Controlling the morphology of poly(ethylene glycol)-

b-poly(lactide) (PEG-PLA) self-assemblies in

solution: Interplay of homopolymer additive and
kinetic traps
PQ Lim
NTU
AN Parikh
UCD
S Venkatraman
NUS
bczarny (  [email protected] )
NTU

Research Article

Keywords: Block copolymer, self-assembly, homopolymer, kinetics, morphology, micelle, vesicle

Posted Date: June 16th, 2023

DOI: https://doi.org/10.21203/rs.3.rs-3066290/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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Additional Declarations:
Competing interests is : The authors declare no competing interests.

Supplementary Discussions not available with this version.

Controlling the morphology of poly(ethylene glycol)-
b-poly(lactide) (PEG-PLA) self-assemblies in
solution: Interplay of homopolymer additive and
kinetic traps

PQ Lim a, AN Parikh b, S Venkatraman c, B Czarny a

a
School of Materials Science & Engineering, Nanyang Technological University, Singapore 639798, Singapore
b
Departments of Biomedical Engineering and Materials Science & Engineering, University of California,
Davis, Davis, California 95616, United States
c
Material Science & Engineering, National University of Singapore, Singapore 117546, Singapore

Abstract

Block copolymer (BCP) self-assembly involves various components in the system interacting in diverse and
intricate ways to result in a final state. This paper explores the how homopolymer additives and kinetic traps can
be used to control the morphology of self-assembly of poly(ethylene glycol)-b-poly(lactide) (PEG-PLA)
nanostructures in water. By kinetically trapping PEG-PLA nanostructures formed in the presence of a non-
adsorbing PEG homopolymer, we reveal how different concentrations and molecular weights of the added PEG
was found to be able to induce micelle-to-vesicle transitions. This phenomenon was ascribed to a change in the
molecular geometry of the underlying BCP induced by the added PEG homopolymer due largely to excluded
volume screening effects. In addition, we demonstrated the reverse vesicle-to-micelle transition upon
homopolymer removal, and the kinetic nature of the system rendered the transition effect time and temperature
dependent. Analysis of the intermediate transition structures reinforces our proposed mechanism involving the
change in the underlying molecular geometry of the PEG-PLA BCP. As proof of concept, we showed how our
understanding of the PEG-PLA system allowed us successfully utilize the PEG-PLA vesicles thermoresposive
delivery systems. We showed how the vesicles was able to retain dye at low temperature (4℃) and the release
can be triggered upon heating (37℃). Overall, the work here introduces a novel means of regulating the
morphology and behavior of BCP nanostructures, which can have important downstream applications.

Keywords: Block copolymer, self-assembly, homopolymer, kinetics, morphology, micelle, vesicle

Introduction

Block copolymers (BCP) are known to be able to self-assemble into a myriad of nanostructures in solution [1],
which can subsequently be used for plethora of applications including drug delivery [2], template synthesis [3],
sensing [4], and catalysis [5]. Because the properties and functions of these BCP nanostructures are inextricably
tied to their morphology, intensive research have been undertaken to elucidate the factors can potentially influence
the final self-assembled shape. The morphology of the final nanostructure in a selective solvent is dependent on
the molecular geometry of the component BCP molecule, a property that is determined by the proportions of the
collapsed solvophobic block (to minimize unfavorable solvent interaction) and the well solvated solvophilic block
[6]. Intuitively, the most direct way of altering the molecular shape of the BCP molecule would be to modify the
relative length of the underlying polymer building block, and this strategy has indeed been widely employed to
tune the morphology of self-assembled BCP in solution [7, 8]. As our understanding of BCP self-assembly
progresses, scientists are beginning to explore how extrinsic factors such as the solvent conditions, temperature,
presence of additives can influence the final BCP self-assemblies [9-11]. Research efforts on this front has helped
scientists better understand the complexities involved in BCP self-assemblies.

