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https://dspace.jaist.ac.jp/

Title

温度応答性両性電解質高分子の液-液相分離の機序解明
および応用

Author(s)廣瀬, 智香CitationIssue Date

2024-06

Type

Thesis or Dissertation

Text version

ETD

URL

http://hdl.handle.net/10119/19334

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

Mechanism of Liquid-Liquid Phase Separation of
Temperature response Polyampholyte and Its
Applications

TomokaHIROSESupervisor: Kazuaki MATSUMURA
Graduate School of Advanced Science and Technology
Japan Advanced Institute of Science and Technology
Materials Science
June 2024

Abstract
Tomoka Hirose
Polyampholytes, a class of polyelectrolytes characterized by the presence of both anionic and cationicgroups, demonstrate unique pH and temperature responsiveness through interactions the polymers and theirsurrounding solutions or among the polymers' own functional groups. Such responsiveness enables
polyampholytes to undergo phase transitions, making them invaluable in diverse applications ranging fromsmart materials to biomedical engineering. Due to its high biocompatibility, it is being actively researched
as an intelligent material suitable for biomaterials such as drug delivery systems (DDS). Understanding themechanisms driving these stimulus-responsive phase transitions is crucial for the optimization ofpolyampholyte-based materials, as it informs the design and functionalization strategies that tailor theirproperties to specific applications. Furthermore, in recent years, attention has been focused on the behaviorof liquid-liquid phase separation (LLPS) in living organisms such as organelles without membranes. This
interest is driven by the role of LLPS in critical life processes, including metabolism, where it facilitates
the compartmentalization of biochemical reactions without the need for physical barriers. Polyampholytes
are also being studied as model compounds for proteins, offering insights into the molecular dynamics
underlying phase separation biology.
In this study, I explore the synthesis and evaluation of ampholyte polymers that exhibit both liquid-liquid
phase separation (LLPS) behavior and temperature responsiveness. We confirmed that by introducing abenzene ring into an ampholyte polymer that causes phase separation behavior due to electrostaticinteractions, stable phase separation behavior can be induced even in salt solvents. Multi-scalemeasurements revealed that the benzene rings play a crucial role during droplet formation and growth stages
likely due to the π-π interaction, thereby stabilizing LLPS behavior. Leveraging the temperature
responsiveness of these polymers, we designed a novel DDS carrier using a composite material withphotothermal properties derived from liquid metal. This approach aimed to ensure a stable, self-sufficient
supply of trigger stimuli, addressing a significant limitation of current DDS technologies. Cell-based
experiments demonstrated the carrier's ability to concentrate drugs effectively, suggesting its potential tominimize side effects associated with drug delivery. These findings not only contribute to our understandingof the fundamental principles governing LLPS in ampholyte polymers but also open new avenues for thedevelopment of safer, more efficient drug delivery technologies. Future research will focus on further
characterizing the interaction mechanisms at play and exploring the clinical applicability of our novel DDS
carrier.
Keywords: Polyampholytes, Liquid-Liquid phase separation, Drug delivery system, Temperature
response polymers, Liquid metals, Biomaterials

2

Acknowledgment
In conducting this research, I would like to thank my supervisor, Prof. Dr. Kazuaki Matsumura,for his careful guidance and advice on everything from research preparation to writing andpresentation of the paper. Also, if I had any questions, I could always go to him for advice, and he wasalways available to help me even though he was very busy. It is thanks to Prof. Matsumura guidance
that I was able to carry out and complete my research. I would like to express my deepest gratitude
and appreciation.
I would like to express my sincere gratitude to Prof. Dr. Masayuki Yamaguchi (JAIST), Assoc.
Prof. Dr. Miyako Eijiro (JAIST), Prof. Dr. Motoichi Kurisawa (JAIST), and Assoc. Prof. Dr. Shinichi
Yusa (University of Hyogo) for reviewing my thesis and providing useful advice.
In Chapter 2, I would like to express my gratitude to Dr. Masaru Nakada (Toray Research Center,Inc.), Mr. Hironobu Uchiyama (Toray Research Center, Inc.), and Mr. Shinya Seki(Toray ResearchCenter, Inc.) for their great cooperation in measurements and analysis during the joint research.
In Chapter 3, I received careful and appropriate guidance from my co-researcher, Assoc. Prof. Dr.
Miyako Eijiro (JAIST). I would also like to thank the members of the Miyako Laboratory for kindly
lending me experimental equipment and providing guidance on how to use it.
Asst. Prof. Dr. Rajan Robin and Ms. Keiko Kawamoto of the Matsumura Laboratory taught me a
lot and enabled me to carry out my research. Also, everyone in the laboratory helped me in various
situations. I would like to express my deepest gratitude.
This work was supported by JST SPRING, Grant Number JPMJSP2102.

Tomoka Hirose

3

Table of Contents

4Chapter 1 ...................................................................................................................................... 8
1-1 Polyelectrolytes ................................................................................................................ 9
1-1-1 Zwitterionic Polymers ---------------------------------------------------------- 10
1-1-2 Polyampholytes ------------------------------------------------------------------ 11
1-2 Liquid-liquid phase separation .................................................................................... 12
1-3 Temperature responsive polymer ................................................................................ 13
1-3-1 Upper Critical Solution Temperature (UCST) Polymer ---------------- 15
1-3-2 Lower Critical Solution Temperature (LCST) Polymer ---------------- 16
1-4 Drug Delivery System (DDS) ....................................................................................... 17
1-5 Metal nanoparticles ...................................................................................................... 20
1-5-1 Liquid metals.......................................................................................... 20
1-6 Research purpose .......................................................................................................... 21
1-7 References ...................................................................................................................... 22
Chapter 2................................................................................................................................. 34
2-1 Introduction ................................................................................................................... 35
2-2 Experiment .................................................................................................................... 37
2-2-1 Materials -------------------------------------------------------------------------- 37
2-2-2 Preparation of temperature responsive polyampholytes --------------- 37
2-3 Characterization ........................................................................................................... 37
2-3-1 UV-Vis spectrophotometer ---------------------------------------------------- 38
2-3-2 Laser scanning microscope---------------------------------------------------- 39
2-3-3 Dynamic Light Scattering (DLS) Measurements ------------------------ 395

2-3-4 Ultra-Small-Angle/Small-Angle X-ray Scattering (USAXS/SAXS)
measurements --------------------------------------------------------------------------- 40
2-3-5 Neutron scattering measurement -------------------------------------------- 40
2-4 Results and discussion .................................................................................................. 41
2-4-1 Synthesis of temperature-responsive polyampholytes ------------------ 41
2-4-2 UV-Vis spectrophotometer ........................................................................................ 43
2-4-3 Laser scanning microscope---------------------------------------------------- 46
2-4-4 Dynamic Light Scattering (DLS) Measurements ------------------------ 49
2-4-5 Ultra-Small-Angle/Small-Angle X-ray Scattering (USAXS/SAXS)
measurements --------------------------------------------------------------------------- 51
2-4-6 Neutron scattering measurement -------------------------------------------- 56
2-5 Conclusion ..................................................................................................................... 60
2-6 References ...................................................................................................................... 63
Chapter 3 ................................................................................................................................ 68
3-1 Introduction ................................................................................................................... 69
3-2 Experiment .................................................................................................................... 71
3-2-1 Materials -------------------------------------------------------------------------- 71
3-2-2 Synthesis of temperature-responsive polymer ---------------------------- 71
3-2-3 Characterization of the polyampholyte ------------------------------------ 72
3-2-4 Particularisation of LM -------------------------------------------------------- 72
3-2-5 Characterisation of composites ---------------------------------------------- 73
3-2-6 Cell viability assay -------------------------------------------------------------- 73
3-3 Results and discussion .................................................................................................. 75
3-3-1 Characterization of temperature-responsive polymer ------------------ 75
6

3-3-2 Fabrication of compositees ---------------------------------------------------- 80
3-3-3 Characterisation of composites ---------------------------------------------- 84
3-3-4 Cell viability assay -------------------------------------------------------------- 88
3-4 Conclusion ..................................................................................................................... 92
3-5 References ...................................................................................................................... 93
Chapter 4 ................................................................................................................................ 97
4-1 General Conclusion....................................................................................................... 98
4-2 Achievements ............................................................................................................... 101

7

Chapter 1
General introduction8

1-1 Polyelectrolytes
Polyelectrolytes can be broadly classified into three categories 1–3: polymers with either anions
or cations, zwitterionic polymers, and amphoteric polyelectrolytes (Figure 1).
Polyelectrolytes have both polymer and electrolyte properties, and since they have dissociative
groups in their repeating units, they exhibit various properties in solution due to electrostaticinteractions between low molecular weight ions in solution and charged groups in the polymer
chain4,5.
When the degree of dissociation changes due to electrostatic interactions, the effective size ofthe chains also changes, leading to significant changes in the conformation of macromolecules
such as coil-globule transitions6. This also significantly changes the physicochemical propertiesof the solution, such as its viscosity. It is possible to cause changes in physical properties bychanging external conditions such as pH, temperature, and the addition of salt. Taking advantageof these properties, it can be used as a thickener7 or dispersant5 in industrial applications, or as a
biological material. Research is being carried out on biosensors8,9 and drug delivery systems(DDS)10. Materials with similar properties are often found in biomolecules, such as polyaminoacids, DNA, and cell membranes11. In recent years, they have been studied as model compounds
for proteins because they have similar properties12.

