport of trehalose is made possible. In the heat based method, trehalose is transported
into the cytoplasm by incubating the cell suspension for about 10 minutes and thenplacing the suspension at 0 and 39 °C for up to 2 hours every 10 minutes.80). In theosmotic method, trehalose can be transported by incubating it for up to 7 hours in
extracellular trehalose at an isotonic pressure or higher81). This method may cause cellmorphological changes due to prolonged exposure to hypertonic solutions. The electricalmethod is called electroporation, and it can temporarily and reversibly increase the cell18

membrane by applying a short external electric field pulse37). However, as with othermethods, reduced membrane stability can make it vulnerable to osmotic shock duringfreezing and thawing.

1.5.2 TRET1
Some organisms that utilize trehalose to enable survival in cold environments have atrehalose transporter known as TRET1 that allows trehalose to accumulate within cells
under environmental stress. By expressing TRET1 in cells to be cryopreserved, trehalose
can be taken into the cells, and desiccation tolerance and cryoprotection effects areenhanced compared to cells that do not express TRET138). However, because genetic
modification of the cells is required, frozen and thawed cells may not be able to be used
for actual clinical applications.

1.5.3 Microinjection
Microinjection is the most direct method of introducing trehalose into cells. Eroglu et al.
successfully delivered trehalose to discarded human oocytes and frozen them in thepresence of extracellular trehalose, and subsequent studies of trehalose-containing
oocytes showed good survival rates after cryopreservation82). However, this method is notsuitable for other cells that are smaller and need to be cryopreserved in large quantities.

1.5.4 Nanoparticles
19

Nanoparticles are also used in the DDS field, and in the cryopreservation field, they aremainly used to transport trehalose into cells. For example, Rao et al. used pH responsive
genipin-cross-linked Pluronic F127-chitosan nanoparticles as nanoparticles to transportand cryopreserve trehalose into primary human adipose-derived stem cells83). It has beenreported that when trehalose is transported using these nanoparticles, the cell survival
rate after thawing is comparable to that of DMSO. In addition, Zhang et al. used low
temperature responsive poly(N-isopropylacrylamide-co-butyl acrylate) as nanoparticles
for cryopreservation84). Unlike Rao et al. nanoparticles, which required 24-hour
incubation before cryopreservation, these nanoparticles can transport trehalose into cells
in about four hours. It was also shown that even when these nanoparticles were used, thecell survival rate was comparable to that of DMSO. However, methods using such
nanoparticles utilize cellular endocytosis, which poses problems such as escape fromlysosomes.

1.5.5 Intracellular delivery of polymers
Methods for introducing polymers into cells include methods in which polymer micelles
are taken into cells via endocytosis, and methods such as cationic polymers andtransmembrane peptides (CPP) that enter cells by making minute holes in the cell
membrane84, 86). However, when a cationic polymer or CPP is used, it creates minute pores20

in the cell membrane, which may cause excessive dehydration during freezing and affectcell growth after thawing. When it is taken up via endocytosis, it is taken up into lysosomes
and does not spread throughout the cell. Poly(SPB), which is a betaine polymer, andpoly(MPC/BuMA), which is a copolymer of 2-methacryloyloxyethyl phosphorylcholine(MPC) and butyl methacrylate (BuMA), penetrate the entire cytoplasm without going
through endocytosis87-89). Poly(SPB) has also been reported to have a cryoprotective effect,
so it can be said to be very useful for intracellular transport of CPAs72).

1.6 Spheroid
Most studies in cell biology are performed using cells of interest cultured in 2D
monolayers. However, since the actual cellular environment is three-dimensional, a 2D
monolayer cannot simulate the complexity and dynamic interactions of the
microenvironment90). Therefore, three-dimensional cell structures such as spheroids,
which can better reproduce the in vivo environment, are attracting attention91-93). Since
serial cell culture consumes many resources, long term preservation of three-dimensional
cell structures is important for industrial use. However, while cryopreservation ofsuspended cells has been achieved by various CPAs, cryopreservation of threedimensional cellular constructs is more difficult due to their complexity. There have beensome reports on cryopreservation of spheroids, but these require the use of ice nucleating
21

agents or small molecules CPAs94, 95).

1.7 Research objects
As mentioned above, the details of the cryoprotective mechanism of polyampholytes
such as poly(DMAEMA/MAA), which have a high cryoprotective effect, are unknown. A
more detailed understanding of the relationship between the cryoprotection mechanismand polymer structure will provide important guidance for the molecular design of CPAs.
Therefore, in Chapter 2, the cryoprotective effects of poly(DMAEMA/MAA) andpolymers with different side chain structures are correlated with their structures using
variable temperature solid-state NMR, with the aim of elucidating the cryoprotection
mechanism. In Chapters 3 and 4, I took advantage of the property of zwitterionicpolymers to permeate into cells and developed a new approach to use sulfobetaine
polymers as intracellular permeating CPAs. Furthermore, I attempted cryopreservationof spheroids to achieve cryoprotection of three-dimensional cellular constructs.
Understanding the cryoprotective mechanisms of these polyampholytes and the use ofzwitterionic polymers as intracellularly permeable CPAs will be of great importance forthe strategic synthesis of CPAs and for the cryopreservation of more complex tissues.

22

1.8

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31

Chapter 2
Relationship between side chain structure ofpolyampholytes and cryoprotective effect
2.1 Introduction
Understanding the cryoprotection mechanism of polyampholytes and correlating theirstructure and function will provide important knowledge for developing new
cryoprotectants. Furthermore, the possibility of application of polyampholyte type
cryoprotectants to regenerative medicine, etc. increases. Fluorescence staining withfluorescein

isothiocyanate

has

shown

that

carboxylated

polylysine(PLL),apolyampholyte, is a non-membrane permeable substance and that its cryoprotective effectis expressed outside the membrane1). In addition, temperature-variable solid-state NMR
has shown that PLL traps water and salt during freezing, forming a polymer matrix thatprevents a rapid increase in osmotic pressure and intracellular ice formation due tocontact with extracellular ice crystals2). Another polyampholyte that has been reported tohave a high cryoprotective effect is poly(2-(dimethyl amino)ethyl methacrylate(DMAEMA)/methacrylic acid (MAA)) (pD)3-5). Although the interaction between thepolymer and cell membrane is thought to be important for this polyampholyte, its
cryoprotective mechanism is not clear, which hinders the optimal molecular design of32

cryoprotectants4).
Therefore, in Chapter 2, I first synthesized four types of polyampholytes with different
side chain structures. The first is the previously reported pD. The second is poly(2acrylamido-2-methyl

propanesulfonicacid(AMPS)/(3-acrylamidopropyl)trimethyl

ammonium chloride (APTAC)) (pA), which has a higher degree of dissociation than pD
because the anion is sulfonic acid and the cation is quaternary ammonium. In addition,poly(vinyl acetate (VA)/acrylic acid (AA)/2-(dimethyl amino)ethyl acrylate (DMAEA))(pV) and poly(methyl vinyl ether (MVE)/AA/ DMAEA) (pM), which have the same
dissociation degree as pD but different molecular weight, were synthesized. Also, while
pD is a random copolymer, pV and pM have cations and anions arranged alternately. Afterpolymer synthesis, physicochemical properties of solvents and solutes under freezing
were observed using temperature-variable solid-state Magic-Angle-Spinning (MAS)NMR to correlate solution properties with cryoprotective effects and side chain structures.
Correlating the polymer structure and cryoprotective effect in this way will deepen our
understanding of the cryoprotection mechanism and lead to the optimal molecular designof cryoprotectants.

33

2.2 Materials & methods
2.2.1 Materials
AMPS, APTAC (74~76 % in water), 2-(dimethylamino)ethyl methacrylate, vinyl
acetate monomer, maleic anhydride (MA), 2-(dimethyl amino)ethanol, potassium
ethylxanthate and benzyl bromide, succinic anhydride(SA) were purchased from Tokyo
Chemical Industry Co. (Tokyo, Japan); MAA was purchased from Fuji Film Wako Pure
Chemical Co. (Osaka, Japan); 25 %(w/w) -poly-l-lysine was purchased from JNC Co.
(Tokyo,Japan);Poly(MVE-alt-MA)(average

Mw~216000),

2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) and 4,4'-azobis(4cyanovaleric acid) (V-501) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
All the reagents were used as received without further purification.

2.2.2 Characterization
H NMR for synthesized materials characterization was measured using a Bruker

1

Avance III 400. The deuterated solvent used was purchased from Sigma-Aldrich. The
NMR data were analysed using Topspin 3.5 software. The molecular weights anddistributions (polydispersity index; PDI) of the polymers were determined by gel
permeation chromatography (GPC; BioSep-s2000, Phenomenex, Inc., CA, USA) using a
Waters Alliance HPLC system. A PBS was used as the mobile phase (flow rate, 0.75
34

mL/min) and pullulan as the standard.

2.2.3 Synthesis of pA
Scheme 1 shows the pA synthesis. AMPS (0.829 g, 4 mmol) was dissolved in 3 ml ofwater, and AMPS was neutralized by adding 780 l of 5M NaOH (AMPS-Na). APTAC(1.102 g, 4 mmol), DDMAT (0.0729 g, 0.2 mmol), and V-501 (0.0112 g, 0.04 mmol)
were dissolved in 12 ml of methanol and added to AMPS-Na. After N2 bubbling for 1 hour,
the reaction was carried out at 70 °C for 1 hour. After the reaction was completed, asample was taken out and the monomer conversion was determined by 1H NMR. The
polymer was obtained by dialysis against distilled water for 24 hours using a dialysis
membrane (Mw 3500, Repligen Corp., Waltham, MA ,USA) and then freeze-drying. The
structure of the compound was determined by 1H-NMR and 13C NMR using D2O as asolvent.

Scheme 2.1 Synthesis of pA.