An interesting parameter that can be employed to influence BCP self-assembly is homopolymer additives. While
homopolymer additives have been widely investigated for their ability to tune the self-assembly of BCP in the
bulk [12-14] phase, studies that examine the effects of these homopolymer on corresponding systems in solution
are comparatively limited [15]. A better understanding of homopolymer effects can allow more scientists to take
advantage of this potentially facile approach to manipulate BCP morphology in solution for various applications.
In addition, the knowledge generated on this subject can potentially lead to insights on how self-assembled
systems behave in complex, multicomponent, real-life settings.

We are interested to explore the effects of non-adsorbing homopolymer additives on BCP self-assembled
nanostructures in a solvent that is selective for the homopolymer and one of the blocks in the BCP. Previous
reports involving these homopolymers and BCP micelle mixtures revealed how the addition of homopolymers
disrupt the ordered packing of BCP micelles [16], leading to rheological changes and the “melting” of the soft
solids formed by the micelles [17]. The addition of the homopolymer was also found to be able to induce
macroscopic aggregation, leading to gelation [18] and phase separation [19] effects. However, to the best of our
knowledge, very few studies directly address the concept of effecting morphological changes in the BCP through
the addition of these non-adsorbing homopolymers. In an earlier report by Abbas et al [20], the authors
investigated the effects of homopolymer additions on the hexagonally packed cylindrical micelles. Using a system
comprising of poly(styrene-b-isoprene) BCP and poly(styrene) homopolymer in dibutyl phthalate solvent that is
selective for the poly(styrene) blocks, they were able to observe changes in the hexagonal lattice packing, order-
order phase transitions and even phase separation given different compositions of BCP and homopolymer.
However, the focus of their study was on the long range ordered phases formed by the high concentration of BCP
used (<32% by volume). Morphological changes on dispersed BCP nanostructures, typically formed at lower BCP
concentrations, was not explored. Furthermore, their study was limited to a rather low range of homopolymer
additive concentrations (>6% by volume). More recently, Yang et al [21] showed the possibility using repulsive
homopolymers to influence the morphologies of discrete nanoparticles formed in solution using AB diblock and
BAB triblock copolymers through computational methods. In their work, they showed how the addition of the
repulsive homopolymers at sufficiently high concentrations could cause simple dispersed spherical micellar
structures to transition into alternative morphologies like cylindrical micelles, vesicles and even helical structures.
However, these effects have yet to be corroborated with experimental data.

This paper sets out to address some of the abovementioned knowledge gaps and will examine the effects of
homopolymers on the morphologies of discrete nanoparticles for a wider range of homopolymer concentrations
through experimental methods. In our study, we will make use of a system comprising of poly(ethylene glycol)-
b-poly(lactide) (PEG-PLA) (a di-block copolymer) and poly(ethylene glycol) (PEG) (a non-associating
hydrophilic homopolymer) in water. The materials were chosen in part due to their structural simplicity and
widespread availability. The biocompatibility of the polymers, biodegradability of PLA block and high aqueous
solubility of PEG can also facilitate potential downstream translational efforts for biomedical and consumer
products [22], inevitably rendering this PEG-PLA and PEG system a compelling model and target for
investigation. More importantly, use of PEG-PLA also affords us a unique advantage: The glassy nature [23, 24]
of the PLA block at room temperature allows us to kinetically trap the BCP structures formed; this provides us
with a strategy to experimentally investigate the effects of high homopolymer additive concentrations using
conventional imaging and scattering techniques post homopolymer removal.