Figure.1

Polyelectrolytes and their classification9

1-1-1 Zwitterionic Polymers
Among polymer electrolytes, those containing both anions and cations are called zwitterionicpolymers. (Figure 2) Typical zwitterionic polymers include sulfobetaine and carboxybetaine ingeneral, betaine-based zwitterionic polymers are often named after the anion moiety13. In thispaper, I define zwitterionic polymers as having anionic and cationic charges in the same monomer
unit to distinguish them from ampholyte polymers described later. The solution behavior ofzwitterionic polymers is often opposite to that of polyelectrolytes, which is called theantipolyelectrolyte effect. Chain extension is highly dependent on the addition of low molecularweight electrolytes, chemical structure, and composition14. Research is being conducted into
disinfectants, emulsifiers15, and cryoprotectants16,17.

Figure 2 Sulfobetaine polymer structure101-1-2 Polyampholytes
Ampholyte polymer is a type of polyelectrolytes, and its original definition refers to a polymer
that has both acidic and basic groups18. For the sake of clarity in this paper, zwitterionic polymers
in which different repeating units have opposite charges are referred to as ampholyte polymers.
(Figure 3) Among electrolyte polymers, they have many similarities with biomolecules such asproteins, and are expected to be applied to biomaterials12,19. The structure and properties ofampholyte polymers are influenced by the Coulombic attraction between anionic and cationicspecies on different monomer units, and the response in solution is highly dependent on thechemical structure and composition of the polymer20,21. It has amphoteric properties, accepting
protons under acidic conditions and releasing protons under basic conditions, meaning that its
charge fluctuates with changes in pH22,23. Because it has temperature and pH responsiveness24,25,
it is being researched to be applied to biomaterials such as drug delivery systems 26,27. Ourlaboratory has already reported on ampholyte polymers as cryoprotectants16,28.

Figure 3 Structure diagram of ampholyte polymer (PLL-SA) with cryoprotective effect11

1-2 Liquid-liquid phase separation
Liquid-liquid phase separation behavior (LLPS) refers to the physical phenomenon in which asolution does not mix homogeneously and separates into two phases due to changes inintermolecular interactions29. (Figure 4) Generally, during phase separation, a dilute phase and aconcentrated phase are separated, and the concentrated phase formed at this time is called adroplet, a concentrate, a coacervate, etc30–32. depending on the size. This phenomenon is often
observed in the natural world and has recently attracted attention as a behavior in which various
intracellular metabolisms are carried out by proteins and RNA in living organisms33–38.
Regions in cells that have no membranes but are enriched with proteins and RNA are called
membraneless organelles, and they vary in size from large ones such as nucleoli and stressgranules to small and temporary ones39,40. A state of liquid-liquid phase separation exists withincells. Those with particularly low fluidity are called gels, and those with larger sizes are
sometimes called protein tertiary structures or aggregated granules38,41,42. This liquid-liquid phaseseparation in living organisms is called biological phase separation, and it has become clear thatit plays an important role in biological phenomena43–45.
During metabolism, droplets formed by LLPS contribute to the concentration and eliminationof molecules depending on their affinity, which leads to the promotion and inhibition ofbiochemical reactions during metabolism. Furthermore, understanding LLPS is important fortreatment of diseases. Aggregation of specific proteins is one of the causes of neurodegenerative
diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic cord sclerosis(ALS)46–49. It has been revealed that such protein condensation tends to occur in the concentrated
phase of liquid-liquid phase separation, and elucidation of the mechanism of liquid-liquid phaseseparation behavior may lead to precise control of protein aggregation inhibition or promotion.

12

Figure 4 Liquid-liquid phase separation behavior

1-3 Temperature responsive polymer
A temperature-responsive polymer is a type of external stimulus-responsive polymer and is apolymer that changes its properties and form in response to temperature changes50–52. It can bedivided into two types: upper critical solution temperature (UCST) type and lower critical solution
temperature (LCST) type. As solubility changes with temperature changes, UCST type polymers
become soluble at a certain temperature, and LCST type polymers become insolubilized at a
certain temperature53,54. These reactions are largely caused by the affinity between the polymers
and the solvent; when the interactions between polymers become dominant, they become
insolubilized, and when they become recessive, they become soluble. (Figure 5) The temperature
at this point is called the phase transition temperature (Tc). Examples of interactions includehydrophilic-hydrophobic interactions and electrostatic interactions. There are also various formsof change, and there are polymers that exhibit solid-liquid phase separation and polymers thatexhibit liquid-liquid phase separation behavior. Taking advantage of its properties, it is a material
that is being studied as a reactive material and biomaterial. Because UCST type polymers are
difficult to reproduce under physiological conditions due to their characteristics, LCST type
polymers are commonly studied as biomaterials.

13Figure 5 Illustration of phase separation behavior54

14

1-3-1 Upper Critical Solution Temperature (UCST) Polymer
Poly(allylamine-co-allylurea) (PAU) is a typical UCST type polymer. (Figure 6) PAU is a typeof ureido polymer that is often used as a UCST type polymer, and is synthesized by adding
potassium cyanate, a ureidation reagent, to polyallylamine55. In general, UCST-type phaseseparation behavior is often caused by hydrogen bonds, so many have a high Tc56. However, PAU
has a wide range of Tc from 5 to 65℃ by controlling the ureidation rate and molecular weight.
The phase separation behavior is due to the strong hydrogen bonding of the ureido groups, and as
mentioned above, LCST-type polymers are mainly studied as biomaterials, and there are only
reports that focus on UCST-type polymers53,56. The current situation is that the number ofmolecules is small. However, in recent years, research has been conducted on the application of
PAU to carriers such as protein capture, temperature sensing in combination with fluorescentprobes, actuators, etc55,57.

Figure 6 Structural diagram of poly (allylamine-co -allylurea) (PAU)15

1-3-2 Lower Critical Solution Temperature (LCST) Polymer
Poly(N-isopropylacrylamide) (PNIPAM) is the most common LCST type polymer58–61. (Figure7a) Tc of PNIPAM exhibits liquid-liquid phase separation behavior at 31-33°C due to interaction
between hydrophilic and hydrophobic groups. The PNIPAM chain, which is normally in a coiled
state in solution, changes its shape into a sphere as the hydrophobic interaction between thehydrophobic skeleton and the isopropyl group becomes dominant when the temperature exceeds
the LCST62. It is a polymer that has been widely studied as a biomaterial because it has a Tc close
to body temperature and has a relatively simple structure63,64. However, since Tc does not dependon the polymer concentration, when considering applications with different Tc, it is necessary toincorporate structural factors into the polymer structure that strengthen the interaction between
the polymer and water. When applied as a carrier for biomaterials, especially DDS, it is generally
used in the form of a gel 65.
Our laboratory has reported an ampholyte polymer that exhibits new LCST properties bymodifying the free amino groups of ε-poly-L-lysine (PLL) with succinic anhydride (SA) (PLLSA)66,67 (Figure 7b). The phase separation behavior is due to electrostatic interactions and exhibits
LLPS. It has been found that Tc is highly dependent on the degree of substitution of SA in thepolymer and on the concentration of the polymer solution. Compared to PNIPAM, it is easier toadjust Tc, and it can be applied over a wide temperature range, including body temperature, bysimply adjusting the concentration of the polymer. Furthermore, one of the raw materials,
polylysine, is a material that is also used as a food additive, and its low biotoxicity is an advantage
when considering it as a biomaterial68. However, there is a problem in that phase separation
behavior is suppressed in solvents containing salts such as body fluids because electrostaticinteractions are inhibited66.

16

Figure 7 LCST type polymer structure (a: PNIPAM/ b: PLL-SA)

1-4 Drug Delivery System (DDS)Drug delivery system (DDS) is a general term for technology that delivers the necessary drug
to the appropriate site at the required time and is considered important in the drug discovery fieldfrom the perspective of reducing side effects and improving drug efficacy69,70. Generally, drugs
themselves do not have site-selectivity, so in order to produce a pharmacological effect at the siteof action, it is necessary to administer a large amount with consideration to absorption outside thesite of action. Although large doses of drugs are the main cause of side effects, it is difficult toobtain pharmacological effects at doses below the prescribed values. DDS, which is one of themethods to solve this problem, can be classified by purpose and can be broadly divided into three
categories71: release inhibition, absorption improvement, and target targeting. Regarding releasesuppression, by designing for sustained release, the drug concentration in the blood is stabilized
and fluctuations are suppressed, which leads to stabilization of pharmacological effects andstabilization of side effects.
To improve absorption, the effective drug itself is modified to suit the environment at the siteof action71. This suppresses absorption in areas other than the target site and is expected to reduceside effects72. Finally, regarding target orientation, the purpose of this is to design a drug to bedelivered only to the target and is an important perspective for drugs that have particularly strong
side effects, such as anticancer drugs73,74. A typical example of target-directed DDS in anticancer
17

drugs is the so-called Enhanced Permeability and Retention (EPR) effect. This is because theholes in the blood vessel walls surrounding cancer cells are larger than in normal cells, so it was
hoped that by enlarging the size of the particles by modifying them with polymers, they would
accumulate only in cancer cells75. DDS is being applied and researched in a wide variety of ways
for each of these purposes. The most classic and familiar application of DDS is sugar-coatedtablets.
In recent years, a technology called smart DDS, which has stimulus responsiveness and can
improve the effectiveness of drug targeting and reduce side effects, has been actively
researched74,76–78. Examples of smart DDS materials include liposomes, metals/metal oxides79–81,
and external stimuli-responsive polymers74. External stimuli-responsive polymers are a generalterm for polymers that change their physical properties and structure in response to theaforementioned external stimuli such as temperature, magnetism, and pH82 (Figure 8). Many
responsive polymers are being investigated as DDS carriers, depending on the treatment method,
purpose, and environment of the target site. LCST type polymers are commonly used as
temperature-responsive polymers83. While thermal stimulation is easy to supply, it is also a
stimulus that is difficult to supply stably over a long period of time due to the homeostatic functionof the living body. For this reason, conventional research into DDS carriers using temperatureresponsive polymers has focused on how quickly and sensitively they can react84.

