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Modulating the Selectivity and Stealth Properties of
Ellipsoidal Polymersomes through a Multivalent Peptide
Ligand Display
Kristel C. Tjandra, Chelsea R. Forest, Chin Ken Wong, Sheilajen Alcantara,
Hannah G. Kelly, Yi Ju, Martina H. Stenzel, Joshua A. McCarroll, Maria Kavallaris,Frank Caruso, Stephen J. Kent, and Pall Thordarson*
nanomedicine therapies already in themarket such as Doxil,[1] a liposomal formulation of doxorubicin, Abraxane,[2]an albumin-bound paclitaxel nanoparticle, and NanoTherm,[3] an iron oxide
nanoparticle.[4] These therapies have shown
improved pharmacokinetics, biodistribution, and better drug tolerance compared to
the free drug. However, the extensive areaof nanoparticle characteristics, such as size,
shape, active-cell targeting, as well as chemical properties that can be manipulated has
not been exhausted, leaving a large room
for improved nanoparticle synthesis foreﬃcient drug delivery.
Both nanoparticle shape and their ligand
density have been suggested to play a major role in eﬀective drug delivery.[5–7] Whilemost earlier studies have been done on spherical nanoparticles,
recent work have suggested that nanoparticle shape can aﬀect
their cellular uptake, biodistribution, and cytotoxicity.[8,9] The
circulating explanation is that the higher aspect ratio of nonspherical particles and their geometrical orientations allows for

There is a need for improved nanomaterials to simultaneously target cancer
cells and avoid non-speciﬁc clearance by phagocytes. An ellipsoidal
polymersome system is developed with a unique tunable size and shapeproperty. These particles are functionalized with in-house phage-display
cell-targeting peptide to target a medulloblastoma cell line in vitro. Particle
association with medulloblastoma cells is modulated by tuning the peptide
ligand density on the particles. These polymersomes has low levels of
association with primary human blood phagocytes. The stealth properties ofthe polymersomes are further improved by including the peptide targeting
moiety, an eﬀect that is likely driven by the peptide protecting the particles
from binding blood plasma proteins. Overall, this ellipsoidal polymersome
system provides a promising platform to explore tumor cell targeting in vivo.

1. Introduction
In the last 40 years, the ﬁeld of nanomedicine has mademany strides toward making drug therapies more potent, more
selective, and most importantly less toxic. There are several
Dr. K. C. Tjandra, C. R. Forest, Dr. C. K. Wong, Prof. M. H. Stenzel,
Prof. P. Thordarson
School of Chemistry
The University of New South Wales
Sydney, NSW 2052, Australia
E-mail: p.thordarson@unsw.edu.au
Dr. K. C. Tjandra, C. R. Forest, Dr. C. K. Wong, Dr. J. A. McCarroll,Prof. M. Kavallaris, Prof. P. Thordarson
Australian Centre for Nanomedicine
The University of New South Wales
Sydney, NSW 2052, Australia
Dr. K. C. Tjandra, C. R. Forest, Dr. C. K. Wong, S. Alcantara, H. G. Kelly,
Dr. Y. Ju, Dr. J. A. McCarroll, Prof. M. Kavallaris, Prof. F. Caruso,
Prof. S. J. Kent, Prof. P. Thordarson
ARC Centre of Excellence in Convergent Bio-Nano Science andTechnology
Australia

The ORCID identiﬁcation number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.202000261

DOI: 10.1002/adhm.202000261

Adv. Healthcare Mater. 2020, 2000261

S. Alcantara, H. G. Kelly, Prof. S. J. Kent
Department of Microbiology and Immunology
The University of Melbourne at the Peter Doherty Institute for Infection
and Immunity
Parkville, VIC 3000, Australia
Dr. Y. Ju, Prof. F. Caruso
Department of Chemical Engineering
The University of Melbourne
Parkville, VIC 3010, Australia
Prof. M. H. Stenzel
School of Chemistry
Centre for Advanced Macromolecular Design (CAMD)The University of New South Wales
Sydney, NSW 2052, Australia
Dr. J. A. McCarroll, Prof. M. Kavallaris
Translational Cancer Nanomedicine Theme
Children’s Cancer Institute
Lowy Cancer Research Centre
The University of New South Wales
Sydney, NSW 2031, Australia
Dr. J. A. McCarroll, Prof. M. Kavallaris
School of Women’s and Children’s HealthFaculty of Medicine
The University of New South Wales
Sydney, NSW 2052, Australia