In this paper, we will explore the effects of PEG homopolymer additive and the kinetic traps afforded by the PLA
block in the BCP to manipulate the morphologies of the final nanoparticles. We first report how different
concentrations and molecular weights (MWs) of PEG homopolymer can induce multiple morphologies from a
single in PEG-PLA polymer, an effort that was facilitated by kinetically trapping the BCP nanostructures.
Subsequently, we demonstrate the reverse vesicle-to-micelle transition upon homopolymer removal and show
how this process is influenced by the kinetically trapped nature of the system. We also explored how these vesicles
can be used as thermoresposive delivery system. Overall, the work here shows how careful consideration of BCP
self-assembling system and its surroundings can open the possibility of generating interesting and novel material
behaviors and properties, which can then be used for practical applications.
Results and Discussion

Addition of homopolymer PEG induces shape transition in PEG-PLA self-assembly

The self-assembly of PEG-PLA (Mn of PEG block = 5000 g/mol, Mn of PLA block = 10000 g/mol) from a dried
thin film in solution was induced with the help of probe sonication. Due to the glassy nature of the PLA block at
room temperature, the sonication process was used to provide sufficient heat and energy to overcome the kinetic
barrier for BCP re-organization to occur. After the sonication step, the sample was allowed to cool to room
temperature which causes PEG-PLA to return to its kinetically trapped state. The presence of the kinetic trap
provided us the opportunity to remove the added PEG homopolymer post BCP self-assembly without affecting
PEG-PLA structures formed in the preceding step. Consequently, this allowed us to explore the effects of high
concentrations of homopolymer additives without having to resort to complex characterization techniques or data
analysis methods typically required for concentrated polymer solutions. Further details of the kinetically trapped
nature of the PEG-PLA used is presented in Supplementary Discussion 1.

The effect of different concentrations and MWs of PEG homopolymer on the self-assembly of PEG-PLA was
explored. In this paper, the PEG homopolymer concentration used will be expressed in terms of weight ratio
between the added homopolymer and water. These homopolymers are of MW 2000 g/mol, 6000 g/mol and 10000
g/mol; which will hereon be referred to as PEG2k, PEG6k and PEG10k respectively. The morphology of the
resultant PEG-PLA structures obtained was characterized using transmission electron microscopy (TEM). The
results obtained are presented in Figure S1-S3 and summarized in Figure 1. We observed two characteristic effect
of the PEG homopolymer addition on the BCP self-assembly. First, a concentration-dependent micelle-to-vesicle
shape transition was apparent irrespective of the MW of PEG used. At low concentration of PEG homopolymer,
micelle formation (both spherical and cylindrical) was observed. As PEG concentration was increased past a
certain limit, the self-assembly tended towards the formation of vesicles. Second, the shape transition occurred at
lower concentration when a higher MW of PEG was used. While transition towards vesicular structures was
observed upon the addition of 1.2 w/w PEG2k homopolymer, only 0.7 w/w of the higher MW PEG10k was needed
to induce a similar effect.
Figure 1: Diagram summarizing the morphological transition of PEG-PLA structures when PEG of different concentration and MW was used.
Morphology was determined using TEM. The blue area of the bars indicate the samples in which micelles (either spherical or cylindrical)
were observed, while the red areas of the bars highlight samples in which vesicles were observed. Between these 2 areas, there is a transition
region where both micelles and vesicles coexist in the sample. The dotted line represents the estimated boundary where this transition occurs
across the different PEG concentration and MW.

Shape transition mechanism likely to be combination of excluded volume screening

effects, interfacial effects and depletion effect

A schematic of the proposed mechanism for the PEG-PLA BCP micelle-to-vesicle shape transition observed
previously is presented in Figure 2.
 Figure 2: Schematic showing the proposed mechanism by which the shape transition occurs – screening of excluded volume effect. The
diagram illustrates how size of the PEG chains and the molecular geometry of the BCP changes with the concentration of PEG added.
Components indicated in red represent the hydrophobic PLA block on the copolymer while components indicated in blue represent the
hydrophilic PEG block on the copolymer. The added free PEG homopolymer is represented in green.

When little or no PEG homopolymer was added, the solution remained dilute and all PEG chains (including both
the added homopolymer and the hydrophilic block on the BCP) existed as swollen and separate chains [25]. This
is due to excluded volume effects which minimized interactions within chain segments and between different PEG
chains. Overall, the expanded conformation of the hydrophilic PEG head group on PEG-PLA BCP could have
resulted in a more conical packing shape that favored micellar formation, which would explain the prevalence of
these structures in the absence or at low concentrations of the PEG homopolymer additive.