18

Figure 8 Typical external stimuli

191-5 Metal nanoparticles
Metal nanoparticles are materials that are being applied and researched in industrial and biologicalmaterials due to their characteristics such as localized surface plasmon resonance and catalytic
properties depending on the particle size. By definition metal particles of 100 nm or less are called
metal nanoparticles. There are various methods for controlling size and shape, and they are used
depending on the purpose. Typical metal nanoparticles include gold and silver. Applications inmany fields, including sensing and optical devices that utilize optical properties that absorb andscatter light at specific wavelengths, magnetic materials and devices that utilize magnetic
properties, nanoscale materials that utilize mechanical properties, and catalysts 85. Phototherapy
in cancer treatment uses surface plasmon resonance to absorb light and generate heat, making useof its photothermal properties86, which have been widely applied in the medical field, such as indestroying cancerous tissue. In the field of DDS, drug targeting and efficient delivery is possible
by combining drugs with nanoparticles and accumulating them in tumor tissue using the EPR
effect79,86.

1-5-1 Liquid metals
It is a general term for metals that are in a liquid state at room temperature, with mercury beinga typical example. It is a well-known fact that mercury has excellent physical and chemical
properties, but it is also widely known that mercury is highly toxic. However, in recent years,
liquid metals based on gallium have been attracting attention. In addition to the excellent
properties of liquid metals, gallium-based liquid metals have low cytotoxicity, and are therefore
being actively researched in the field of nanomedicine39,87. Since it can be made into particles bya simple method such as ultrasonication, it can also be applied as metal nanoparticles88.

20

1-6 Research purpose
In this study, I synthesized a temperature-responsive ampholyte polymer that exhibits liquidliquid phase separation behavior, incorporating hydrophobic groups to enable stable phaseseparation behavior even in biological environments, and explored the mechanism of its phaseseparation behavior. and its application to DDS. In Chapter 2, I focus on the phase separation
behavior of the synthesized temperature-responsive ampholyte polymers and evaluate thecharacteristics of the synthesized polymers and measure the liquid-liquid phase separation
behavior on various scales to determine the presence or absence of hydrophobic groups. In
Chapter 3, I focused on temperature responsiveness, created a composite with liquid metal as aconventional next-generation temperature-responsive carrier, and evaluated its characteristics as
a DDS carrier with two-stage responsiveness. Chapter 4 provides an overview of this research
and future prospects. Chapter 4 provides an overview of this research and future prospects.

21

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33

Chapter 2
Elucidation of the liquid-liquid phaseseparation mechanism of Polyampholytes342-1 Introduction
As mentioned in Chapter 1, liquid-liquid phase separation behavior is intricately related tovarious biological functions including metabolism in living organisms, and analyzing whatstrongly affects the behavior is important for phase separation. It has the potential to become thekey to understanding life from a biological perspective and new treatments for diseases 1-6. Sincea wide variety of proteins already exist, it is expected that new compounds exhibiting LLPS
behavior3, 4 will continue to be synthesized and increase in number.
Since the properties of ampholyte polymers, which are also model compounds for proteins,change depending on the combination of acidic and basic groups, it can be assumed that countless
types of ampholyte polymers will be synthesized and researched7. In addition, not only from theperspective of phase separation biology, but also because ampholyte polymers have highbiocompatibility and responsiveness to external stimuli, they have the potential to be applied tobiomaterials as intelligent materials8-10. One of the important points when applying materials as
intelligent materials is how precisely their properties can be controlled11. The precision of polymer
synthesis is also an effective factor in controlling properties, but the presence or absence of basic
research on what strongly causes these properties has a wide impact, from the design of thepolymer to the setting of the application environment12.
In general, when properties are acquired through structural changes such as phase transitions, it
is not due to only one effect, but it can be said that interactions with other properties of the polymer
also have an influence13. Therefore, even if they have similar structures, their properties maychange significantly by modifying a part of them. PNIPAM, which was mentioned in Chapter 1,
is one of them, and its phase separation temperature changes significantly by modifying part ofits structure or increasing its molecular weight14, 15. The same is true for ampholyte polymers, and35

it can be said that it is necessary to study the mechanism for each compound that exhibits LLPS
behavior.
Currently, the experimental and measurement methods necessary to study the mechanism ofliquid-liquid phase separation behavior have not yet been established. This is also related to thefact that, as mentioned earlier, there is a strong possibility that the mechanisms differ for eachcompound that exhibits LLPS. Therefore, in this chapter, I focused on the droplet generation stage,
which is the first stage in which LLPS is triggered, and performed multi-scale measurements.
Phase separation behavior was evaluated by measuring on a scale from the occurrence of LLPS
behavior to droplet formation. The ampholyte polymers used were PLL-SA, which was used inprevious research, and carboxylated phthalic anhydride polylysine (PLL-PA). PLL-PA is a
product in which a benzene ring is introduced for stabilization through hydrophobic interactionin order to overcome the problem that phase separation behavior is resolved when inhibited bysalts present in the solvent of PLL-SA. Both are compounds having an amino group as an acidic
group and a carboxyl group as a basic group, and have the same degree of polymerization. I
evaluated by comparing and measuring how the introduction of a hydrophobic site caused a
difference in the initial stage of phase separation.

36

2-2 Experiment2-2-1 Materials
A 25% ε-polylysine solution was procured from JNC Corporation (Tokyo, Japan). Anhydrous
succinic acid and anhydrous phthalic acid were obtained from Nacalai Tesque Inc. (Kyoto, Japan).
The powder form of phosphate-buffered saline (PBS (-)) was purchased from Shimadzu
Diagnostics Corp. (Tokyo, Japan).

2-2-2 Preparation of temperature responsive polyampholytes
An aqueous solution of 25% PLL was prepared, and an appropriate amount of SA or PA wasadded to the solution. The mixture was stirred at 60°C until complete dissolution of reagents. Fifty
per cent of the amino groups of PLL carboxylated with SA or PA, were denoted as PLL-SA50
and PLL-PA50, respectively. The polymer solution obtained was lyophilised and stored in vacuum
until usage. Sample solutions were prepared in arbitrary solvents and concentrations for thevarious measurements.

2-3 Characterization
For various measurements, PLL-SA50 and PLL-PA50 and polymers containing equal amountsof acidic and basic groups were used.
In USAXS/SAXS measurements, DLS measurements, and observations using a scanning laser
microscope, PLL-SA50 and PLL-PA50 were prepared in an aqueous solution with theconcentration adjusted so that the phase separation temperature was as close as possible to 3335°C. For comparison purposes, an aqueous solution sample and a PBS solution sample with the37same polymer concentration were also prepared and measured. A table listing sample conditions
is shown in Table 1.
Table 1 Sample conditions used for measurement

solvent
Polymer

water

PBS

PLL-PA50 PLL-SA50 PLL-PA50 PLL-SA50

Tc(℃)

35

33

47

Concentration(wt/v%)

19

40

19

40

In neutron scattering measurements, the polymer concentration was fixed at 20 w/v% andadjusted using three types of solvents: pure water solvent (H2O), mixed solvent of heavy waterand pure water (M2O), and heavy water solvent (D2O).

2-3-1 UV-Vis spectrophotometer
The temperature responsiveness and phase separation temperature were determined using atemperature-controlled UV-Vis spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan).
Polymer solutions were placed in 2 mL quartz cells, and optical data were collected continuously
at a fixed wavelength of 550 nm, while varying the temperature. The transmitted light intensityof the homogeneous solution state prior to phase separation was set as 100%, and the temperature
at which the transmitted light intensity reached 50% was defined as the phase separation
temperature.
The Tc of the sample solutions used in the following characteristic evaluations was measured
and adjusted in the same manner.

38

2-3-2 Laser scanning microscope
Using a heated slide glass (Figure 9b) with a scanning laser microscope (LaSCOPE LM9001
manufactured by Astrodesign) (Figure 9a), the process of solution/droplet formation before andafter phase separation was observed at elevated temperatures.