2.2.4 Synthesis of pD
Scheme 2 shows the pD synthesis. MAA (0.344 g, 4 mmol) and DMAEMA (0.629 g, 435

mmol) were added to 20 ml of water and methanol mixed solvent (1:1 [v/v]). Then
DDMAT (0.0729 g, 0.2 mmol) and V-501 (0.0112 g, 0.04 mmol) were added. After N2
bubbling for 1 hour, the reaction was carried out at 70°C for 24 hours. After the reaction
was completed, a sample was taken out and the monomer conversion was determined by
H NMR. The polymer was reprecipitated against 2-propanol, recovered by

1

centrifugation, and dried in vacuum. The structure of the compound was determined by

Scheme 2.2 Synthesis of pD.

H-NMR using D2O as a solvent.

1

2.2.5 Synthesis of S-benzyl-O-ethyl dithiocarbonate
S-benzyl-O-ethyl dithiocarbonate was synthesized according to a previous report6)(Scheme 2.3). Specifically, first, Potassium ethylxanthate (0.8 g, 5.0 mmol) was dissolvedin 10 ml of ethanol at 50 °C. Benzyl bromide (590 l, 5.0 mmol) was added as is, andthe reaction was allowed to proceed for 5 hours. After the reaction was completed, 30 mlof water was added, and the mixture was extracted three times with 25 ml of diethyl ether.
It was dried over MgSO4 and the solvent was removed on a rotary evaporator (0.75 g,

36

yield 70.6%). 1H NMR (CDCl3, 400 MHz):  -7.55-7.20 (m, 5H, Ar-H), 4.68 (q, 2H,

Scheme 2.3 Synthesis of poly(VA-alt-MA).

OCH2), 4.39 (s, 2H, SCH2), 1.44 (t, 3H, CH3).

2.2.6 Synthesis of poly(VA-alt-MA)
Scheme 4 shows the synthesis of poly(VA-alt-MA). VA (1.0 g, 11.6 mmol), MA (1.96 g,
20 mmol), S-benzyl-O-ethyl dithiocarbonate (80 mg, 0.337 mmol) and V-501 (15.1 mg,
0.054 mmol) were dissolved in 8 ml of dioxane. N2 bubbling was performed for 1 hour,
and the reaction was carried out at 80 °C for 16 hours. After 16 hours, the reaction was
terminated by immersing the flask in liquid nitrogen. After returning to room temperature,
the product was reprecipitated in diethyl ether. The polymer was collected bycentrifugation and dried under vacuum overnight. The structure of the compound wasdetermined by 1H-NMR and 13C NMR using DMSO-d6 as a solvent.

Scheme 2.4 Synthesis of poly(VA-alt-MA).
37

2.2.7 Modification of poly(VA-alt-MA) and poly(MVE-alt-MA) with
2-(dimethyl amino)ethanol
The synthesis of alternating array polyampholytes are shown in Scheme 2.5. 2 g ofpolymer was dissolved in 50 ml of THF and heated to 50°C. After dissolution, excess
addition of 2-(dimethyl amino)ethanol (4 g, 44.9 mmol) produced a pink or purple solid,
which was stirred for 30 minutes. 50 ml of water was added to dissolve the product andallowed to react overnight. After the reaction was completed, dialysis was performed
against distilled water for 48 hours using a dialysis membrane (14 kDa, Sekisui ChemicalCo., Ltd., Tokyo, Japan or 3500 Da, Repligen.), followed by freeze-drying to obtain apolymer. The structure of the compound was determined by 1H-NMR using D2O as asolvent.

Scheme 2.5 Modification of poly(VA-alt-MA) and poly(MVE-alt-MA). a) pV, b) pM.
38

2.2.8 Modification of -poly-l-lysine with SA
PLL was synthesized according to a previous report (Scheme 2.5). PLL was obtained bydissolving SA (1.27 g, 12,67 mmol) in 25 w/w% -poly-l-lysine and reacting at 50 °C for
1 hour. The introduction rate of carboxyl groups was confirmed by 1H NMR using D2O
as a solvent.

Scheme 2.6 Modification of PLL with SA.

2.2.9 Cell culture
Mouse fibroblasts (L929; American Type Culture Collection, Manassas, VA, USA) wereprepared in Dulbecco's modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO,
USA) supplemented with 10 % foetal bovine serum (FBS). Cells were cultured in an
incubator at 37 °C with 5 % CO2. Passaging was performed when the cells exceeded
80 % of the bottom of the dish. For passaging, the cells were detached by treatment withtrypsinsolution

(0.25

%

[w/v]

trypsinethylenediaminetetraacetic acid in PBS).

39

containing

0.02

%

[w/v]

2.2.10 Cryopreservation
The polymer was dissolved in 0.3 M NaCl solution to 15 w/w%. The solution was
sterilized through a 0.22 m filter to obtain a cryoprotective solution. L929 cells were
adjusted to 1 × 106 cells, suspended in 1 ml of polymer solution, and stored at -80 °C
without controlling the cooling rate. After 24 hours, the samples were immersed in a
37°C water bath to thaw the cells. The cell suspension was then diluted 10 times withDMEM and centrifuged at 1000 rpm for 4 min. The recovery rate was determined bysuspending the cell pellet in a small amount of DMEM and staining with trypan blue (Eq.
2.1).
Recovery rate (%) =Living cell× 100
Frozen cell

(Eq. 2.1)

2.2.11 Ice Recrystallization Inhibition (IRI) assay
The solution for IRI assay was prepared in the same manner as for cryopreservation. 2
l of the prepared solution was sandwiched between two microcover glasses (diameter =15 mm). The sample was then placed in the low temperature stage and the temperature
was lowered to -40 °C at 20 °C/min and then increased to -6 °C at 10 °C/min.
Thereafter, the material was annealed for 30 minutes in a nitrogen atmosphere at thattemperature, and the ice crystals were photographed after 0 and 30 minutes. ImageJ was40

used for image analysis, and the average of the long sides of 10 randomly selected ice
crystals was taken as the maximum particle size.

2.2.11 Temperature variable solid state Magic-Angle-Spinning(MAS)NMR measurements
Temperature-variable solid-state NMR measurements were performed on a 700 MHz
JEOL ECZ spectrometer (JEOL). The cryoprotectant-containing solution samples weresealed in a DSI internally sealed cell for the XC4 rotor, and the samples were rotatedbetween 3.6 and 5.8 kHz and measurements were taken from room temperature to around-40 °C. In the temperature change experiment, I first cooled the sample slowly to below-40℃, left it in that state for over an hour to stabilize it, and then started measurements.
Next, the temperature was increased to around 2 to 3 degrees, and the following
measurements were performed. The samples in the cell were allowed to stand for about
10 minutes until they reached each measurement temperature, and then measurements
were taken. All data were processed using Delta Ver. 6.1 (JEOL).

41

2.3 Results & discussion
2.3.1 Polymer characterization
In order to polymerize poly(VA-alt-MA), a precursor of pV, by reversible additionfragmentation chain transfer (RAFT) polymerization, I synthesized S-benzyl-O-ethyl
dithiocarbonate, a chain transfer agent (Fig. 2.1).

Figure 2.1 1H NMR spectrum of S-benzyl-O-ethyl dithiocarbonate.

pA, pD and poly(VA-alt-MA) were synthesized by RAFT polymerization. When thesamples of pA and pD at the completion of the reaction were confirmed by 1H NMR, lossof the vinyl proton of the monomer appearing at around 5.2 ppm to 6.2 ppm was observed,
confirming that polymerization of the polymer had progressed. For poly(VA-alt-MA), an
42excess amount of MA was added to VA to ensure the realization of an alternating array ofpoly(VA-alt-MA), and the reaction was terminated regardless of the conversion rate 7).
When the composition ratio of the polymers pA and pD was confirmed by 1H NMR, it
was shown that the composition of pD was approximately 49.1% DMAEMA, which wasable to be polymerized at a composition ratio of approximately 1:1 (Figures 2.2 & 2.3).

Figure 2.2 1H NMR spectrum of pA.

43

Figure 2.3 1H NMR spectrum of pD.

However, the composition ratio of pA could not be determined because the peaks ofpendant methylene and pendant methyl protons of AMPS and APTAC overlapped in acomplicated manner, and the peaks of methine and methylene protons in the main chain
overlapped in a complicated manner. In addition, the pendant amide protons around 7.5
ppm to 8.3 ppm overlapped, so an accurate integral ratio could not be obtained. In aprevious study, the composition was determined by measuring reverse gated decoupled
C-NMR, so a similar method was used in this study8). The 13C-NMR results are shown13

in Figure 2.4.

44

Figure 2.4 13C NMR spectrum of pA.

The composition ratio of AMPS and APTAC was determined from the integral ratio of
methylene carbon around 57 ppm and 64 ppm. The results showed that the content of
AMPS was approximately 49.9%, and it was possible to synthesize it at a composition
ratio of 1:1, similar to pD. In addition, pA and pD showed narrow dispersity by size
exclusion chromatography (Table 2.1).
The structure of poly(VA-alt-MA) was also measured using reverse gated decoupling
C-NMR in the same way as pA (Figure 2.5).

13

45

Table 2.1 Characteristics of polyampholytes prepared via RAFT polymerization.
Conposition
Molar ratio

in feedpAin polymera)in feedpD
in polymer
pVb)

Cation

Anion

50

50

49.9

50.1

50

50

49.1

50.9

50

50

c)

Mn

d)

Mw/Mn

100 : 1 : 0.2

7156

1.03

100 : 1 : 0.2

9719

1.42

93 : 1 : 0.16

7419

1.95

13

1

a) Determined by inverse gated decoupling C NMR, b) Determind by H NMR, c) [monomer]:[CTA]:[initiator], d)determined by GPC.

Figure 2.5 13C NMR of spectrum poly(VA-alt-MA).

46d)Since the integral values of a, d, b derived from VA and the integral value of e' derived
from MA are approximately 1:1, this polymer was shown to be an alternating array of VA
and MA7).To obtain ampholyte polymers, 2-(dimethyl amino)ethanol was used as anucleophile to open the rings of (VA-alt-MA) and poly(MVE-alt-MA). By 1H NMR, themethine peak around 3.8-4.2 ppm disappeared, confirming the success of ring opening(Figures 2.6 & 2.7).

Figure 2.6 Confirmation of ring opening by 1H NMR. Red; poly(VA-alt-MA), Blue; pV.