At sufficiently high concentrations of the added PEG homopolymer, a combination of several factors could have
lead to the shape transition. We believe that the driving factor for this transition is excluded volume screening
effects. A crowded solution increases the likelihood of the PEG chains interpenetrating with each other, and this
chain overlap screens the excluded volume interactions that was previously present at lower PEG concentrations
[25]. This excluded volume screening effects have even been exploited as a strategy to increase the grafting
density of polymer brushes onto surfaces [26]. Regardless, the reduction of excluded volume interactions will lead
to a corresponding decrease in PEG polymer coil size, a phenomenon that has been experimentally observed using
complex small-angle neutron scattering (SANS) techniques by Gurnev et al [27]. Overall, the condensed
conformation in the PEG head group on the PEG-PLA copolymer would lead to a more cylindrical packing shape
which allowed the BCP to pack into vesicles.

This excluded volume screening effect would also explain the second observation that a lower concentration was
required for vesicle-to-micelle transition when a higher MW PEG homopolymer was used. Longer polymer chains
typically exist as large coil (i.e. have large radius of gyration) in a good solvent due to the additive effects of the
excluded volume among all the chain segments and allow them to experiencing chain overlaps at lower
concentration [25]. Consequently, this can allowed the proposed excluded volume shielding effects to set in at
lower concentrations, resulting in the observation that vesicle transition occurred at lower concentration when a
higher MW of PEG was used.

A secondary factor that could have supplemented PEG is less polar than water excluded volume effects. Since
PEG is less polar than water [28], its presence near the hydrophilic-hydrophobic interface could help reduce the
interfacial curvature and support the formation of more planar lamellar constructs such as the bilayer structure in
vesicles. However, we acknowledge that this mode of action may only be limited to PEG homopolymer additives
of lower molecular weight, since it is generally found that large homopolymers have more difficulty in penetrating
the corona of the micelles [19].

Depletion effects could also contribute partially towards the shape transition effect. In general, the addition of
non-adsorbing homopolymers have been found to be able to induce a net attractive force between suspended
colloidal particles and this phenomenon is known as depletion effect. For self-assembled BCP colloidal structures,
numerous studies have reported that the depletion attraction caused by these homopolymers can result in an
increase the aggregation number of the micelles formed [19, 29]. This will lead to an increase in the core size of
the micelles formed which requires the hydrophobic block of the constituent BCP to adopt a more stretched
conformation. However, as reasoned by Mai and Eisenberg [1], this stretching cannot continue indefinitely due to
the decrease in entropy of the core-forming hydrophobic chains. At a certain threshold, the system will compensate
for the stretching of the hydrophobic chains by inducing the shape transitions, which parallels the observations
for our system. However, we were unable to replicate the shape transition with other depletant homopolymer
additives (See Supplementary Discussion 2) which led us to the following conclusion: While depletion effect
from the added PEG homopolymer may be present in our system and can exert some effects, it is unlikely to be
the driving factor for the shape transition.

Shape transition is reversible upon homopolymer removal and is time and temperature
dependent
Based on the proposed mechanism, it is predicted that the micelle-to-vesicle shape transition is reversible upon
removal of the triggering PEG homopolymer. The kinetic nature of the system also suggested some form of time
and temperature dependency in the vesicle-to-micelle shape transitions. To validate this, the PEG-PLA vesicles
were incubated at different temperatures (4℃, 37℃ and 60℃) for 1 h and 24 h and the resultant structures were
observed under the TEM; the results are presented in Figure 3. Samples stored at 4℃ showed no significant
changes in their vesicular structures as regardless of the incubation time. In contrast, samples kept at 37℃ and
60℃ showed changes in their morphology. But there was a marked difference in the rate of transition between
the samples: While both samples completely reverted to their micellar form after 24 h, only the 60℃ contained
micelles at the 1 h timepoint; the sample kept at 37℃ appeared to be in the transition process after 1 h of incubation,
containing structures that appeared to look like vesicles that have been broken and flattened with the edges fraying
off. Overall, the results here confirm the reversibility and the time and temperature dependencies of shape
transition process.