Figure 9 Scanning laser microscope used for observation (a)/heated slide glass (b)2-3-3 Dynamic Light Scattering (DLS) Measurements
The DLS measurements analyzed the dynamics of the solution. Our goal was to evaluate thedroplet formation and growth range, which is larger than the range that can be measured byneutron scattering or USAXS/SAXS.

39

2-3-4 Ultra-Small-Angle/Small-Angle X-ray Scattering (USAXS/SAXS)
measurements
The measurements were performed using the BL08B2 equipment at Spring-816, 17, and the results
were analyzed and evaluated. It has a temperature range of 100-1100K, and in this measurement,
the temperature was raised from 10℃ to 60℃.

2-3-5 Neutron scattering measurement
I used the BL-21 high-intensity total scattering apparatus (NOVA) located at J-Parc18, 19. (Figure10)
NOVA has a wide Q range. Here, I conducted measurements to measure the interactions andfluctuations between the polymer and solution before droplet formation. In addition to different
solvents, the samples were filled with two types: a homogeneous solution before phase separation anda concentrated phase after phase separation, and measurements were taken while increasing thetemperature.

Figure10 NOVA sample insertion port used for measurement and image of the measured sample

40

2-4 Results and discussion

2-4-1 Synthesis of temperature-responsive polyampholytes
Temperature-responsive polyampholytes, PLL-SA and PLL-PA, were synthesised by adding SA
and PA to a PLL solution, according to Scheme 1.

Scheme 1Synthesis of PLL-SA and PLL-PA
The synthesis was performed under heating and stirring conditions, resulting in the formation ofcarboxylated polylysine solutions. Degree of substitution was calculated from integration values
using 1H NMR spectroscopy and utilizing the following equation. (Figure 11 and Table 2)

Table2 Characterization of polyampholytes
composition

composition(in feed)

(NMR)

NH2

COOH

NH2

COOH

PLL

100

0

100

0

PLL-PA50

50

50

49

51

41

Figure 11 1H-NMR of PLL-PA50(solvent：D2O)

42

2-4-2 UV-Vis spectrophotometer
To assess the temperature responsiveness of PLL-PA in both pure water and saline solution, I
conducted measurements of transmitted light intensity using a temperature-variable UV-Vis
spectrophotometer. In case of PLL-SA50, phase separation was observed only in pure aqueous
solution, as shown in Figure 12(A); the phase separation temperatures for different concentrationsof PLL-SA50 in pure water solvent were as follows: 3.2, 11 and 41°C at 12, 12.5 and 15%transmittances, respectively. No change in the transmitted light intensity was observed at any
concentration with PBS as solvent. The phase separation temperatures at 12% and 15% polymer
concentrations were 3.2°C and 41°C, respectively, indicating that phase separation can be induced
over a wide range of temperatures by adjusting polymer concentration. In contrast, phaseseparation temperatures for different polymer concentrations of PLL-PA50 in pure water solvent
were as follows: 11.5, 19 and 34°C at 22.5, 25 and 30% transmittances, respectively. In PBS
solvent, the temperatures were 27.5, 37 and 50°C at 20, 25, 30% transmittances, respectively.
PLL-PA exhibited phase separation in aqueous solution and PBS (Figure 12（B)）, suggestingthat PLL-PA can be used over a wide temperature range by adjusting polymer concentration in
the physiological salt environment. The phase separation temperature curves obtained from theseexperiments confirm the presence of LCST-type phase separation and demonstrate that the phaseseparation temperature can be tuned over a wide range by adjusting polymer concentration.
This measurement enables the evaluation of both temperature responsiveness and the behaviorof liquid-liquid phase separation. It is established that the range of liquid-liquid phase separation
behavior measurable through transmitted light is 1 nm or greater20. Generally, the particle sizes atwhich transparency decreases are observed to be 1 nm or greater, with size of 100 nm or more
resulting in pronounced cloudiness. In this study, the temperature at which the intensity oftransmitted light intensity reduces to 50% is defined as the phase separation temperature. On a
43

macroscopic level, the behavior of separation becomes visually discernible as the solution turns
cloudy and separates into two distinct phases. Nevertheless, the determination of transparency isinherently qualitative, necessitating the need for quantitative evidence for rigorous analysis.
Therefore, the measurement of transmitted light intensity emerges as a critical method for
acquiring quantitative data on the behavior of liquid-liquid phase separation.

44

Figure 12 Changes in Transmittance Intensity Across Different Temperature Ranges (UV-vis,
λ=550nm; 50% Transmittance Indicates Phase Separation Temperature) (A) The phaseseparation temperatures for different polymer concentrations of PLL-SA50 in pure water(B) The phase separation temperatures for different polymer concentrations of PLL-PA50 inpure water and PBS

45

Generally, the particle sizes at which transparency decreases are observed to be 1 nm or greater,
with size of 100 nm or more resulting in pronounced cloudiness21. In this study, the temperature
at which the intensity of transmitted light intensity reduces to 50% is defined as the phaseseparation temperature. On a macroscopic level, the behavior of separation becomes visuallydiscernible as the solution turns cloudy and separates into two distinct phases. Nevertheless, thedetermination of transparency is inherently qualitative, necessitating the need for quantitative
evidence for rigorous analysis. Therefore, the measurement of transmitted light intensity emerges
as a critical method for acquiring quantitative data on the behavior of liquid-liquid phaseseparation.

2-4-3 Laser scanning microscope
The video demonstrates the droplet formation process in PLL-SA water solvent and PLL-PA
water/PBS solvent near the phase separation temperature captured during heating observation
using a scanning laser microscope (Figure 13). As mentioned above, the presence of PBS solvent
for PLL-SA suppresses phase separation behavior due to salt interface, resulting in the absence ofobservable droplet formation. Droplet formation and growth were observed in all samples thatexhibited phase separation behavior due to temperature increase. In PLL-SA, fine droplets ofirregular size were continuously formed and subsequently aggregated, whereas in PLL-PA system,
droplets of somewhat uniform size were rapidly formed and then enlarged. The rate ofcoalescence of droplets in the PLL-PA was also slower than that of PLL-SA, indicating enhanced
stability of PLL-PA during the droplet growth stage.
It is already known that the phase separation behavior of PLL-SA is caused by the electrostaticinteractions between amino groups and carboxy groups22. The newly synthesized PLL-PA ,despite having the same ratios of these functional groups and a molecular weight difference of46

approximately 770, exhibits distinct phase separation behavior in the physiological salt solution.
This divergence is likely attributable to π-π interactions facilitated by the aromatic ring introducedin the PLL-PA structure, particularly in the presence of salt solvents22-24. Such interactions are notprevalent in the PLL-SA system, highlighting the critical role of molecular structure and specificintermolecular forces in dictating phase separation dynamics.

47

Figure 13

Captured image of the heating process of a polymer solution using a scanning laser48

2-4-4 Dynamic Light Scattering (DLS) Measurements
In my investigation of droplet formation dynamics, DLS measurements provided insight into thechanges at a finer scale than could be observed by laser microscopy. The DLS measurement
results are shown in Figure 14.
Excluding the PLL-SA/PBS system, where phase separation behavior is suppressed, a notablevariation in the autocorrelation coefficient with increasing temperature was observed. It can be
confirmed that the PLL-SA system changes continuously with temperature increasing regardlessof the phase separation temperature. In contrast, in the PLL-PA system, significant changes in the
autocorrelation coefficient were observed beyond the phase separation temperature. Thisdistinction aligns with the previously noted behavior where PLL-SA exhibits continuous
formation of unstable and small-sized droplets, whereas PLL-PA is characterized by the rapid
formation of droplets of a relatively uniform size.

49

Figure 14 Autocorrelation coefficient and temperature dependence50

Although the precise minimum diameters of droplets formed by PLL-SA and PLL-PA
remain undetermined, it is hypothesized that they are akin to proteins with analogous properties,
which these compounds model. Given that vesicle sizes within biological cells span fromseveral hundred nanometers to a few micrometers25-27, the observed continuous decrease in the
autocorrelation coefficient for PLL-SA prior to reaching the phase separation temperature may
not necessarily indicate droplet formation28. Instead, this trend could suggest fluctuations
occurring in advance of droplet emergence29. This phenomenon was not evident in the PLL-PA
system, hinting at the possibility that, despite superficially similar phase separation behaviors at
the macroscopic level, the underlying mechanisms may differ significantly.
The observation that the autocorrelation coefficient does not drop to zero but stabilizes at a
low value beyond the phase separation temperature is attributed to measurements being
conducted in regions where droplets were actively growing and undergoing coacervation. Thispersistence of a non-zero autocorrelation coefficient underscores the dynamic nature of droplet
formation and growth, reflecting continuous structural changes at the microscopic scale.