47

Figure 2.7 Confirmation of ring opening by 1H NMR. Red; poly(MVE-alt-MA), Blue; pM.

Figure 2.8 shows the 1H NMR peak of the prepared PLL. The SA introduction rate wasapproximately 67% based on the peak integral ratio of the methine proton adjacent to theamino group.

Figure 2.8. 1H NMR signal assignments for PLL-SA.
48

2.3.2 Cryopreservation
To confirm the cryoprotective effect of the synthesized polymer, L929 cells were frozen
at -80 °C by dissolving the polymer at 15 w/w% in a 0.3 M NaCl aqueous solution(Figure 2.9). pV and PLL had a high cryoprotective effect and a cell recovery rate ofabout 85% was confirmed. In addition, the cell recovery rate for pD was approximately
47%, which was lower than previous reports, but in previous studies, the cell viability
was calculated as the ratio of the number of viable cells after thawing to the number ofcells recovered after thawing. In this study, the cell recovery rate was calculated from thenumber of viable cells after freezing and the total number of cells at the time offreezing3, 9). It is not possible to recover 100% of dead cells after cryopreservation, andcell survival rates tend to be low with this calculation method, so there is no problem
with the results. High molecular weight (Mw 226000) pM and pA had poor
cryoprotective effects, with cell recovery rates of 26.6% and 14.4%, respectively.

49

Figure 2.9 Cell Recovery and of the polymer against L929 cells.

2.3.3 IRI activity
It has been reported that adding a polymer with high IRI activity during
cryopreservation suppresses ice crystal fusion and improves cell recovery. Therefore, IRI
activity was measured to investigate the cause of the difference in cryoprotective effect ofeach polymer. Splat cooling assays are often used to examine IRI activity, but theboundaries become vague due to overlapping ice crystals. Therefore, this time I used asandwich method in which a polymer solution was sandwiched between two cover glasses.
Figure 2.10 a) is an image of ice crystals annealed with a polymer solution on a -6 °C
low temperature stage for 30 minutes. When the polymers were added, the ice crystal size
was smaller compared to the 0.3 M NaCl aqueous solution, but the mean large grain size
(MLGS) was not significantly different between each polymer (Figure 2.10 b)). In

50

addition, with poly vinyl alcohol whose IRI activity contributes to cryoprotection, ice
crystals are smaller compared to the polymer synthesized this time even at a lower
concentration of 1 mg/ml. It has also been reported that there is a difference in the
cryoprotective effect of AFP with similar IRI activity, so it is thought that there is no
significant correlation between the cryoprotective effect and IRI activity10-13).

Figure 2.10 Polynucleated ice crystals after 30 minutes annealing -6 °C. a) Microscope
image, b) Mean large grain size (MLGS) with 0.3 M NaCl aq.
51

2.3.4 NMR measurements
NMR enables real-time observation of information on the structure, chemical
environment, amount, and dynamics of functional groups from information such as
resonance positions (chemical shifts), intensities, and line widths obtained from spectra.
Furthermore, since the motion of molecules is related to the relaxation process, it ispossible to detect changes in molecular motion by observing this. During freezing,
residual water provides space to the cells, which may protect them from mechanicaldamage. Furthermore, the increase in osmotic pressure caused by freeze concentration
causes serious damage to cells. In NMR spectra, it is possible to compare the broad peaksof solid components with the narrower peaks of solution-based molecules14). Therefore, I
discussed the freezing mechanism by investigating the physicochemical properties of apolymer solution during freezing using temperature-variable solid-state Magic Angle
Spinning (MAS)- NMR. A typical 1H NMR signal is shown in Figure 2.11. Figure 2.12
shows the amount of residual water during freezing. Using a semi-logarithmic plot
representation, we found that the residual water decreases with different time constants
at the high and low temperature sides (Figure 2.12 g)). This phenomenon can beinterpreted as the freezing of free water on the high temperature side and the freezing of
coordination water on the low temperature side. In the pA solution, which has the lowest
52

freezing protection effect, the freezing speed of the free water was clearly faster than thatof the other polymer solutions. Comparing the amount of water remaining at -10°C,
80% of the water in the pA solution froze, leaving only 20% as residual water, whereas
the other polymers left approximately 30% of water as residual water. pA is an amphotericelectrolyte polymer that has quaternary ammonium as a cation and sulfonic acid as ananion. Sulfobetaine polymers (polySPB) with similar ion pairs have close charge densities
of cations and anions, making it easy to form intramolecular salts 15). In addition,poly(SPB) has salt response and blocks the electrostatic interactions that form internal
salts with NaCl, allowing it to be used as a polymer brush hydrated in salt solutions 16, 17).
However, it has been reported that hydration water within poly(SPB) brushes forms a
network structure similar to hydrogen bonds in the bulk state 18). In other words, it isthought that this rapid decrease in the amount of residual water occurred in pA because
the network structure of hydrated water is similar to that of bulk water.
Since the line width of the peak mainly reflects the mobility of molecules, the line width(full width at half maximum; FWHM) of water protons in the polymer solution wasmeasured while changing the temperature (Figure 2.13). The line width begins to widen
in the temperature range where freezing of bulk water ends and freezing of water
coordinated to the polymer begins. The temperatures were pA; -17 °C, pD; -19 °C,53

pV; -21 °C, pM; -27 °C, PLL; -18 °C, respectively. The temperature at which the bulk
water freezes and the coordinated water freezes is around -20 °C, except at very highmolecular weight pM. In addition, as in previous reports, the line width of PLL broadened
significantly, eventually increasing to around 3440 Hz (Figure 2.13 e))2). The linewidthsof pV and pM also broadened to some extent, increasing to 2000 and 1500 Hz,
respectively (Figure 2.13 c) & d)). Furthermore, I investigated the temperaturedependence of the spin-lattice relaxation time (T1) by conducting water relaxation
experiments (Figure 2.14). 1H relaxation experiments can give an indication of therotational diffusion rate of residual water. It was confirmed that for all polymers, T1 has aminimum value at the switching temperature from bulk water to residual water freezing,
and T1 rises again as the temperature decreases. Furthermore, when T1 rose again, asharp increase in the line width of the residual water signal was simultaneously observed,
indicating a decrease in mobility and an increase in viscosity 19, 20). The degree ofbroadening of residual water was particularly remarkable in PLL, followed by pV and pM.
(Figure 2.14 c), d), f)).

54

Figure 2.11 1H NMR signal of water. a) pA, b) pD, c) pV, d) pM, e) PLL, f) Comparison
between each polymer.

55

Figure 2.12 Temperature dependent residual water ratio. a) pA, b) pD, c) pV, d) pM, e)
PLL, f) Comparison between each polymer.
56

Figure 2.13 Temperature dependent full-widths-at-half-maximum of the proton signals.
a) pA, b) pD, c) pV, d) pM, e) PLL, f) Comparison between each polymer.

57

Figure 2.14 Water proton T1 relaxation times as functions of temperature. a) pA, b) pD,c) pV, d) pM, e) PLL, f) Comparison between each polymer.

58

Na NMR was used to investigate the behavior of Na ions in polymer solutions during

23

freezing (Figure 2.15). Figure 2.16 shows that the chemical shift value of the Na ion peakchanges with temperature. It was revealed that all polymers have a maximum chemical
shift value at approximately the same temperature as the freezing of bulk water. For PLL
and pV, minimum values exist in even lower temperature ranges (Figure 2.16 c & e)). A
report on the vitrification ability of Trehalose-agarose gel observed by Fourier transform
infrared spectroscopy (FT-IR) positioned three transitions of glycosidic bond peaks
during melting as glass melting of frozen concentrate, rapid growth of ice crystals due torecrystallization, and melting of ice crystals, respectively21). In this study, the melting
process of the polymer solution was observed by NMR. The increase in the mobility of
the glassy water increases the amount of coordinating water, and thus the peaks that had
shifted to the high-field side are shifted to the low-field side due to reorientation to ions,which are considered to be the PLL and pV minima.
Previous studies have shown that the Na peak in polymer solutions is divided into two
types, central transition and satellite transition, as freezing progresses 2, 22, 23)(Figure 2.14f)). Na ion in aqueous solution has a single peak due to the presence of a surrounding
hydration shell, but the freezing of bulk water reduces the number of water moleculesaround the Na ion, causing the polymer chain to penetrate into the second hydration shell,59

resulting in an asymmetric transition and two separate peaks, one broad and one sharp.
Broad peaks are satellite transitions, and this time I will only discuss sharp peaks (centertransition) without making them the subject of discussion. Figure 2.17 shows thetemperature dependence of the intensity of the sharp component of the Na ion peak. The
first large drop in the intensity of each peak corresponds to the broadening and separationof the satellite peaks from the main peak due to the breaking of the electric field symmetryaround the Na ion, indicating that the polymer approaches around the Na ion in thistemperature range and affects the orientation of the coordination water. The
temperatures were pA; -7 °C, pD; -10 °C, pV; -7 °C, PLL; -11 °C, respectively foreach polymer. Since the temperature for all polymers is roughly the same as that at whichbulk water freezes, it was suggested that the decrease in bulk water around Na causes thepolymer chains and Na ions to approach each other, causing a break in the electric fieldsymmetry of the Na ions. Interestingly, at pM, the intensity of the Na ion peak is low at
0 °C. Since water is hardly frozen at around 0 °C, it is thought that the polymer is more
likely to coordinate with Na at pM (Figure 2.17 d)). In addition, the decrease in peakintensity starting around -35 °C is the temperature at which Na ions of the polymer
chain penetrate into the first hydration shell, that is, coordinate to the polymer chain. For
pV and pM with cations and anions introduced regioregularly and PLL the peak intensity
60

decreases significantly after -35°C, so it is thought that more Na ion coordination occurs
than for pA and pD (Figure 2.17 c-e)).
Figure 2.18 compares the half-widths of the sharp peaks (central transitions). Chemical
exchange between the two states of Na ions, namely asymmetric and symmetric states ofthe electric field around the Na ion broadens the peaks, and when the chemical shiftdifference between the central and satellite transitions and the exchange rate between thetwo states are comparable, a local increase in line width (of the peak) is observed. There
were no major differences in local maximum values among the polymers, but local
minimum values appeared for PLL, pV, and pM (Figure 2.18 c-e)). Similar to thediscussion on chemical shift positions earlier, this is thought to be because in thesepolymers, the mobility of Na ions increases first due to glass melting, and then water
rearranges. T1 of Na ions also gives an indication of the rotational diffusion rate of Na
ions, as in the water relaxation experiment. At low temperatures, there is no significant
difference among the polymers, but there is a difference in the relaxation time at 0 °C,
suggesting that pV and pM have a higher ability to trap Na ions than the other polymers
because the motion of Na ions is more inhibited than the other polymers (Figure 2.19).