Figure 3: Series of images showing the resultant structures that were formed after PEG-PLA vesicles were incubated at different
temperatures (4℃, 37℃ and 60℃) for different lengths of time (1h and 24h).

This time and temperature dependency in the vesicle-to-micelle shape transition could also be characterized in-
situ using optical methods. Turbidity have been shown to correlate with colloid particle size with larger sizes
exhibiting higher turbidness [30]. Since turbidity can be measured by light absorbance, in-situ by measurements
of the sample absorbance at 600 nm throughout the heating process can be used to effectively track the vesicle-
to-micelle shape transition [31, 32]. Samples of the PEG-PLA vesicles were heated for 30 mins at different
temperatures (30℃, 40℃ and 50℃) and pictures of the resultant solutions together with their corresponding
absorbance profiles are shown in Figure 4. From the results, negligible change in turbidity was observed at
incubation temperature of 30℃. In contrast, the samples heated to 50℃ showed a rapid decrease in turbidity
indicating the rapid rate of shape transition, with the intensity reaching a plateau at 7 min timepoint signaling the
end of the shape transition process. For the samples maintained at 40℃, a slower decrease in turbidity was
observed as compared to the samples heated at 50℃, which is indicative of the slower transition rate. An
interesting sharp downward step-like profile was observed during the first part of the absorbance reading for the
samples incubated at 40℃ and 50℃. This could be because vesicle-to-micelle transition does not occur with a
monotonic decrease in size; the size decrease could be larger during the initial segment of the transition as
compared to the rest of the process, which would result in a sharp drop in the early absorbance readings.

Figure 4: In-situ monitoring of the sample turbidity upon incubation at various temperatures (30℃, 40℃ and 50℃) for 30mins. (a) Photos
showing the turbidity of PEG-PLA samples before and after the incubation process. (b) Plot of normalized absorbance at 600nm (to track
turbidity changes) against time for each of the different samples.

Transition intermediates for vesicle-to-micelle can be linked back to underling packing

geometry changes

To better understand the transition process, PEG-PLA vesicles were incubated at 37℃ to induce the shape
transition and aliquots were removed at specific timepoints and quenched in 4℃ water. These samples were
imaged using the TEM and results are presented in Figure 5. Several unique structures were observed, including
the porated vesicle structure at 15 min, “jellyfish” structure at 30 min and irregular clumps with extended
structures at 3 h. It is interesting to note how such a process can potentially be used a strategy to generate
interesting morphologies.

Figure 5: Series of TEM images taken of PEG-PLA vesicles incubated at 37℃ over a period of 6 h.

Using the above information, a detailed pathway of the vesicle dissociation was conceptualised and illustrated in
Figure 6. In the initial stages, pore formation was observed on the vesicles which resulted in a curved open lamella
structure. This structure gradually evolved into a “jellyfish” configuration: The pore continued to widen to form
a more open bilayer structure, which gave rise to the “cap” of the “jellyfish”; the edges of the pore frayed into
elongated protrusion, forming the “tentacles” of the “jellyfish”. Subsequently, the elongated protrusion continued
to grow while the bilayer structure receded and flattened out, resulting in the appearance of irregular-shaped
bilayer regions with elongated structures extending from them. In the final stage, the bilayer structure was no
longer observed, and only micellar structures remained. No further shape transformation was expected given that
micellar structures were the most stable configuration in water at room temperature.
Figure 6: Schematic showing the proposed pathway by which PEG-PLA vesicles transition into micelles. The hydrophilic PEG block and
hydrophobic PLA block of the BCP is denoted by blue and red lines respectively.