2-4-5 Ultra-Small-Angle/Small-Angle X-ray Scattering (USAXS/SAXS)
measurements
In order to investigate the phase separation behavior at a more detailed scale, USAXS/SAXS
measurements were performed using a similar sample. The data obtained by the measurements
are shown below. (Figure 15)51

Figure 15 USAXS/SAXS measurement results52After confirming that both USAX/SAXS measurements were capable of resolving structureson the nanometer scale, I proceed to analyze the relationship between correlation length andtemperature as well as relaxation time and temperature. The analysis results are shown in Figure
16. Analysis was performed using the Olstein-Zernike equation30, 31 in the USAX measurement
region and the Debye-Bueche equation32, 33 in the SAXS region. The following formula was used
for this fitting.
𝐼(𝑄) =

𝐼DB
𝐼𝑂𝑍
+
+ 𝐵𝐺
2
2
2
2
(1 + 𝜉𝐷𝐵 𝑄 )1 + 𝜉OZ
𝑄2

I(Q): scattering intensity
IDB: intensity of the Debye-Bueche component
ξDB: correlation length of the Debye-Bueche component
IOZ: stands for the intensity of the Ornstein-Zernike component
ξOZ: correlation length of the Ornstein-Zernike component
BG" corresponds to the scattering background.

In the high-q region (q > 10-2 Å-1), the scattering intensity decays with q and converges to atemperature-independent curve. This phenomenon highlights the diminishing influence ofthermal fluctuations on scattering at smaller length scales. In the mid-q region (10-3 Å<q<10-2 Å1) a plateau in scattering intensity is observed, which becomes more pronounced with decreasing

temperature. This plateau indicates enhanced concentration fluctuations within a homogeneousstate, directly linked to the thermal conditions of the system. In the low-q region (q < 10-3 Å-1) the
scattering intensity becomes independent of temperature again , suggesting a return to uniformity
over larger scales. This observation underscores the scale-dependent nature of scattering
phenomena. The Ornstein-Zernike equation is adept at describing scattering from concentration
fluctuations in both the intermediate and high q regions34, 35, offering a theoretical framework for53

understanding the observed patterns. Conversely, in the low q region, where scattering is
influenced by random non-uniformities, the Debye-Bueche equation provides a more accurate
description. By integrating the Ornstein-Zernike and Debye-Bueche equations, a comprehensivemodel can be developed to fit the scattering characteristics across the entire range of q values.
This approach allows for a unified understanding of scattering behavior, highlighting the interplay
between thermal fluctuations, concentration variations, and structural non-uniformities.

54

Figure 16 Analysis results regarding temperature of correlation length and relaxation time
The concept of correlation length is pivotal for elucidating changes in the phases ofsubstances, including phase transitions and critical phenomena. It is defined as the distance overwhich the correlation function decreases to a certain value, serving as a measure of the extent towhich particles or phases are correlated with each other across distance36-38.
A smaller correlation length suggests that order within the system is localized, indicatingthat the likelihood of observing critical phenomena is reduced. Conversely, a divergence in thecorrelation length signifies that the system is nearing a critical point, heralding an imminent
phase transition. This behavior underscores the correlation length's role in predicting thesystem's approach to criticality and its transition from one phase to another.

55

2-4-6 Neutron scattering measurement
Neutron scattering experiments were conducted to investigate the dynamics of polymer
solutions and their interaction with water based on a series of trends. PLL-SA50 and PLL-PA50
were prepared using three types of solvents: pure water, a mixed solvent of pure water and heavy
water, and heavy water. The experimental setup aimed to leverage the contrasting neutron
scattering lengths of hydrogen and deuterium to differentiate the polymer-solvent interactions
under varying hydration levels and solvent compositions. Sample identifiers, used to distinguishbetween the different solution conditions, are provided adjacent to Figure 17 for reference.

100

transmittance/ ％━━━━━ PAH━・━ PAM・・・ PAD━━━━━ SAH━・━ SAM・・・ SAD

0
40

50

60

Temperature/ ℃
Figure17 Sample label and phase separation temperature used for neutron scattering
measurements56

To ensure comparability across experiments, the concentration of the polymers were adjusted
to normalize the phase separation temperature when dissolved in pure water. PLL-SA50 was
prepared at a concentration of 20wt/v%, resulting in phase separation temperature of 47.9℃,
while PLL-PA50 was set at 40wt/v%, achieving a phase separation temperature of 49.9℃. Similar
polymer concentrations were utilized for samples involving intermediate mixtures of water andheavy water, facilitating a direct comparison across solvent systems. Measurements of transmitted
light intensity, as depicted in Figure 17, indicate a notable decrease in phase separation
temperature with an increased proportion of heavy water, despite the uniform polymer
concentration. This phenomenon, consistently observed in both PLL-SA and PLL-PA samples, isattributed to the distinct density differences between heavy water and pure water, which
presumably affect the phase behavior of the polymer solutions. To enhance the precision of ourdata, initial measurements were conducted at a fixed temperature of 300K, below the phaseseparation threshold, with a heightened number of integrations for accuracy. The fortuitous
availability of additional machine time permitted further exploration of phase behavior in heavy
water solutions. Specifically, SAD and PAD measurements were carried out at 35°C and 50°C for
one hour each, alongside detailed PAD analyses conducted at 1°C increments ranging from 39°C
to 44°C, each for a duration of one hour. The outcomes of these meticulous measurements arepresented in Figure 18.

57

Figure18 Neutron scattering measurement results for 1 hour in temperature range

58

Alterations in the structural factors of the hydrated sections were specifically noted in SAD before
and subsequent to phase separation. Initial analysis across various scales suggested the presenceof a consistent trend. However, the requisite adjustment of polymer concentrations to equalize
phase separation temperatures introduced a significant variation that complicates straightforward
comparison and discussion of these observations. Therefore, it was concluded that a directcomparison of the results, within the framework of this measurement approach, is not feasible.

592-5 Conclusion
This chapter delves into the study of two types of polymers: PLL-SA, which is a temperatureresponsive ampholyte polymer that exhibits liquid-liquid phase separation behavior, and PLL-PA,
which has a hydrophobic moiety introduced into the PLL-SA structure. The phase separation
behavior was compared and discussed. It was confirmed that PLL-PA can exhibit stable phaseseparation behavior even in salt solvents by introducing an aromatic ring. It was suggested thatby changing the concentration conditions of the polymer, it could be applied as an intelligent
material over a wide temperature range.
Using a scanning laser microscope and a stage that can be heated, I succeeded in clearlyobserving the formation and growth process of droplets on a scale of several micrometers in apolymer solution around the phase separation temperature. The PLL-SA system was observed to
grow through the continuous formation and coalescence of minute droplets, whereas the PLL-PA
system demonstrated the formation of droplets a certain size from the onset, which then underwent
growth. It was confirmed that the phase separation behavior, which appeared to be the same on
the scale of transmitted light intensity measurement, had its own characteristics. In DLS
measurements, the phase separation behavior was measured on a smaller scale from the viewpointof autocorrelation coefficient and temperature by performing measurements while increasing thetemperature. In PLL-SA, the autocorrelation coefficient decreased continuously as thetemperature increased, but in the PLL-PA system, the autocorrelation coefficient decreased
significantly after reaching the phase separation temperature. PLL-SA changed continuously
without reaching the phase separation temperature, suggesting that some structural changes may
have occurred before droplet formation. While the exact minimum size of emerging PLL-SA
droplets remains undetermined, drawing parallels to the dimensions of biological droplets, suchas vesicles and lipid droplets, allows us to hypothesize that similar pre-formation structural
60

changes could be detectable. Together with the observation results using a laser microscope, it
was suggested that the phase separation and droplet formation mechanisms of PLL-SA and PLLPA may be due to different effects. This contrast not only highlights the compositeity of polymer
behavior in response to temperature changes but also underscores the potential for diverse
applications of these materials based on their unique phase separation dynamics.
Following our preliminary observations, I engaged in USAXS/SAXS measurements toinvestigate the phenomena on a nanoscale level. These measurements aimed to validate ourhypotheses regarding pre-droplet formation changes by identifying specific patterns or data
indicative of such alterations. In the analysis of correlation length and temperature, the phaseseparation behavior was considered to be a phase transition at a critical point because thecorrelation length diverged in both cases. Analysis in the SAXS region shows that droplet
formation occurs concurrently with phase separation temperature and the divergence of thecorrelation length. This suggests that the continuous decrease in the autocorrelation function
observed in DLS might be attributed to minute structural changes, distinct from droplet formation.
Moreover, our multi-scale measurement results elucidate a notable stability in the phaseseparation behavior of the PLL-PA system, contrasting with PLL-SA. Despite observable changes
surrounding the phase separation temperature in PLL-PA, no continuous alterations were detected,
underscoring a fundamental difference in the phase separation dynamics between the twopolymers.
To further delineate the observed trend in phase separation behavior, neutron scatteringexperiments were conducted under controlled conditions, ensuring the phase separation
temperatures of PLL-SA and PLL-PA remained constant. These experiments revealed a change inthe hydration structure factor with water molecules in PLL-SA before and after the phaseseparation temperature. However, the significance of this observation towards clarifying the61underlying mechanism is tempered by the potential influence of varying polymer concentrations
within the measurements.Contrastingly, the phase separation behavior of PLL-PA demonstrated
remarkable stability across different scales of measurement. This stability is attributed to the π-πinteractions facilitated by the aromatic ring incorporated for enhancing phase separation underphysiological conditions. Typically, electrostatic interactions between amino and carboxyl groups
are expected to predominate over π-π interactions. This unexpected finding prompts areevaluation of the conditions under which hydrophobic groups, capable of engaging in π-πinteractions, are introduced. Such a reassessment could elucidate the balance of interactions that
contribute to the stabilization of phase separation behavior in PLL-PA.