61

Figure 2.15 23Na NMR signal. a) pA, b) pD, c) pV, d) pM, e) PLL, f) Center transition andsatellite transition

62

Figure 2.16Temperature dependence of Na ion peak shift position. a) pA, b) pD, c) pV, d)pM, e) PLL, f) Comparison between each polymer.

63

Figure 2.17 Temperature dependence of Na ion sharp peak shift intensity. a) pA, b) pD,c) pV, d) pM, e) PLL, f) Comparison between each polymer.

64Figure 2.18 Temperature dependent full-widths-at-half-maximum of the Na ion signal. a)pA, b) pD, c) pV, d) pM, e) PLL, f) Comparison between each polymer.

65

Figure 2.19 Na ion T1 relaxation times as functions of temperature. a) pA, b) pD, c) pV,d) pM, e) PLL, f) Comparison between each polymer.

66

The physical meanings of the important parameters correlated between the behavior ofwater and the Na ions of each polymer are outlined in Table 2.2. In the case of pA, thewater hydrated in the polymer has a hydrogen bond close to that of bulk water, so theamount of residual water decreases rapidly, and the amount of residual water at -10 °C
is 20.2%, about 10% lower than that of other polymers. In addition, the temperature atwhich Na ions coordinate with the polymer chain is around -31 °C, which is the same asother polymers. This is thought to cause a sudden increase in osmotic pressure, leading
to excessive cell dehydration and a decrease in the cryoprotective effect 24, 25). Furthermore,since the Na ion intensity at -45 °C is similar to that of pD, which has a moderatecryoprotective effect, it is thought that rapid dehydration immediately after freezing leads
to the low cryoprotective effect of pA. For pV and PLL, the minima in the chemical shift
position of Na around -40 °C and the increase in the line width of water suggest that a
rapid increase in viscosity (vitrification) is occurring, and water is expected to become
amorphous during freezing (Figures 2.14 c), d) & 2.16 c), d)). Furthermore, since the Na
ion intensity of these polymers at around -45 °C is lower than that of pA and pD, it isthought that the polymer chains coordinate more Na ions. In other words, polymers witha high cryoprotective effect are thought to have an ability not only to vitrify the solution
but also to prevent an excessive rise in osmotic pressure during freezing. PD, which has
67

a similar structure to pV, has a bulkier methyl group in its main chain, which reduces thedegree of freedom in rotation and orientation of the polymer structure, making it more
likely to generate ice nuclei25, 26). Therefore, it is thought that it does not have thevitrification ability that was seen with pV. The higher Na trapping capacity of pV is also
expected to be due to the 1:1 and alternating sequence of anions and cations in pV,
whereas in pD the anions and cations are completely random. pD has a specifictemperature dependence of residual water and Na ion signals compared to other polymers,
and both hardly decrease from around -30 °C. In addition, since the peak of polymer
chain of pD is broad in the higher temperature region, it is thought that a cryoprotective
effect can be obtained by forming a polymer matrix and isolating water and Na ions(Figure 2.20). From the T1 measurement results for Na, it can be seen that pV and pM
have the same degree of motility (in a low state) at 10.3 ms and 8.3 ms, respectively, atnear 0 °C (Fig. 2.17 c), d)). In fact, satellite transitions appear from around 0 °C in
pM, suggesting that polymer-Na interactions occur at higher temperatures in pM.
Furthermore, since the decrease in residual water is about the same as that in pV and PLL,
it is thought that trapping of Na ions occurs at higher temperatures, resulting ininsufficient cell dehydration and a decrease in the cryoprotective effect27, 28). This isconsistent with the fact that the switching temperature from freezing bulk water to68freezing coordination water is significantly lower at -27°C at pM than for other polymers.
Also, in pM, the line width of the water proton peak appears to broaden as in pV and PLL.
However, since the increase in T1 does not occur in parallel, means no increase in theviscosity, it can be seen that the broadening is not caused by vitrification. Since pM has aweight average molecular weight of 216,000, which is extremely large compared to otherpolymers, it is thought that the decrease in residual water may have increased chain
entanglement, making the gaps in the matrix coarser. Although this study only measured
temperatures down to around -50 °C, general cryopreservation uses

-80 °C or lower

temperature, so polyampholytes with no or lower vitrification capacity are likely to cause
cryoinjury because salt and water are difficult to isolate from the cells. Therefore, in order
for a polyampholytes to exhibit a cryoprotective effect, it is thought that salt concentration
adjustment during freezing by trapping salt or water and vitrification ability are necessary.

69

Figure 2.20 Change in peak intensity of polymer due to temperature change. a) pA, b)pD,c) pV, d) pM, e) PLL.

70

Table 2.2 Various data for each polymer obtained by temperature-variable solid-state NMR.

T1 of water Chemical shift change

T1 of Na

Water proton peakintensity

Water proton line width

Around -Around -Temperature

10°C

45°C

at which

residual

residual

widening

water / %water / %starts / °C

pA

20.2

8.4

-17

740

0.56

-18

No

-7

0.33

-31

No

22

pD

28.3

9.9

-19

590

0.58

-21

No

-10

0.3

-31

No

16

pV

34.1

9.9

-21

1430

0.72

-19

-44

0.23

-32

-42

10.3

pM
PLL

33.4
29.7

10.2
7.4

-27
-18

1070
3440

0.49
0.92

-21
-18

No
-40

-7
≧ 0

0.24
0.21

-32
-28

-42
-39

8.3
14.3

Line width
at around 45 °C / Hz

protonLongitudinalrelaxation
time around-45 °C / sof Na

Na signal strength
Satellite

First

Secondpeak

inflection

inflection

appearance

point / °C

point / °C

temperature
/ °C

71

-11

Na signal line width

signalNa signal

Maximum

Secondintensityline width

widening of relaxation

around -temperature line widthtime around

45°C

/ °C

0 °C / ms/ °C

Longitudinal

2.4 Conclusion
In Chapter 2, I synthesized polyampholytes with different side chain structures andcorrelated their cryoprotective effects and structures. pA with stronger anions and cations
and pM with higher molecular weight and polydispersity had poor cryoprotective effects.
The IRI assay revealed that there was no significant difference in the ability of eachpolymer to inhibit ice crystals, and that there was no clear correlation between the
cryoprotective function and IRI activity of these polymers. Temperature-variable solidstate MAS-NMR confirmed a rapid decrease in the amount of residual water immediately
after freezing in pA. This is thought to be due to the fact that the hydrogen bonds of thehydration water in pA are relatively close to the bulk water. For polymers with highcryoprotective effect such as pV and PLL, vitrification and coordination of Na ions to thepolymer chain were observed at higher temperatures, suggesting that, as in previous
studies, vitrification is an important factor for cryoprotective effect. In the future, it isbelieved that a deeper understanding of polymer structure and vitrification ability will
enable the molecular design of more effective cryoprotectants.

72

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M. Bonn and K. Meister, Biomacromolecules, 2022, 23(3), 1214-1220.
14) D. W. Aksnes and L. Gjerdåker, J. Mol. Struct., 1999, 475(1), 27-34.
15) Q. Shao, L. Mi, Z. Han, T. Bai, S. Liu, Y. Li and S. Jiang, J. Phys. Chem. B, 2014,
118(24), 6956-6962.
16) T. Wang, X. Wang, Y. Long, G. Liu and G. Zhang, Langmuir, 2013, 29(22), 65886596.
17) Y. Chang, S. Chem, Z. Zhang, and S. Jiang, Langmuir, 2006, 22(5), 2222-2226.
18) Y. Higaki, Y. Inutsuka, T. Sakamaki, Y. Terayama, A. Takenaka, K. Higaki, N. L.
Yamada, T. Moriwaki, Y. Ikemoto and A. Takahara, Langmuir, 2017, 33(34), 84048412.
19) N. Bloembergen, E. M. Purcell and R. V. Pound, Phys. Rev, 1948, 73, 679-712.
20) I. Solomon, Phys. Rev, 1955, 99, 559-565.
21) M. Wang, A. Mahajan, A. Mahajan, J. S. Miller, D. H. McKenna and A. Aksan, ACS

Appl. Bio Mater, 2023, 6(6), 2226-2236.
7422) D. J. Morales and S. Greenbaum, Int. J. Mol. Sci., 2020, 21(9), 3402.
23) C. Naumann and P. W. Kuchel, Polym, Chem., 2010, 1, 1109-1116.
24) N. E. Hoffmann and J. C. Bischof, Urology, 2002, 60(2), 40-49.
25) J. H. Crowe, J. F. Carpenter, L. M. Crowe and T. J. Anchordoguy, Cryobiology, 1990,
27(3), 219-231.
26) M. Mousazadehkasin and J. G. Tsavalas, Biomacromolecules, 2020, 21(11), 46264637.
27) P. Mazur, J. Gen. Physol. 1963, 47(2), 347-369.
28) P. Mazur, S. P. Leibo and E. H. Y. Chu, Exp. Cell. Res., 1972, 71(2), 345-355.