This dissociation pathway could be understood from the perspective where the system attempts to eliminate
unfavorable bilayer structures. The removal of the added PEG homopolymer brough about a consequential
elimination of the excluded volume screening effect. PEG block on the BCP tended to revert to its more extended
configuration which resulted in a change in packing shape from cylindrical to conical that favored micelle
formation. The initial pore formation of the PEG-PLA vesicles was the result of the system trying to generate
micellar edges with higher curvature to better accommodate the new conical packing shape. Subsequently, the
reduction in the overall volume of lamella structures and corresponding increase in the formation of elongated
micellar protrusion throughout the transformation process all served to help the system adapt to the new packing
shape of the underlying PEG-PLA BCP. The final system was one void of any unstable lamellar structures, leaving
behind only micellar constructs

It was noted that the transformation pathway presented seemed to parallel the shape transitions that have been
reported by researchers working on polymerization-induced self-assembly (PISA) [33, 34]. A typical PISA
process involves the gradual addition of monomer of a hydrophobic block to a soluble homopolymer chain to
form a BCP. As the chain extends, it increases the hydrophobic to hydrophilic ratio of the BCP of the system to
drive in situ changes in self-assembly, typically causing a micelle-to-vesicle transition. Modifications of the PISA
process can also result shape transition in the reverse direction [35]. But regardless of the direction of the shape
transition, the intermediate structure reported in PISA such as the “jellyfish” structure and other flat bilayer
structures was extremely similar to the ones observed in the current study. However, while the PISA technique
involves changes in the fundamental self-assembling polymer entity (which requires synthetic effort and involves
the use of solvent and monomers precursors), the shape transitions presented here can achieve a similar
polymorphism effect simply from a single underlying BCP.

Implications of this study

To the best of our knowledge, this is the first study that experimentally shows how the addition of non-adsorbing
homopolymers (at high concentrations) can affect the morphology of discrete self-assembled BCP nanoparticles
in solution. This technique of using homopolymer addition to manipulate the final shape of self-assembly
nanoparticle is extremely versatile; it allows users to generate a range of structures from just a single underlying
BCP. This method circumvents the need to undertake judicious tuning of the relative solvophobic to solvophilic
block lengths of the BCP in order to form different shapes for applications.

In addition, the learnings that were generated in this work can help us better understand how self-assembled
structures behave in complex practical systems. For instance, we could use this understanding to better evaluate
the performance of self-assembled nanostructures in multi-component formulations (especially polymeric co-
solutes containing ones) found in industrial [36], consumer [37] and pharmaceutical [38] products. In addition,
this knowledge may even be extended to help us gain insights to how macromolecules in the crowded biological
milieu [39, 40] can potentially influence the conformational changes of biological self-assembled structures,
which can have significant biological consequence.

Furthermore, the reverse vesicle-to-micelle transition exhibited by PEG-PLA structures explored in the later part
of this paper represents a unique strategy to generate stimuli-responsive particles. Stimuli-responsive particles can
change properties when an external stimulus (such as pH, temperature, light, magnetic field, and light) is applied.
There is growing interest in using these nanoparticles to develop smart delivery systems that can afford better
spatiotemporal control over the release of an active cargo which the potential to influence and improve the
application outcome [41]. An example of one such system in the literature are the thermo-responsive vesicles
fabricated by Qin et al. using PEG-poly(N-isopropylacrylamide) (PEG-PNIPAAM) [10]. Since PNIPAAM
changes its conformation upon cooling below its lower critical solution temperature, they were able to induce
vesicle dissociation upon cooling to 25℃ from 37℃. This structural change was used to induce release of
hydrophilic anticancer drug doxorubicin. In another example, Cerritelli et al. synthesized PEG-disulfide-
poly(propylene sulfide) used it to form vesicles [42]. As the disulfide bond joining the hydrophilic and
hydrophobic block is sensitive to redox reaction, the presence of reducing agent caused the cleavage of the
disulfide bond and lead to the disassembly of the vesicle. However, a common thread that was noted among these
reports was that the preparation of such nanoparticles typically requires the fundamental BCP to intrinsically
possess stimuli-responsive characteristics. This requires careful selection and design of polymers that may pose
as a challenge from a synthesis point of view. The strategy explored here can circumvent this problem: By trapping
the self-assembly in a non-equilibrium state, the strategy presented here made use of the nanostructures intrinsic
drive to transit towards a different conformation at a lower energy state when provided with sufficient stimulus to
induce a response. This approach allows stimuli-responsive behavior to be expressed in nanostructures that are
constructed from BCP that do not fundamentally possess stimuli-responsiveness (such as PEG-PLA) and can help
to expand the repertoire of materials that can be used to generate smart nanoparticle systems. This increase in the
flexibility of BCP material choice can potentially facilitate downstream application.