62

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67

Chapter 3
Application of DDS by

68

polyampholytes

3-1 Introduction
In Chapter 2, we focused on the liquid-liquid phase separation behavior of ampholyte polymers
and compared the behavior on multiple scales by conducting and evaluating measurements at
different scales. This chapter focuses on the high biocompatibility and temperature
responsiveness of ampholyte polymers and describes their application as carriers in drug delivery
systems.
Many of the materials that have been studied in recent years as carriers of DDS are materials
with functions and properties called smart materials or intelligent materials1-5. Stimuli-responsive
polymers are one type of such polymers, and as mentioned in Chapter 1, temperature-responsive
polymers6-8 such as PLL-SA and PLL-PA are also being studied in the DDS field as temperatureresponsive carriers.
The high reactivity to thermal stimuli required of conventional temperature-responsive carriers
stems from the fact that it is difficult to continue stably supplying heat from outside the body due
to the homeostatic function of the living body9, 10. It is extremely difficult to continuously supply
heat to the in-vivo environment around the carrier precisely in 1°C increments due to external
stimuli. Therefore, one of the goals of conventional temperature-responsive carriers was how
quickly and sharply they could respond to temperature changes. Although many studies have beenreported on overcoming challenges through precision synthesis of polymers11, 12, etc., I thought itwould be effective to first design a carrier that is not affected by the homeostatic functions of theorganism and that can utilise temperature responsiveness.
I hypothesised that the primary challenge associated with the application of temperatureresponsive polymers in DDS, which is their high sensitivity to fluctuating thermal stimuli, could
be effectively addressed by designing composite materials. These composites would integratematerials with diverse properties, enabling a self-regulating mechanism rather than relying on
69

external control for drug delivery. Near-infrared light is appropriate as a source of heat generation
by light stimulation because of its high penetration into living organisms and its use in medical
practice13, 14. Therefore, materials with a photothermal effect on near-infrared light were appliedin this study.
Many metallic nanoparticles have been reported as materials with photothermal effects, withgold nanoparticles being a typical example. Gold nanoparticles absorb near-infrared light and areused as heat-generating materials in thermotherapy15-17. Metals such as gold and silver arecharacterised by their ability to produce particles with well-defined diameters and allow easy
surface modification. Liquid metals (LMs) are also a type of metal nanoparticles that show
photothermal effect with near-infrared light18-23. Mercury, a typical LM, has several excellent
mechanical properties, including high electrical conductivity and flexibility, but it is also known
to have high biotoxicity. However, gallium-based LMs, which have been attracting attention inrecent years, are chemically stable and have extremely low biotoxicity in addition to the propertiesof conventional LMs. Furthermore, compared to other solid metals such as gold and silver
nanoparticles, gallium-based LMs have a lower melting point, which makes it easier to break theoxide film formed on the metal surface and transform it into particles20, 23. Particulation can also
be achieved by a simple method such as sonication, which is ideal for processing composite
materials.
The LM temperature-responsive polymer composite is a physical, stimulus-responsive carrier,triggered by light. In this Chapter, the anticancer drug doxorubicin hydrochloride (DOX), wasselected as the drug to be delivered, assuming that it is particularly suitable for anticancer drug
therapy in spite of strong side effects. DOX is a widely used anticancer drug that inhibits DNA
and RNA synthesis24, 25. Combined with near-infrared laser therapy, it is expected to exert its
effects locally at the target site with minimal side effects.
70Based on the above, the present study aimed to capitalise on the temperature-responsive attributesof the polyampholyte PLL-PA and harness the photofunctionality of LMs, to engineer a composite.
This composite was designed to exhibit a two-step responsive behaviour—a two-step stimuliresponse—wherein stable heat generation, upon triggering with near-infrared laser light, induced
phase separation. Through this approach, I endeavoured to fabricate, assess, and characterise an
innovative, intelligent composite entity that exhibits a sequential response mechanism.

3-2 Experiment3-2-1 Materials
A 25% ε-polylysine solution was procured from JNC Corporation (Tokyo, Japan). Anhydrous
succinic acid and anhydrous phthalic acid were obtained from Nacalai Tesque Inc. (Kyoto, Japan).
The powder form of phosphate-buffered saline (PBS (-)) was purchased from Shimadzu
Diagnostics Corp. (Tokyo, Japan). The Gallium-Indium eutectic alloy was employed using
products from Alfa Aesar (Ward Hill, MA, USA). 2-iminothiolane hydrochloride was procured
from Toronto Research Chemicals Inc. (Toronto, Canada) and doxorubicin hydrochloride wasprocured from Wako (Osaka, Japan).

3-2-2 Synthesis of temperature-responsive polymer
An aqueous solution of 25% PLL was prepared, and an appropriate amount of PA was added tothe solution. The mixture was stirred at 60°C until complete dissolution of reagents. Fifty per centof the amino groups of PLL carboxylated with PA, was denoted as PLL-PA50, respectively. To
increase the affinity of PLL-PA with LM, 2-iminothiolane hydrochloride was added and the
mixture was stirred at 25°C for 1 h. In this process, 40% of the amino group of PLL was71carboxylated with PA and 20% was thiolylated with 2-iminothiolane hydrochloride (PLL-PA(SH)). The polymer solution obtained was lyophilised and stored in vacuum until usage.

3-2-3 Characterization of the polyampholyte
The synthesised polymers were subjected to1

H nuclear magnetic resonance (NMR)

spectroscopy using a 400 MHz NMR instrument from Bruker. The NMR data acquired wereanalysed using the Topspin 3.6.5 software to calculate the compositional ratios based on the
integration values.
The temperature responsiveness and phase separation temperature were determined using atemperature-controlled UV-Vis spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan).
Polymer solutions were placed in 2 mL quartz cells, and optical data were collected continuously
at a fixed wavelength of 550 nm, while varying the temperature. The transmitted light intensityof the homogeneous solution state prior to phase separation was set as 100%, and the temperature
at which the transmitted light intensity reached 50% was defined as the phase separation
temperature.

3-2-4 Particularisation of LM
Particularisation of the LM was achieved through ultrasonic treatment. Changes in particle
size were observed using a transmission electron microscope (TEM) (HF-7650, Hitachi HighTech Corp., Tokyo, Japan) to assess the effects of processing time and thiol functionalisation.
Microgrids (NS-C15, Stem, Tokyo, Japan) were utilised for the observations. A diluted sample
solution was deposited onto the grid with a droplet, dried at 50°C, washed with distilled water forsalt removal, and subsequently dried again in an oven.

723-2-5 Characterisation of composites
Confirmation of the photothermal effect of the composite and verification of control were
performed by irradiating 1 ml of sample solution in a cell with a laser beam with a wavelength of
785 nm (BRM-785-1.0-100-0.22-SMA; B&W Tek, Newark, DE, USA, 6.29 W/cm2), taking
thermographic (FLIR i7: FLIR Systems) images, and evaluating with a thermocouple
thermometer (AD-5601A: A&D Company, Tokyo, Japan).
The two-step reaction was observed using a laser scanning microscope (IX73: Olympus, Tokyo,
Japan), by placing a droplet of the sample solution on a glass slide, covering it with a coverslip,
and irradiating it with 808 nm laser light for 3 s.
DOX dissolved in PBS was used as the solvent in the polymer solution preparation, and thecomposite sample was prepared by adding LM followed by sonication, as described previously.
The sample solution was adjusted to phase separation at 41°C, and the final concentration of DOX
was calculated using a calibration curve.
The release rate of DOX was determined by irradiating a 1 mL sample solution in a cell with
808 nm laser light (LSR808-5W-FCH: LASEVER) for 3 min, to induce phase separation (15.3
W/cm2). The concentrations of DOX in the upper and lower phases were calculated based on thecalibration curve obtained, using a microplate reader (Infinite 200 PRO M Nano+: Tecan,
Männedorf, Switzerland) at 480 nm.

3-2-6 Cell viability assay
To assess the functionality of the composites and the efficacy of drug action, an 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. The
polymer concentration was adjusted and fixed at 25 wt% to achieve phase separation at 41°C.

73

Composites were prepared either containing DOX (1µg/mL) or without DOX. Cytotoxicity

was compared with and without laser irradiation (808 nm, 3 W, 5 min) (15.3 W/cm2).
Human colon adenocarcinoma HT-29 cells (from DS Pharma Biomedical (Tokyo, Japan)) were
cultured and 500 µL of cell suspension was added to 24-well plates at a density of 6000 cells/mL.
After 60 h of incubation, the samples (500 µL) were added, followed by laser irradiation andcontinued incubation. After 3, 6, and 24 h, samples were removed, and residual samples were
washed with PBS. Subsequently, MTT solution dissolved in DMEM without foetal bovine serum(FBS), at a concentration of 100 µg/mL, was added in 500 µL aliquots and incubated for 4 h. The
liquid was then removed, and 500 µL of DMSO was added. To completely remove the LM, thesolution obtained was transferred to microtubes and subjected to centrifugation (20°C, 1000 rpm,
5 min), and the resulting supernatants were transferred to a 96-well plate, in 100 µL aliquots, formeasurement using a microplate reader (λ = 540 nm).