75

Chapter 3
Enhancement of cryopreservation with intracellularly
permeable zwitterionic polymers
3.1 Introduction
Polymeric cryoprotectants are often studied as alternatives because they are usually large
and cannot easily pass through the cell membrane. Polymers that inhibit ice crystallization,such as polyvinyl alcohol, antifreeze glycoproteins, glycopeptides, and polyampholytes(that are composed of carboxylated polylysine or N-dimethylamine and carboxylic acids),
have been extensively studied as alternative polymeric cryoprotectants. 1–5) Studies havealso been conducted on small-molecule cryoprotectant structures in polymer side chains.
Burkey et al. reported that polymers with a polyol structure in the main chain and a
DMSO-like methyl sulfinyl structure in the side chain provide higher cryoprotection thanDMSO6). Similarly, a methacrylate polymer with a methyl sulfoxide structure has beenreported, but its cryoprotective properties are inferior to those of methacrylate polymers7).
A notable difference between the two polymers is that the methacrylate form does not
penetrate intracellularly, whereas the polyol form is taken up by the cell via endocytosis.
Anotherpromisingmembrane-impermeable

cryoprotectant

thatenhances

cryoprotection upon intracellular permeation is trehalose8, 9). Outside the cell, trehalose76

inhibits ice formation via hydration, while inside the cell, trehalose interacts withmembrane lipids and contributes to membrane stabilisation. Furthermore, it has beenreported that the low-molecular-weight polyethylene glycol (PEG) can permeate cells andimprove cryoprotection by suppressing the increase of osmotic pressure during freezing,stabilising cell membranes, and inhibiting ice formation, suggesting that the intracellular
introduction of membrane-impermeable cryoprotectants is crucial for improving
cryoprotective efficacy10). Zwitterionic polymers are polymers that contain both cations
and anions in their molecules. Zwitterionic polymers have been the subject of muchresearch in applications such as drug delivery systems, antifouling coatings, andhydrogels11-15).

Zwitterionicpolymers,

such

as

sulfobetaine

polymersandpoly(MPC/BuMA), a copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC)
and butyl methacrylate (BuMA), have been shown to permeate the entire cytoplasm
without requiring endocytosis16–18). Because sulfobetaine polymers have also beenreported to have a cryoprotective effect, the development of a cell-permeable copolymer
with a sulfobetaine polymer and a DMSO structure is expected to improve thecryoprotective effect19).
In chapter 3, copolymers of 3-((3-acrylamidopropyl)dimethylammonio)propane-1sulfonate (SPB) and 2-(methyl sulfinyl)ethyl methacrylate (MSEMA), which has the77same methyl sulfinyl structure as DMSO, were synthesised, and their cryoprotective effectwas confirmed. After investigating the penetration and reverse diffusion of thefluorescent-modified polymers into cells, the effectiveness of the zwitterionic polymers as
the carriers of membrane-impermeable cryoprotectants was confirmed by performing
cryopreservation with the polymers penetrating the cells. Poly(SPB) enhanced
cryoprotection via intracellular penetration, indicating that it could be used as a novel
cryoprotectant carrier. Expanding upon established cryoprotective properties of SPB, ourinnovative approach integrates DMSO-mimicking monomers through strategic
copolymerization, highlighting the potential to exploit SPB's intrinsic intracellularpermeability for superior cryoprotection of 3D tissue constructs. To the best of ourknowledge, this work represents the first incorporation of methylsulfinyl motifs into thezwitterionic polymer structure, generating a material with significantly enhanced
properties and indicating a substantial advancement in the cryoprotection field.

78

3.2 Materials & methods
3.2.1 Materials
Methacryloyl

chloride,

2-(methylthio)ethanol,

n-butylamine,

and2,2'-

azobis(isobutyronitrile) (AIBN) were purchased from Tokyo Chemical Industry Co.
(Tokyo,Japan);

fluoresceino-acrylate,triethyl

amine

(TEA)

and

2-

(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) were purchased from
Sigma-Aldrich (St. Louis, MO, USA); tris(2-carboxyethyl)phosphine hydrochloride and3-(4,5-dimethylthial-2-yl)-2,5-diphenyltetrazalium bromide (MTT) were purchasedfrom FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan); dichloromethane(DCM), dimethyl sulfoxide (DMSO), methanol and hydrogen peroxide (30%) was
purchasedfrom

NacalaiTesque,Inc.

(Kyoto,acrylamidopropyl)dimethylammonio)propane-1-sulfonate

Japan);
wasandsuppliedby3-((3Osaka

Organic Chemical Industry, Ltd (Osaka, Japan). All the reagents were used as received
without further purification.

3.2.2 Characterization
H NMR was measured using a Bruker Avance III 400. The deuterated solvent used was1purchased from Sigma-Aldrich. The NMR data were analysed using Topspin 3.5 software.
FT-IR was measured using JASCO FT/IR-6X in attenuated total internal reflection mode.
79

Measurements were performed with 16 scans per spectrum and a resolution of 4 cm -1.
The molecular weights and distributions (polydispersity index; PDI) of the polymers were
determined by gel permeation chromatography (GPC; BioSep-s2000, Phenomenex, Inc.,
CA, USA) using a Waters Alliance HPLC system. A 0.1 M sodium bromide aqueous
solution was used as the mobile phase (flow rate, 1 mL/min) and pullulan as the standard.

3.2.3 Synthesis of 2-(methylthio)ethyl methacrylate (MTEMA)
The synthesis of MTEMA is shown in scheme 3.1. TEA (4.46 g, 44.1 mmol) and 2(methylthio)ethanol (3.64 g, 39.5 mmol) were added to 50 mL of DCM and kept at 0 °C.
Then, methacryloyl chloride (4.60 g, 44.0 mmol) was added dropwise and the mixture
was allowed to react for 10 h. After completion of the reaction, the precipitates were
filtered and washed thrice with a saturated aqueous sodium hydrogen carbonate solution.
They were then washed with saturated saline and dehydrated using magnesium sulphate.
The solvent was removed using an evaporator and dried under a vacuum (5.71 g, yield
52.0 %). 1H NMR (CDCl3, 400 MHz): δ –6.13 (s, 1H, Hcis), 5.59 (s,1H, Htrans), 4.33 (t,2H, –O–CH2), 2.77 (t, 2H, –CH2–S–), 2.17 (s, 3H, –S–CH3), 1.95 (s, 3H, –CH3).

Scheme 3.1 Synthesis of MTEMA.
80

3.2.4 Synthesis of MSEMA
The synthesis of MSEMA is shown in scheme 3.2. MTEMA (4.0 g, 25 mmol) was cooled
to 0 °C in an ice bath, and N2 was bubbled through for 30 min. Then, hydrogen peroxidewater (3.12 g, 27.5 mmol) was added to the mixture dropwise to raise its temperatureback to 25 °C for reaction to proceed for 24 h, following which 50 mL distilled water wasadded to terminate the reaction. The product was then extracted with DCM thrice, andthe collected organic layers were removed by evaporation and dried under vacuum (3.44g, yield 78.3 %). 1H NMR (CDCl3, 400 MHz): δ –6.15 (s, 1H, Hcis), 5.64 (s, 1H, Htrans),
4.59 (m 2H, –O–CH2), 3.07 (m, 2H, –CH2–SO–), 2.67 (s, 3H, –S–CH3), 1.96 (s, 3H, –CH3).

Scheme 3.2 Synthesis of MSEMA.

3.2.5 Synthesis of zwitterionic polymer
The synthesis of zwitterionic polymer is shown in scheme 3.3. Poly(SPB) was preparedby dissolving SPB (5.57 g, 20 mmol) in DDMAT (72.9 mg, 0.2 mmol), and AIBN (6.57
mg, 0.04 mmol) in 40 mL of a 1:3 water/methanol solution. When synthesizing81

copolymers with MSEMA, MSEMA was added to the reaction mixture at 10, 20, and 30 %of the total polymer content. For example, when the introduction rate was 10 %, SPB(5.01 g, 18.0 mmol), MSEMA (0.352 g, 2.0 mmol), DDMAT (72.9 mg, 0.2 mmol), andAIBN (6.57 mg, 0.04 mmol) were dissolved in 40 mL of 1:3 water/methanol solution. N2
bubbling was performed for 1 h, and the mixture was reacted at 70 °C for 6 h. Samples
were subsequently taken for 1H NMR analysis. Monomer conversion was determinedusing vinyl protons near 5.8 ppm and 6.2 ppm based on the methylene peak of thepolymer near 2.9 ppm. After completion of the reaction, the sample was dialysed against
methanol and water for 24 h (MWCO of 3.5 kDa, Spectra/Por® 3 Dialysis Membrane,
Spectrum Labs, Inc., Rancho Dominguez, CA, USA) and freeze-dried. The chemical
structure of the compound was confirmed by 1H NMR spectroscopy, using D2O as thesolvent.

Scheme 3.3 Synthesis of zwitterionic polymer.

82

3.2.6 Synthesis of fluorescence-modified polymers
The synthesis of fluorescence-modifies polymer is shown in scheme 3.4. Each polymer
(0.76

mol)

was

dissolved

in

1

M

NaCl

aqueoussolution,andtris(2-

carboxyethyl)phosphine hydrochloride (17.4 mol) was added. N-butyl amine (0.76mmol) was added, reacted at room temperature for 2 h, dialysed for 7 d (MWCO of 3.5
kDa), and freeze-dried. The obtained polymer (6.08 mol) was dissolved in PBS
containing a small amount of tris(2-carboxyethyl)phosphine hydrochloride. Fluoresceino-acrylate (18.2 mol) was dissolved in DMSO, mixed with the polymer solution, andallowed to react at room temperature for 10 h in the dark. Following the completion of
the reaction, the solution was dialysed for 7 days (MWCO of 3.5 kDa) and then freezedried.

Scheme 3.4 Synthesis of fluorescence-modified polymers.
833.2.7 Cell culture
Mouse fibroblasts (L929; American Type Culture Collection, Manassas, VA, USA) wereprepared in Dulbecco's modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO,
USA) supplemented with 10 % foetal bovine serum (FBS). Cells were cultured in an
incubator at 37 °C with 5 % CO2. Passaging was performed when the cells exceeded
80 % of the bottom of the dish. For passaging, the cells were detached by treatment withtrypsinsolution

(0.25

%

[w/v]

trypsincontaining

0.02

%

[w/v]

ethylenediaminetetraacetic acid in PBS).