Furthermore, from an applications point of view these vesicles can be used as thermo-responsive cargo delivery
systems. To demonstrate this, PEG-PLA vesicles were loaded with a hydrophilic sodium fluorescein (NaFluo)
dye and the dye release was evaluated at 4℃ and 37℃. From the DLS results comparing the nanostructures before
and after the release experiments (Figure 7a), only the samples that were kept at 37℃ showed significant changes
in size (indicating the shape transition) after the release experiment; samples stored at 4℃ showed no significant
difference with the original sample. These observations support the results earlier in the chapter which indicate
that shape transitions can only be triggered above PLA glass transition temperature. From a practical perspective,
it also implies that samples can be stored without much morphology changes in a 4℃ fridge. In terms of dye
release (Figure 7b), it was noticed that only the sample maintained at 37℃ showed significant overall dye release.
This is expected since vesicle-to-micelle shape transitions would have occurred under this temperature, leading
to the release of hydrophilic dye sequestered in the aqueous cavity of the vesicles. The sample kept at 4℃ retained
most of the encapsulated dye, albeit a small amount of dye leakage (>10%) which could be attributed a little
portion of dye that may not have been properly encapsulated during the fabrication process. Overall, the work
here represents an initial proof of concept that the kinetically-trapped PEG-PLA vesicles engineered using the
homopolymer-induced effect can serve as thermo-responsive delivery systems where cargo release can be
thermally triggered and controlled via time and temperature change. More work would still need to be done to
optimize these structures for specific applications in the future.
Figure 7: Results of release experiment for PEG-PLA vesicles incubated at different temperatures. (a) DLS results showing the Z-Avg and
PDI of the samples before and after the release experiment of 72h. (The symbol * is used to denote two data sets are significantly different (p
≤ 0.05), while the symbol # is used to denote two data sets do not show statistically significant differences (p >0.05).) (b) Release profile of

NaFluo dye from the vesicles. Initial release rate was calculating using datapoints from the first 3 h.

Conclusion
In this paper, we explored how homopolymer additive and kinetic traps work together to influence the morphology
and behavior of self-assembling BCP in solution, specifically using of a system comprising of PEG-PLA BCP
and PEG homopolymer in water. The work presented here reveal a new and versatile tool that researchers can use
from their toolkit of techniques to tune the morphology self-assembly BCP nanoparticles in solution. It also
reinforces the importance of understanding of BCP self-assembly in the context of an interplay of the multitude
of components within the system. This can allow scientists to conceptualize more effective and intricate systems,
which could ultimately pave the way for BCP self-assemblies to be a valuable platform for enabling the production
of next generation materials with potent applications.

Experimental Methods

Materials
PEG-PLA (Mn of PEG block = 5000 g/mol, Mn of PLA block = 10000 g/mol) was obtained Advanced Polymer
Materials. Homopolymer PEG of MW 2000 g/mol, 6000 g/mol and 10000 g/mol was purchased from Sigma-
Aldrich; these homopolymers will be referred to as PEG2k, PEG6k and PEG10k respectively. Additional
chemicals such as acetone, NaFluo and ammonium molybdate was obtained from Sigma-Aldrich. All polymers
and reagents used in the experiments were obtained from commercial suppliers and used without further
purification.