74

3-3 Results and discussion

3-3-1 Characterization of temperature-responsive polymer
The temperature-responsive polyampholyte PLL-PA(-SH) was synthesised by adding 2iminothiolane hydrochloride to PLL-PA and expanding at 25 °C for 1 h according to Scheme 2.
Degree of substitution was calculated from integration values using 1H NMR spectroscopy(Figure 19 and Table3).

Scheme 2 Synthesis of PLL-PA(-SH)

752 − 𝐼𝑇(%) =

[1]
2
[1]
𝛿
6 + 𝑃𝐴(%) + 2

× 100

Table 3 Characterization of polyampholytes
composition

composition

PLL

100

0

0

100

0

0

PLL-PA(-SH)

40

40

20

40

42

18

76

Figure 191H-NMR of PLL-PA50(solvent：D2O)

77

The introduction ratio of anions can be easily varied by adjusting the feeding amounts of PA. In
order to obtain phase separation temperatures relevant for biomaterial applications, anintroduction ratio in the range of 40-60% is considered suitable. When utilised as carriers in thisstudy, polymers in a state where the ratio of anions (-COOH) to cations (-NH2) was equimolar
were synthesised and employed for PLL-PA(-SH), PLL to PA to SH ratio of 40:40:20).
To assess the temperature responsiveness of PLL-PA(-SH) in both pure water and saline solution,
we conducted measurements of transmitted light intensity using a temperature-variable UV-Vis
spectrophotometer.
The same behaviour was confirmed for PLL-PA(-SH) (Figure 20). The phase separation
temperature curves obtained from these experiments confirm the presence of LCST-type phaseseparation and demonstrate that the phase separation temperature can be tuned over a wide range
by adjusting polymer concentration.

Figure 20 Phase separation curves of PLL-PA at 40-60% PA incorporation in water and PBS.

78

The phase-separation behaviour of PLL-PA(-SH) in salt solutions is thought to be due to the ππ interactions caused by the introduction of the benzene ring. I also confirmed that the phaseseparation temperature decreased at the same concentration, as more hydrophobic moieties wereintroduced (Figure 21). This could be due to the fact that the main phase separation behaviour iscaused by the electrostatic interaction between the amino and carboxyl groups, whereas an excess
of anionic carboxyl groups causes an imbalance in the interaction.
These results confirm that PLL-PA(-SH) are polyampholytes capable of stable phase separation
at salt concentrations encountered in physiological conditions; this is necessary for temperatureresponsive biomaterials.
With cancer treatment as the target application, I decided to employ PLL-PA(-SH) with a 25
wt% polymer concentration and a phase separation temperature of 41°C as the designated DDS
carrier.

79

Figure 21 Changes in transmittance intensity across different temperature ranges (UV-vis,
λ=550 nm; 50% transmittance indicates phase separation temperature) The phase separation
temperatures for PLL-PA(-SH) at various concentrations in PBS.

3-3-2 Fabrication of compositees
As the next step, I explored the optimal conditions for the particle formation of the LM, the othercomponent of the composite. LM was added to a polymer solution (10 ml)

adjusted to the

optimal concentration, and variations in conditions were introduced through sonication. Pulsed
sonication was chosen due to the simplicity of the method and uniformity in the particle sizeachieved during particle formation for composite preparation. The changes in particle size bysonication time and the dispersion effect resulting from thiolation of the polymer were evaluatedby TEM observation of the composite after sonication with a 10-fold dilution (Figure 22). The
results showed that relatively large-sized LM nanoparticles (LMNPs) with sizes ranging from 200
to 500 nm were obtained with a sonication time of 5 min (Figure22(A)). By extending the80

sonication time to 10 min (Figure 22(B)), a reduction in the particle size of LMNPs to 200-100
nm was confirmed. Since heat generation during sonication in pulse mode, can cause potential
thermal damage to the polymer, a sonication time of 10 min was deemed appropriate in this study.
However, for both sonication times, aggregation of LMNPs was observed more frequently with
PLL-PA50 than without PLL-PA50.
The effect of thiolation on the polymer, to increase the affinity as a composite, was evaluated by
preparing a similar sample using PLL-PA(-SH) (Figure 22(C)). Although the average particle size
was 200-100 nm, more monodisperse particles were observed compared to the PLL-PA50 sample,
and smaller monodisperse particles were observed even when the duration of sonication was same.
The findings indicate that the addition of thiol groups improves the dispersibility of LMNPs,
underscoring the benefits of thiolation in the preparation of the composite.

81

82

(Figure 22) TEM images demonstrating LM nanoparticle formation and thiolation via pulse
sonication with 2 seconds of irradiation per pulse (20 kHz, Ice Bath, 10 ml Polymer, 30 μL LM):(A) 5-min on PLL-PA50, (B) 10-min on PLL-PA50, (C) 10-min on PLL-PA(-SH)

83

3-3-3 Characterisation of composites
Following the assessment of the compatibility between the polymer and LM for use as acomposite, I further characterised the composite as such. Exploration of the photo-thermal
characteristics of the composite and its control was carried out through thermographic
measurements (Figure 23(A)) and thermal cycling (Figure 23(B)).
The thermographic images in figure 23(A) show results of the composite being heated byirradiation with a near-infrared laser (λ = 785 nm). In the control PBS solution in the upper panel,
the temperature increased by only 1.8°C after 3 min of irradiation. However, the temperature of
the composite solution increased by 5.5°C after 3 min of irradiation.
In figure 23(B), a 96-well plate containing 100 µL each of PBS solution and composite, wasirradiated with the same near-infrared laser for each well, and the change in temperature wasrecorded using a thermocouple thermometer. The rate of change in temperature at each elapsed
time was measured (Figure 23B-1). The results indicate almost no correlation between the laser
irradiation time and the rate of temperature increase in the PBS solution, but a clear correlationin the composite containing LM. Fig. 23B-2 shows the thermal cycles of repeated irradiation andcooling, for the composite irradiated with different laser outputs. The range of temperature rise in
the cycles were 2.8 ± 0.12 °C, 5.6 ± 0.41°C and 7.9 ± 0.62°C, with laser outputs of 250, 500 and1000 nW, respectively. The high reproducibility suggests that the durability of the material andtemperature can be controlled by the power photo-thermal characteristics of the composite andeffectiveness of laser output and irradiation time on control and confirm the sufficient heat
resistance of the material.

84

Figure 23 Characterisation of the photo-thermal properties of the composite under nearinfrared laser (λ=785 nm) exposure: (A) Thermography images: top row - PBS solution (1 mL),bottom row - composite (1 ml) at 0, 1 and 3 min of irradiation. (B) Temperature changes insamples (PBS or composite) in a 96-well plate measured via thermocouple: (B-1) Temperature
variations for both samples during irradiation, and (B-2) Thermal cycling of the composite
across 5 cycles at different power levels.

85

Next, I investigated if the polymer solution component could undergo liquid-liquid phaseseparation by utilising the heat generated through the photothermal characteristics as a carrier for
DDS. Using laser microscopy, I observed the behaviour of the composite material in real-time
during laser irradiation (Figure 24). For understanding the core-shell formation of the composite,
I also studied samples involving the addition of LM to a thiol-bearing, 16-mercaptohexadecanoic
acid (MHDA) solution, followed by ultrasonic treatment. While the MHDA solution exhibitedslight changes around LMNPs due to laser irradiation, no further changes were observed.
Conversely, in the case of the composite, a behaviour was distinctly observed as LMNPs
generated heat in response to near-infrared laser light, triggering core-shell formation of thepolymer solution around the particles, which then spread. As a result, I confirmed that thecomposite heated by near-infrared laser exhibited an increase in the overall temperature as theirradiation time was prolonged. The results from laser microscopy indicate the capability tocapture the initial-stage changes of the composite.
Based on these observations, I confirmed that the composite possessed a two-step
responsiveness, as intended, where it generates heat in response to light stimulation and utilises
this heat to induce phase separation behaviour.

86Figure 24: Laser Microscopy Images (λ=808nm, Irradiation: 3 seconds, Scale: 20 μm):(A) MHDA + LMNPs Solution, and (B) Composite.

87

3-3-4 Cell viability assay
Following the confirmation of the desired properties of the composite, actual drug release
experiments were conducted by adding the anticancer drug DOX, to the composite. The drug
release rate was calculated by measuring three different states: the initial homogeneous state of
the composite solution, the upper phase, and the lower phase after laser irradiation-induced phaseseparation, using a microplate reader. Due to the scattering effect caused by the presence of
LMNPs, it was not feasible to directly measure the DOX concentration26, 27. Therefore, a polymer
solution with an equivalent concentration to the composite without LM was prepared andmeasured for this purpose. Figure 25 shows the sample after phase separation. Due to the intricate
nature of the liquid-liquid phase separation behaviour exhibited by this sample, achieving acomplete separation between the upper and lower phases proved to be challenging. Compared to
the DOX concentration in the homogeneous sample before phase separation, the DOX
concentration in the upper phase after phase separation was about 20%, while that in the lower
phase was up to 150%.
From the aforementioned results, it was evident that the composite exhibited a behaviour where
it concentrated the drug in the lower phase during phase separation, rather than releasing the drug
upon phase separation. This characteristic is unique, differing from that of the conventionaltemperature-responsive carriers designed for drug-release6-8, 28-31. Furthermore, given the
maximum concentration enhancement factor of 1.5, I deduced that this composite holds thepotential to be a carrier with reduced side effects in drug delivery applications.
By enhancing the localised concentration of the drug at the intended site, this approach affords
the potential to maximise therapeutic outcomes while minimising adverse reactions whenemployed as a DDS carrier.