3.2.8 Cryopreservation
The polymer solution was dissolved in an aqueous 0.3 M NaCl aqueous solution to 10
w/w%. The solution was filter-sterilised through a 0.22 m filter. Then, 1 × 106 L929
cells were suspended in 1 mL of the polymer solution and stored at –80 °C; cooling was
conducted without a controlled cooling rate. After permeation of the cells with thepolymer, 1 × 106 permeated-L929 cells were seeded in a 35 mm dish and cultured for 24
h. Then, the medium was removed and 1 mL of polymer solution was added to the culture,
which was incubated at 4 °C for 10 min. After washing thrice with PBS, the cells weretreated with trypsin and collected by centrifugation (1000 rpm). They were thensuspended in 1 mL of the polymer solution and stored at -80 °C in the same manner.
84

After 24 h, the cells were thawed by immersing the samples in a water bath at 37 °C.
The cell suspension was then diluted 10-fold with DMEM and centrifuged at 1000 rpm
for 4 min. The cells were then suspended in a small amount of DMEM and viability wasdetermined by staining them with trypan blue. Cell viability and recovery were
determined using the following formula (Eq. 3.1).
Recovery rate (%) =Living cell× 100
Frozen cell
(Eq. 3.1)

3.2.9 Mitochondrial membrane potential
Mitochondrial membrane potential was measured using the Tetramethylrhodamine
Methyl Ester Perchlorate ( TMRM, TCI). Before the experiment, 2.5 mg of TMRM wasdissolved in 500 L of DMSO to create a 10 mM DMSO stock solution. First, cells wereincubated in 35 mm glass bottom dishes for 24 hours after freeze-thawing. Once themedium was removed, 1 ml of DMEM without FBS was added. Thereafter, 1 mL of
TMRM staining solution diluted to 250 nM was added and left standing in a CO2
incubator for 30 minutes. After staining, cells were washed with DMEM without FBS andobserved

underaconfocalmicroscope.

For

control,

carbonyl

cyanide4-(Trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler for electron transport andoxidative phosphorylation, was added to a final concentration of 20 mM before staining85

with TMRM, and the cells were incubated for 10 minutes. Depolarization was performed.

3.2.10 Cytotoxicity test
L929 cells were dispensed into 96-well plates at 1 × 103 cells/100 L and cultured in an
incubator for 3 days. Samples were prepared by dissolving in DMEM so that theconcentrations of the polymer and DMSO ranged between 0 and 20 %, followed by filter
sterilisation. After culturing, 100 L of each prepared sample was dispensed and further
incubated again in an incubator for 2 days. MTT was dissolved to 0.3 mg/mL in DMEM
without FBS, and 100 L of the solution was dispensed and incubated for 3 h.
Subsequently, all solutions were removed from the plate, 100 L of DMSO was dispensed,
and the absorbance was measured at  = 540 nm using a microplate reader (Infinite 200
PRO M Nano, Tecan).

3.2.11 Confocal Microscopy
For fluorescence observation, 1 × 106 L929 cells were seeded in a 35-mm glass-bottom
dish and cultured for 24 h. Then, the medium was removed, 1 w/w% fluorescencemodified polymer solution was added dropwise, and the mixture was incubated at 4 °C
for 10 min. After removing the polymer solution and washing with PBS thrice, the cells
were treated with PBS again and observed under a confocal laser microscope (FV1000D,
Olympus).
86

3.2.12 Observation of counter diffusion
To observe the counter diffusion, 1 × 106 L929 cells were seeded in a 35 mm dish andcultured for 24 h. Subsequently, the medium was removed, 1

w/w% of the fluorescence-

modified polymer solution was added dropwise, and the mixture was incubated at 4 °C
for 10 min. After washing thrice with PBS, the cells were trypsinised and collected bycentrifugation. The harvested cells were suspended in 10 mL of medium, and 1 mL of thesuspension was plated on a 35-mm glass-bottom dish and incubated at 37 °C.
Fluorescence observations were performed every 10 min using BZ-X800 (Keyence, Osaka,
Japan).

3.2.13 Differential scanning calorimetry
Differential scanning calorimetry (DSC) was performed using the EXTAR SII-6200(Seiko Instruments, Japan). After cooling from 5 °C to –50 °C at a cooling rate of
1 °C/min under a nitrogen flow of 50 mL/min, the temperature was maintained for 5
min and then heated to 20 °C at 1 °C/min.

87

3.3 Results & Discussion
3.3.1 Polymer characterization
MSEMA was synthesised in two steps. First, 2-(methylthio)ethanol was reacted withmethacryloyl chloride to prepare MTEMA (Figure 3.1). Then, MTEMA was oxidisedwith 1.1 equiv. of hydrogen peroxide to obtain MSEMA; 1H NMR results confirmed thecomplete oxidation of the MTEMA20, ,21) (Figure 3.2). In addition, in the Fouriertransform infrared spectrum, the peak near 1300 cm–1 showed no significant change andthe S=O stretching vibration of sulfone could not be observed, suggesting that excessiveoxidation did not occur (Figure 3.3).

Figure 3.1 1H NMR spectrum of MTEMA.
88

Figure 3.2 1H NMR spectrum of MTEMA.

Figure 3.3 FT-IR spectra of MTEMA and MSEMA.
89

Poly(SPB) and poly(SPB/MSEMA) were synthesised via reversible addition–
fragmentation chain-transfer polymerisation, using DDMAT and AIBN as the chaintransfer agent (CTA) and initiator (In), respectively. Polymerisation was performed
with an initial feed ratio of [monomer]:[CTA]:[In] = 100:1:0.2, aiming at a degree ofpolymerisation of 100. In addition, for poly(SPB/MSEMA10), poly(SPB/MSEMA10),
and poly(SPB/MSEMA10), the ratios of MSEMA were set to 10, 20, and 30 of the total
monomers, respectively. The peak in the NMR spectrum corresponding to the sulfinyl
methyl protons at approximately 2.4 ppm increased with increasing MSEMA feed ratio(Figure 3.4-7). The synthesized polymers exhibited very high monomer conversions ofover 90 % and exhibited narrow size disparities, as demonstrated by size exclusion
chromatography (Figure 3.8, Table 3.1). The incorporation rate of MSEMA wascalculated from the peak of 2.6 - 2.8 ppm (3H of MSEMA) compared to the peak of 2.8- 3.7 ppm (12H of SPB).
Table 3.1 Characterization of zwitterionic polymer.
Composition

Composition

Molar

Conversion

Mn

Mw/

(in feed)(NMR)a)ratiob)/ %c)×104 d)

Mnd)

SPB

MSEMA

SPB

MSEMA

poly(SPB)

100

0

100

0

100 : 1 : 0.2

96.7

1.47

1.25

poly(SPB/MSEMA10)

90

10

89.9

10.1

100 : 1 : 0.2

98.6

1.48

1.29

poly(SPB/MSEMA20)

80

20

81.8

18.2

100 : 1 : 0.2

98.5

1.25

1.24

poly(SPB/MSEMA30)

70

30

75.7

24.3

100 : 1 : 0.2

97.5

1.29

1.27

a) Using the integral value of the NMR peak in Figure 3.4-7, the amount of MSEMEA was calculated by Eq.3.2. b)[monomer]:[CTA]:[initiator].
c) Determined using vinyl protons near 5.8 ppm and 6.2 ppm based on the methylene peak of the1
polymer near 2.9 ppm by H NMR. d) Determined by GPC (Figure 3.8).

90 (2.8 ppm)
MSEMA (%) =3 (3.0 - 3.6 ppm)
12+

 (2.8 ppm)

× 100

3

where I refers to the area under the peak corresponding to the chemical shift

Figure 3.4 1H NMR spectrum of poly(SPB).

91(Eq. 3.2)

Figure 3.5 1H NMR spectrum of poly(SPB/MSEMA10).

Figure 3.6 1H NMR spectrum of poly(SPB/MSEMA20).

92

Figure 3.7 1H NMR spectrum of poly(SPB/MSEMA30).

Figure 3.8 GPC curve of polymers.

93

3.3.2 Cytotoxicity
The cytotoxicity of each polymer is shown in Figure 3.9. During the freeze–thaw cycle,
the cells are exposed to the polymer for a short duration. Because the maximum
intracellular permeation time of the polymer was approximately 1 h, a cytotoxicity test
was conducted by exposing L929 cells to a 10 w/w% polymer solution for 1 h. The results
showed that after 1 h of exposure, the cell recovery rate was comparable to that observed
without the polymer solution, indicating that there was no adverse effect on the cellrecovery rate attributed to polymer toxicity during cryopreservation by intracellularpermeabilisation.

Figure 3.9 Cytotoxicity of each polymer against L929 cells 1 hour after exposure.

94

3.3.3 Intracellular permeability
To confirm permeation of the polymer into the cells, the RAFT ends of the polymer
were treated with butylamine and then modified with fluorescein-o-acrylate via a thiol–ene reaction. Confocal microscopy was then used to confirm that the fluoresceinmodified polymers had permeated the cells. Because intracellular permeation of
sulfobetaine polymers is reported to be enhanced at 4 °C, polymer permeation was
performed at 4 °C for 10 min17, 18) (Figure 3.10 a)). The fluorescence-modified polymer
was also found to diffuse throughout the cell, including the nucleus (Figure 3.10 c)).
Poly(MPC/BuMA) has been reported to diffuse out of cells depending on the
incubation time after intracellular permeation22). As counter diffusion may occur forpoly(SPB) as well, after permeation of the fluorescein-labelled polymer, it was
incubated at 37 °C and the fluorescence of the cells was checked every 10 min using afluorescence microscope. Figure 3.10 b) shows fluorescence microscopy images of thecells incubated at 37 °C after polymer penetration. Following a 30-min incubation
period, the fluorescence of the cells decreased compared to the initial levels observed
immediately after polymer penetration, confirming that the polymers were counterdiffusing, as reported previously. This indicated that these polymers did not requirecomplex manipulations to be removed from the cells after the freeze–thaw cycle.
95

Figure 3.10 Fluorescence images of L929 cells. a) Confocal microscope images offluorescein-labelled poly(SPB) and poly(SPB/MSEMA10) in L929 cells. b) Fluorescence
images of L929 cells at 10-minute intervals after incubating at 37 °C after permeation offluorescein-labelled poly(SPB). Scale bars: 10 m.
96

3.3.4 Cryoprotective effects
To confirm the cryoprotective effect of the synthesised polymers, L929 cells were
cryopreserved at –80 °C in 0.3 M NaCl aqueous solution with 10 w/w% polymers. In
addition, to investigate the cryoprotection effect by the intracellular permeation of thepolymer, 1.0 × 106 L929 cells were pre-seeded and incubated with 10 w/w% polymer
solution at 4 °C for 10 min before freezing. Figure 3.11 a) shows that the cell recovery
rate decreased as the percentage of MSEMA increased. By contrast, when the polymer
was allowed to permeate the cells, the cell recovery rate increased for poly(SPB) andpoly(SPB/MSEMA10); In particular, for poly(SPB/MSEMA10) with a methyl sulfinyl
structure, an increase of more than 20 % was observed. To confirm that the introductionof the polymer improved the cryoprotective effect, I also checked the cryoprotective
effect of counter diffusion at 37 °C for 1 h after polymer infiltration and found that thecell recovery rates were significantly lower for both polymers compared to those at the
infiltration conditions, indicating that the cryoprotective effect was improved bypolymer permeation (Figure 3.11 b)).