Fabrication of BCP self-assembled structures

PEG-PLA was first dissolved in acetone. This solution was then pipetted into a 2 mL vial and left to dry, leaving
behind a 5 mg polymer film. Subsequently, the hydrating solution was added into the vial and the contents were
subjected to 2 min of sonication for 2 cycles using a probe sonicator (Q125, QSONICA) equipped with a 1/8”
probe to promote self-assembly. Depending on the experiment, the hydrating solution can be deionized water or
a pre-prepared aqueous solution containing PEG homopolymer additives as indicated in the main text. In this
paper, the PEG homopolymer concentration used will be expressed in terms of weight ratio between the added
homopolymer and water. After sonication, the sample was allowed to cool to room temperature before they were
diluted with deionized water and subjected post-fabrication purification by dialysis.

TEM characterization
The TEM grids were first treated with glow discharge using hydrophilic treatment device (HDT 400, JEOL
DATUM). A 2uL of the sample (which have been diluted to 0.25 mg/mL using deionized water) was pipetted
onto the TEM grid. After 30 s, a filter paper was used to wick off the excess sample solution. 2 uL of the negative
stain (2 w/v% ammonium molybdate in deionized water) was then pipetted onto the sample and left to sit for 1
min. A filter paper was used to wick off the excess stain solution and the sample was left to dry in air before
imaging under the TEM (Libra 120 Plus, Carl Zeiss) using a 2 k × 2 k CCD camera (Troendle (TRS), Sharpeye).

Heating experiment to induce reverse shape transition

PEG-PLA vesicles were prepared using the above-mentioned method using an aqueous solution containing 1 w/w
PEG6k. The vesicles were then aliquoted into Eppendorf tubes, with each tube containing 200uL. These samples
were then stored in ovens of having different temperatures (4℃, 37℃ and 60℃). At different timepoints (1h or24
h), the aliquots were removed and quenched in ice water to halt any shape transition process. The samples were
then imaged using the TEM conditions described previously.

UV-Vis characterization
The UV-Vis-NIR equipment (Cary 5000, Agilent Technologies) was used to characterize sample absorbance at
600nm at specific temperatures (30℃, 40℃ and 50℃). Absorbance can be used to indicate changes in size of the
nanoparticles in solution. Quartz cuvettes were used to contain the liquid samples. deionized water was used in
the reference cell. For temperature control, the Varian Cary Dual Cell Peltier accessory was used. Each sample
was kept at their respective temperatures for 30 mins and the readings were collected at 10Hz. All measurements
were done in triplicates.

Cargo release from vesicle sample

For experiments involving cargo release from vesicle sample, 50 mg of PEG5k-PDLLA10k was hydrated with 1
mL of 1 w/w PEG6k aqueous solution containing with 50 mg/mL of NaFluo added. This sample was processed
using the same fabrication method as mentioned previously and purified via centrifugation. For the release
experiment, the 1 mL of the dye loaded samples was sealed in 10 kDa regenerated cellulose dialysis bags
(Spectrum Labs). These dialysis bags were then placed in vials containing 10 mL of deionised water as the external
buffer and stored at different temperatures (4℃ and 37℃). At specific timepoint, sample was taken from the
external buffer before the entire buffer was exchanged with fresh 10 mL of deionised water. With the help of an
independently prepared standard curve, fluorescence intensity from the collected sample was used to track the
release of NaFluo. Similarly, the measurements were collected using a microplate reader (Infinite 200, Tecan)
with an excitation wavelength of 480 nm and emission wavelength of 520 nm.

Statistical analysis
All experiments were performed in triplicates. Where applicable, the means and standard deviations were
calculated and student t-test (with p value set at 0.05) was conducted to establish significant difference.
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