88

Figure 25 Composite after phase separation
Following the discovery of this capability of the composite to concentrate the drug from low
concentrations for effective action, this function was evaluated for cytotoxicity using the MTT
assay.
The composite was prepared at a polymer concentration of 25 wt/wt% (10 mL), an LM volumeof 30 µL, and a DOX concentration of 1 µg/mL to demonstrate the phase separation temperatureof 41°C. Human colon adenocarcinoma HT29 cells were used for the experiment and seeded in
24-well plates at a density of 6000 cells/ml per well and employed for the assay after 2.5 days of
incubation. Experimental groups were established, including groups with and without DOX, as
well as groups with and without laser irradiation (Figure 26).

89

Figure 26 Results of Cell Viability Assay using MTT Assay (HT-29cells)
Significance test
Fisher's constrained LSD method
Statistically significant at 0.05 or higher*:P<0.05

90

For all observed time points, the group treated with laser irradiation using the composite with
DOX, exhibited the lowest cell viability. The initial DOX concentration contained in thecomposite was 1 μg/ml, and the lower phase DOX concentration after phase separation is thought
to be a maximum of 1.5 μg/ml, due to the concentration effect. The significant toxicity at thisconcentration is a result consistent with previous reports32. There was no significant difference incell viability between the DOX-containing group without laser irradiation and the group without
DOX, suggesting that the DOX concentration of 1 µg/mL employed in this experiment was a nontoxic, low concentration for cells. The effectively higher concentration after phase separation in
the irradiated Dox group may have led to this result. Furthermore, the absence of a notabledifference in cell viability regardless of laser irradiation in the DOX-free group indicated that thelaser output used in this experiment did not exert a substantial impact on cell viability. It cantherefore, be inferred that the lower cell viability observed only in the group treated with DOXcontaining composite and laser irradiation, is attributed to the property of the composite, where
photothermal stimulation induced by near-infrared laser light triggered heat generation andutilised this heat for phase separation and drug concentration.
From the results of the cell viability assays, I conclude that the composite exhibits the designed
functionality and effectively exerts its intended action.

91

3-4 Conclusion
This Chapter aimed to address the challenges in the application of conventional temperatureresponsive polymers in DDS, by utilising a composite consisting of a highly biocompatible nearinfrared laser-responsive LM. The composite developed in this study is an intelligent material
with two-step responsiveness, possessing the unique ability to concentrate drugs. I firmly believethat this composite is a highly effective and low side-effect next-generation DDS carrier.
The phase separation temperature required for drug-release can be readily adjusted throughvariations in polymer concentration, and the necessary heat can be controlled via the near-infraredlaser light output and irradiation time. The designed composite demonstrates the anticipatedphotothermal characteristics triggered by light stimulation, inducing temperature-responsive
liquid-liquid phase separation. Furthermore, unlike conventional carriers, the composite
concentrates the drug during phase separation, further confirmed in the cytotoxicity test.
Although challenges remain in terms of precise control over LM particle size and the phaseseparation temperature dependence on polymer concentration, practical applications can beenhanced. By modulating parameters like ultrasonic treatment conditions and polymer
concentration, the amphiphilic properties of these polymers can be leveraged to design optimal
DDS carriers in forms such as gels and micelles.

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96

Chapter 4
Summary97

4-1 General Conclusion
In this thesis, I investigated PLL-PA, a polyampholyte, has both temperature responsiveness andliquid-liquid phase separation behavior. Addressing the challenge of diminished phase separation
behavior in PLL-SA within salt solvents, PLL-PA was synthesized with the incorporation of abenzene ring as a hydrophobic moiety. Due to its high biocompatibility and temperature
responsiveness, PLL-PA emerges as a promising candidate for applications in biomaterials suchas DDS. The observed LLPS behavior of PLL-PA, a focal point of recent scientific inquiry,
underscores its potential in shedding light on the mechanisms governing phase separation inpolymeric systems. This thesis delves into the basic properties of PLL-PA, elucidating themechanism of phase separation behavior, evaluating its applicability as a novel, temperatureresponsive DDS carrier.

In Chapter 2, I conducted an in-depth comparative analysis of PLL-SA and PLL-PA throughmulti-scale measurements using to scrutinize the early stages of phase separation behavior,specifically the generation and growth of droplets. No difference was observed in the phaseseparation behavior of PLL-SA and PLL-PA in the measurement range beyond the transmitted
light intensity measurement. However, i a detailed evaluation at various scales revealed distinct
behaviors between the two polymers. As the temperature increases, PLL-SA exhibits a slight andcontinuous change, suggestive of structural fluctuations prior to droplet formation. On the other
hand, PLL-PA showed remarkable stability in all measurements. This stability is attributed to π-πinteractions, facilitated by the incorporation of aromatic rings, which promote phase separation
under physiological conditions. This finding challenges the conventional expectation thatelectrostatic interactions between amino and carboxyl groups would predominate. The resultsfrom SAXS/USAXS measurements support this conclusion, suggesting a re-evaluation of the98conditions under which hydrophobic groups, capable of engaging in π-π interactions, areintroduced into polyampholytes.

In Chapter 3, I explore the development of a novel carrier for the drug delivery system (DDS)
field, utilizing the temperature-responsive properties of a composite material with photothermal
properties derived from liquid metal (LM). This approach aims to address the longstanding issueof achieving a self-sufficient, stable supply of trigger stimuli for phase transitions in DDS carriers.

Our investigation involved the use of a composite carrier incorporating LM, known for its
photothermal capabilities, to reliably induce phase transitions. Cell experiments demonstrated thecarrier's efficacy in concentrating drugs, which implies a potential for creating DDS carriers with
reduced side effects. However, achieving precise control over LM particle size and managing thephase separation temperature's dependence on polymer concentration emerged as significant
challenges.

To address these challenges and enhance the practical application of our findings, I consideredvarious modulation strategies. By adjusting ultrasonic treatment conditions and polymer
concentrations, I aim to exploit the amphiphilic properties of these polymers more effectively.
This approach is anticipated to facilitate the design of optimal DDS carriers, such as gels andmicelles, tailored to specific therapeutic needs.

Despite the hurdles, our work lays the groundwork for future research into leveraging
photothermal properties in DDS. By refining the control over material properties and exploring99

the full potential of temperature-responsive carriers, I move closer to realizing more efficient,safer drug delivery mechanisms.

100

4-2 Achievements-PublicationTomoka Hirose, Robin Rajan, Eijiro Miyako, Kazuaki Matsumura, Liquid metal-polymer
nano-microconjugations as an injectable and photo-activatable drug carrier. Mol. Syst. Des. Eng.,
2024, Advance Article.

-Presentation-[International Conferences]
・Tomoka Hirose, Eijiro Miyako, Kazuaki Matsumura, Hybrid composites of Polyampholytes
and Liquid Metals as next-generation temperature-responsive DDS carriers, The 33rd Annual
Conference of the European Society for Biomaterials, Davos, Switzerland, September 2023.
(Poster)・Tomoka Hirose, Kazuaki Matsumura, Stabilization of polyampholytes coacervates by
PEGylation during phase separation behavior, The 33rd Annual Conference of the European
Society for Biomaterials, Davos, Switzerland, September 2023. (Poster）[Domestic Conferences]
・第４３回日本バイオマテリアル学会大会、「温度応答性を持つ両性電解質高分子の液-液相分離挙動の制御および DDS 分野への応用の検討」、口頭発表、名古屋開催、
令和 3 年 11 月 30 日(https://confit.atlas.jp/guide/event/jsbabmc2021/static/outline?lang=ja)
101

・第 71 回高分子討論会、「温度応答性を有す両性電解質高分子の液-液相分離挙動の評価」、ポスター発表、北海道開催、令和 4 年 9 月 5-7 日(7 日発表)
(https://main.spsj.or.jp/tohron/71tohron/)
・第 44 回日本バイオマテリアル学会大会、「温度応答性両性電解質高分子の疎水性部位導入による液-液相分離挙動の評価」、ポスター発表、東京開催、令和 4 年 11 月 2122 日(21 日発表)
(https://www.kokuhoken.jp/jsb44/)
・第 32 回日本 MRS 年次大会、「類似構造を持つ温度応答性両性電解質高分子の液液相分離挙動の比較」、口頭発表、横浜開催、令和 4 年 12 月 5-7 日(6 日発表)
(https://www.mrs-j.org/meeting2022/jp/index.php)
・第 11 回日本バイオマテリアル学会北陸信越ブロック若手研究発表会、「両性電解質高分子の疎水性部位導入による温度応答性への影響評価」、口頭発表、オンライン開催、令和 4 年 12 月 20 日(http://kokuhoken.net/jsbm/gyouji/gyouji20221220.html)

102