97

Figure 3.11 Cryoprotective properties of poly(SPB) and poly(SPB/MSEMAn). a)
Comparison of cryoprotective effects with and without polymer permeation. b) Cell
recovery after permeation and counter-diffusion of poly(SPB) and poly(SPB/MSEMA10).

When the percentage of MSEMA was increased to 20 % or 30 %, no improvement incell recovery was observed even after intracellular permeabilisation. Cell proliferation
after thawing showed that the number of cells after 7 days was 60–70 %, although the98proliferation rate was lower than that of fresh cells or DMSO, indicating that the growthrate of cells was not affected by intracellular infiltration of the polymer (Figure 3.12).

Figure 3.12 Cell growth curve after freeze–thaw cycle.

3.3.5 Thermal behavior of polymers
As membrane-permeable cryoprotectants such as DMSO and glycerol are known todisplace intracellular water molecules during freezing, which causes freezing-point
depression and vitrification, I used differential scanning calorimetry (DSC) to measure
the freezing point and amount of ice formed during the freezing of a 0.3 M NaClaqueous solution containing 10 w/w% polymers23,24). All the polymers showed less ice

99formation than the 0.3 M NaCl aqueous solution. Notably, there were no significant
differences between the solidification and melting enthalpies of the polymers, and thetotal amounts of ice formed were similar (Fig. 3.13, Table 3.2). This suggests that theamount of antifreeze water did not directly affect the cryoprotective effect. The freezing
point of poly(SPB/MSEMA10) was slightly higher than those of the other polymers,
suggesting that the salt ions in the solution were captured faster, possibly alleviating
osmotic shock.

Table 3.2 Thermal properties of zwitterionic polymers as measured by DSC.

0.3 M NaCl aq.
poly(SPB)

Tfa) (°C)–19.7 ± 6.52
–19.4 ± 0.55

Hfb) (J/g)–259.5 ± 12.20–227.5 ± 5.53

Hmc) (J/g)
225.5± 4.06
200.0 ± 4.50

poly(SPB/MSEMA10)–16.40 ± 2.07

–240.6 ± 3.88

208.3 ± 4.91

poly(SPB/MSEMA20)
poly(SPB/MSEMA30)–19.9 ± 1.26
–17.9 ± 3.36

–233.7 ± 5.20
–235.00 ± 1.59

203.2 ± 3.45
204.1 ± 6.09

a) Tf: Ice nucleation temperature of cryoprotectant solution, b) Hf: solidification enthalpy, c) Hm:
Melting enthalpy.

100

Figure 3.13 DSC trace of polymers.

Based on the two-factor hypothesis proposed by Mazur et al.,24) lethal cell damage
during freezing can be divided into two major categories: osmotic damage due todehydration and damage due to intracellular ice crystal formation. Matsumura et al.
have previously reported the mechanism of cryoprotection by polyampholytes using
solid-state NMR25). The dynamics of the water, salt, and macromolecules duringfreezing were also evaluated. The effects of the inhibition of ice crystal growth by
improving the viscosity of water during freezing, and the control of dehydration by the
dynamics of the salt and polymer chains in solution that inhibit salt concentration
increase during freezing, are important. The mechanism is that the ions in thepolyampholytes interact strongly with the salt ions in the residual water during freezing,101thereby suppressing the rapid increase in osmotic pressure due to the freezing
concentration and dehydration under sufficiently high and mild conditions, thus
suppressing intracellular ice crystal formation and dehydration damage. The degree ofion trapping is affected by the charge density of the amphoteric electrolyte. The
amphoteric portion that can trap salt ions was reduced in poly(SPB/MSEMA20) andpoly(SPB/MSEMA30), which might have resulted in osmotic shock and, consequently,
lower cell recovery rates.

3.3.6 Freezing and thawing of cell pellets.
To confirm that cryoprotection can be achieved with intracellular polymers alone, I
removed the extracellular solution after polymer permeation and cryopreserved the cellpellets (Fig. 3.14).

Figure 3.14 Cryoprotective capacity of trehalose- and poly(SPB)-permeabilized cell pellets.
102For comparison, trehalose, a protective agent that does not penetrate into cells, wasused. After suspension in trehalose solution, the cells were pelleted by centrifugation,
the supernatant was removed and frozen. Poly(SPB) improved the cell recovery rate,
which was approximately 45% compared with the case without permeation into thecells. The above recovery rate is comparable to that of DMSO. Furthermore, after
permeating the cells with 1% fluorescently modified polymer, the cell pellets were
cryopreserved (Figure 3.15). As a result, polymer fluorescence was observed only inliving cell groups that were not stained with trypan blue. In addition, by incubating thecells at 37°C and 5% CO2 for 30 minutes after thawing, intracellular fluorescence wasgreatly reduced, similar to the observation of counter-diffusion. This experimentreconfirmed the enhanced cryoprotective effect of the polymer through intracellular
penetration and its reverse diffusion after thawing.

Figure 3.15 Cryoprotective capacity of fluorescence-modified poly(SPB)-permeabilized
cell pellets.
103Furthermore, the membrane potential of mitochondria was observed by TMRM
fluorescent dye 24 h after thawing of the cell (Figure 3.16).

Figure 3.16 Mitochondrial membrane potential before and after freeze-thaw. Scale bars:
100 m.

Mitochondria play a role in intracellular ATP production and apoptosis, allowing
observation of cell activity after thawing. TMRM is a fluorescent dye has a rhodamine
backbone and is the most commonly used non-cytotoxic cell-permeable fluorescent dye
to assess mitochondrial function. TMRM accumulates in mitochondria in response tomitochondrial membrane potential. However, when the mitochondrial membranepotential is reduced, as in the case of FCCP(+), which depolarizes mitochondria,
TMRM is either not taken up by mitochondria or is released from mitochondria.

104

Therefore, staining cells with TMRM allows one to examine the ATP activity of cells
after thawing. Regardless of the permeation or non-permeation of poly(SPB), redfluorescence due to high mitochondrial activity was observed in both non-frozen andfrozen cells. Therefore, the amount of ATP production after thawing is considered to beequivalent to that of non-frozen cells. However, when the cells were permeabilized withpoly(SPB), their morphology was round; as shown in Figure 3.10, the cells maintained
their spindle shape at the time of poly(SPB) permeabilization, suggesting thatpoly(SPB) in the cells after thawing trapped the magnesium ions necessary for celladhesion In order to use poly(SPB) intracellular permeation for actual clinicalapplication, it is necessary to evaluate cell adhesion and DNA damage in the future.
In contrast, poly(methylglycidyl sulfoxide) (PMGS) demonstrated a higher cellrecovery rate than DMSO. However, it has been reported that while poly(SPB) quickly
diffuses out of cells, PMGS is taken up by cells during freezing and remains within thecells even after 24 hours post-thawing2). Since low molecular weight PEG has beenreported to stabilize cell membranes and improve cryoprotection after permeation into
cells, PEG backbone of the main chain is believed to enhance the cryoprotective
effect10). Similarly, poly(SPB) has been reported to exert cryoprotection by interactingwith the cell membrane19), suggesting that this occurs through membrane protection105

similar to the PEG structure. The fact that poly(SPB) can cryoprotect cells and that the
cryoprotective effect decreases with increasing MSEMA incorporation ratios potentiallycorroborate previous reports emphasizing the significance of PEG in the main chain7).
Additionally, in the case of trehalose, the cells could not be recovered by cryoprotection
using only pellets. Here, the cryoprotective effect was not observed because it isbelieved that trehalose was not present outside the cells. Intracellular trehalose usuallyenhances the cryoprotective effect. Methods for introducing trehalose, a membraneimpermeable cryoprotectant, into cells include the use of nanoparticles and theexpression of trehalose transporter 1 (TRET 1). However, the use of nanoparticles
requires long incubation times and involves the risk of endosomal escape, while theexpression of TRET 1 requires the genetic modification of cells26-28). Poly(SPB) is
therefore an excellent cryoprotectant because it can permeate membranes and can beeasily introduced into cells.

1063.4 Conclusion
In conclusion, I used poly(SPB) and poly(SPB/MSEMA) as intracellular permeable
cryoprotectants because of their ability to permeate membranes, and confirmed thatthey enhanced the cryoprotective effect through intracellular permeation. In contrast,
the cryoprotective effect of only poly(SPB) was observed to be comparable to that of
DMSO. Fluorescence observations showed that, unlike common membrane-permeable
cryoprotectants such as DMSO and glycerol, the developed polymers were easily
removable from cells by counter diffusion and did not affect cell growth after freeze–
thaw cycling. In addition, unlike nanoparticles, which are established carriers ofmembrane-impermeable cryoprotective agents, the polymer itself has a cryoprotective
effect and a membrane-permeating ability that does not involve endocytosis, making
introduction facile. Through this study, I have thus shown that polymers with optimized
molecular configurations can achieve unparalleled cryoprotective efficacy. The proposed
approach elucidates that DMSO integration plays a pivotal role in modulating cellularinteractions. The synergistic interplay between DMSO and SPB underscores theinfluence of chemical structures on bio-interactions, and this is capable of surpassing
conventional cryoprotectants and nanoparticles in both efficacy and potential. By
integrating multifaceted cryoprotectants such as ethylene glycol analogues, trehalose,107

and especially PEG structures, poly(SPB) may strongly enhance membrane elasticity
and cryopreserve three-dimensional structures such as spheroids. The incorporation ofthese cryoprotectant structures will lead to improved safety and efficacy and is expected
to open new avenues for the development of engineered polymers.

108

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DeVries, Chem. Sci., 2010, 1, 538-551.
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5) J. Zhao, M. A. Johnson, R. Fisher, N. A. D. Burke, and H. D. H. Stöver,Langmuir, 2019, 35(5), 1807-1817.
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Sprague, A. M. Rosales and N. A. Lynd, Biomacromolecules, 2020, 21(8), 30473055.
7) T. Ishibe, N. -G. Martinez, P. G. Georgiou, K. A. Murray and M. I. Gibson, ACS

Polym. Au, 2022, 2(6), 449-457.
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Toner, Nat. Biotechnol., 2000, 18, 163-167.
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10911) L. D. Blackman, P. A. Gunatillake, P. Cass and K. E. S. Locock, Chem. Soc.

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15) N. Erathodiyil, H. -M. Chan, H, Wu, J. Y. Ying, Mater. Today, 2020, 38, 84-98.
16) T. Goda, Y, Imaizumi, H. Hatano, A. Matsumoto, K. Ishihara and Yuji
Miyahara, Langmuir, 2019, 35(24), 8167-8173.
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M. Suzuki and F. M. Winnik, Biomacromolecules, 2016, 17(4), 1523-1535.
18) T. Goda, H. Hatano, M. Yamamoto, Y. Miyahara and N. Morimoto, Langmuir,
2020, 36(33), 9977-9984.
19) R. Rajan, F. Hayashi, T. Nagashima and K. Matsumura, Biomacromolecules,
2016, 17(5), 1882-1893.
20) S. Li, S. Chung, A. Simakova, Z. Wang, S. Park, L. Fu, D. C. -Karni, S. Averick,and K. Matyjaszewski, Biomacromolecules, 2017, 18(2), 475-482.
11021) Doğuş Işık, Elisa Quaas and Daniel Klinger, Polym. Chem., 2020, 11, 7662.
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Mater., 2021, 2, 15.
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111

Chapter 4
General Conclusion
4.1 Conclusion
This thesis focused on elucidating the cryoprotective mechanism of polyamoholytes andtheirapplication to cryopreservation using intracellularpermeation. Because

polyampholytes are less toxic than commonly used cryoprotectants such as dimethyl
sulfoxide (DMSO), understanding the cryoprotective mechanism and the transport ofcryoprotectants using polyampholytes are very important for regenerative medicine andother applications. The results obtained in this study are summarized below. In Chapter
2, four types of polyampholyte with different side chain structures were synthesized, andthe correlation between their cryoprotective effects and polymer structures wasinvestigated using temperature variable magic angle spinning (MAS) solid-state NMR.
The cryoprotective effect of poly(vinyl acetate/acrylic acid (AA)/2-(dimethyl
amino)ethyl acrylate (DMAEA) (pV), which has an alternating arrangement of cations
and anions, showed a cell recovery rate of about 85 %, indicating that it is as effective as
carboxylated -poly-l-lysine (PLL) in cryoprotection. Below, cryoprotective effectdecreased in the order of poly(2-(dimethyl amino)ethyl methacrylate/methacrylic acid)(pD), a random copolymer with the same degree of dissociation, poly(methyl vinyl
112

ether/AA/DMAEA) (pM), which has the highest molecular weight with a similar
structure to pV, Highly dissociated poly(2-acrylamido-2-methyl propanesulfonic acid/(3acrylamidopropyl)trimethyl ammonium chloride)(pA). The results of growing ice crystals
at -6 °C suggested that the ice recrystallization inhibition (IRI) activity of these polymers
was relatively low and did not affect their cryoprotective effects. We discussed the
behavior of water molecules and Na ions during freezing of polymer solutions using
temperature-variable solid-state MAS NMR. In pA, which has a low cryoprotective effect,
the hydrogen bonds of hydrated water are relatively close to bulk water, and the amountof antifreeze water at -10 °C is about 10% less than that of other polymers. Therefore,
it was suggested that the cryoprotective effect would be poor due to exposure to osmotic
shock at higher temperatures. In PLL and pV, vitrification and coordination of Na ions topolymer chains were observed at higher temperatures, suggesting that vitrification is an
important factor in the cryoprotective effect, similar to previous studies. In the future, it
is believed that a deeper understanding of polymer structure and vitrification ability will
enable the molecular design of more effective cryoprotectants.
In Chapter 3, we attempted to enhance the cryoprotective effect by utilizing thepenetration of the zwitterionic polymer poly(sulfobetaine (SPB)) into cells. I alsosynthesized a copolymer of 2-(methyl sulfinyl)ethyl methacrylate (MSEMA), which has
113the same methyl sulfinyl group as DMSO, a cell-permeable cryoprotectant, and confirmed
its cryoprotective effect. Cytotoxicity tests revealed that exposure time to the polymer
solution during freezing resulted in little cytotoxicity. Intracellular permeation was
confirmed using the fluorescently modified polymer, and it was found that counter
diffusion was easily achieved by incubating at 37 °C for about 30 minutes after
permeation. Cells were suspended in each polymer solution (10 w/w%) and subjected tofreeze/thaw cycles. As a result, poly(SPB) and poly(SPB/MSEMA10) improved cellrecovery by approximately 20 or 30% due to intracellular permeation. The cell recovery
rate after counter diffusion of polymer at 37 °C is comparable to that before polymer
infiltration, and it is possible to cryopreserve only the cell pellets after polymer
permeation, suggesting that poly(SPB) can improve cryoprotection by intracellular
infiltration. Furthermore, there was no significant difference in cell proliferation afterthawing before and after permeation, suggesting that poly(SPB) is very promising as an
intracellular permeable cryoprotectant. Poly(SPB) is a methacrylate polymer, so
cryoprotectants such as polyethylene glycol and sugars that do not penetrate cell
membranes can be easily incorporated into the polymer, and it is expected to be used forcryopreservation of three-dimensional cell structures such as spheroids in the future.
This study revealed that vitrification and interaction with salt and water are important114

for cryoprotection of polyampholytes. This understanding of the cryoprotection
mechanism will provide important guidelines for the molecular design of cryoprotectantsin the future. Furthermore, the ability of zwitterionic polymers to reversibly permeate
into cells and enhance their cryoprotective effects offers the potential for cryopreservationof more complex three-dimensional cellular structures such as spheroids, and the abilityof artificial polymers to It is expected that this will open up new avenues for development.

115

Achievement
Publication
1. Ryota Yamasaki, Robin Rajan, Kazuaki Matsumura, Enhancement of cryopreservation
with intracellularly permeable zwitterionic polymers, Chem. Commun., 2023, 59,
14001-14004, https://doi.org/10.1039/D3CC04092E.

Misc
1. 山﨑椋太, Robin Rajan, 林文晶, 長嶋敏雄, 松村和明, 側鎖構造の異なる両性電解質
高分子による細胞凍結保護効果, 低温生物工学会誌, 2021, 67(2), 125-128.

Conference
1. 山﨑椋太, Robin Rajan, 林文晶, 長嶋敏雄, 松村和明, 凍結過程におけるポリマーと塩の相互作用, 第 66 回低温生物工学会大会、2021, 5. オンライン
2. 山﨑 椋太, 林 文晶, 長嶋 敏雄, Rajan Robin, 松村 和明, 温度可変固体 NMR を用いた側鎖構造と凍結保護効果の相関, 第 71 回高分子討論会, 2022, 9. 北海道
3. 山﨑 椋太, Rajan Robin, 松村 和明, 両性電解質高分子の構造と凍結保護効果の関係, 第 11 回北信越ブロック若手研究発表会, 2022, 12. オンライン
4. Ryota Yamasaki, Kazuaki Matsumura, Cryopreservation by intracellular permeationof zwitterionic polymers, 33rd Annual Conference of the European Society for
Biomaterials, 2023, 9. Swiss Confederation
116

5. Ryota Yamasaki, Robin Rajan, Kazuaki Matsumura, Relationship Between Structure
and Cryoprotective Effect of Synthesized Polyampholytes Using VariableTemperature Solid-State NMR, 33rd Annual Conference of the European Society for
Biomaterials, 2023, 9. Swiss Confederation

6. Ryota Yamasaki, Robin Rajan, Kazuaki Matsumura, Application of zwitterionicpolymers to cryoprotection through intracellular permeation, 2023, 12. Online

117

ACKNOWLEDGEMENT

I would like to express my deep gratitude to my main supervisor, Professor Kazuaki
Matsumura, for his enthusiastic guidance and encouragement in a wide range of areas,from conducting experiments to writing papers, in carrying out this research.
I would also like to thank Assistant Professor Robin Rajan, who gave me much advice incarrying out my research, including polymer synthesis.
I would like to express my deep gratitude to Professor Noriyoshi Matsumi, Professor
Motoichi Kurisawa, Associate Professor Eijiro Miyako of the Japan Advanced Institute of
Science and Technology, and Associate Professor Shinichi Yusa of the University of
Hyogo, who served as my doctoral thesis review committee.
I would also like to express our deep gratitude to Keiko Kawamoto for providing
guidance regarding cell culture experiments and basic experimental operations.
I would also like to express my deep gratitude to KEF Corporation's Representative
Director Yoshitake Iyoku and Senior Managing Director Hiroyuki Shinbo for theirguidance and support during my internship.
In addition, I would like to express my deep gratitude to Dr. Fumiaki Hayashi and Dr.
Toshio Nagashima of the Advanced NMR Application and Platform Team, NMR
118

Research and Collaboration Group, NMR Science and Development Division, RIKEN for
providing us with much advice and equipment, including variable temperature NMR
measurements.
Finally, I would like to thank all my friends and family for supporting me during mystudent life.
March 2024
Ryota Yamasaki

119