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Scheme 1. Structures of the block copolymers used for the solvent-switch self-assembly formation of multivalent polymersomes bearing medulloblastoma cell-targeting peptides.

an increased interaction with cell surface receptors and oﬀers amore diverse cellular mode of entry.[10–12] However, the extent towhich particle geometry contribute to speciﬁc biological eﬀects
were still debatable, thus highlighting the need for further investigations. Our current study gives insight into the eﬀect ofligand density on targeting using a non-spherical polymersome
platform.
Polymersomes are artiﬁcial vesicles with a bilayer membrane,
made up of amphiphilic block copolymers. This bilayer system
is desirable for drug delivery as it allows the versatility of imbedding hydrophobic drugs in the core of the membrane or a hydrophilic drug to be encapsulated within the aqueous core of
the particle.[13–15] One of the advantages polymersomes have overother nanoparticles is that they can easily adopt several morphologies apart from spherical such as rod-like, ellipsoidal, anddisc using several diﬀerent techniques without the need for post
assembly manipulation or scaﬀolds like particle replication in
non-wetting templates.[16–18]
One downfall of many nanoparticle based therapeutic systems
is the rapid clearance from the blood by phagocytes that leads
to poor tumor biodistribution and pharmacokinetics. This rapid
clearance is mainly due to blood protein interactions with thesurface of the nanoparticle which leads to increased immune
cell association.[19] One approach to evade this large scale immune clearance is by precoating particles with polyethylene glycol (PEG) to create a stealthy proﬁle. Here we aim to determine
if the presence of a cell-targeting peptide on the surface of theparticle changes the immune cell-association of the particle andtherefore predicting their clearance from the blood.
Herein we report for the ﬁrst time the synthesis of a selective and multivalent polymersome system using two blockcopolymers (Scheme 1). We investigated the eﬀect of varying celltargeting peptide density and particle size on both the selectivityof non-spherical polymersomes toward tumor cells and theirstealth property toward primary human immune cells. A suite of
15 ellipsoidal polymersomes (Table 1) were reproducibly generated building on our previous work on self-assembled perylenebearing polymer system.[16,20] Ten of these polymersomes will beAdv. Healthcare Mater. 2020, 2000261

Table 1. Hydrodynamic diameter and polydispersity indices of small andlarge high ligand density (HLD) and low ligand density (LLD) polymersomes in this study as determined through dynamic light scattering (DLS)
measurements.
Size groups

Small

Peptide ligand density(equivalent peptide units)
HLD

LLD

Large

Z-averagea)(Dh [nm])

PdIa)

0% (0)

96 ± 0.5

0.22 ± 0.01

25% (1)68 ± 0.8

0.263 ± 0.02

50% (2)86 ± 0.2

0.25 ± 0.02

75% (3)91 ± 0.8

0.11 ± 0.02

100% (4)109 ± 0.1

0.112 ± 0.03

0% (0)

70 ± 0.3

0.16 ± 0.01

25% (0.375)

69 ± 0.4

0.12 ± 0.00

50% (0.75)

72 ± 0.4

0.13 ± 0.01

75% (1.125)

70 ± 0.6

0.10 ± 0.02

100% (1.5)

70 ± 1.3

0.15 ± 0.05

0% (0)

201 ± 1.2

0.14 ± 0.01

25% (0.375)

191 ± 0.9

0.11 ± 0.03

50% (0.75)

196 ± 1.1

0.14 ± 0.01

75% (1.125)

197 ± 2.0

0.14 ± 0.03

100% (1.5)

193 ± 4.5

0.14 ± 0.05a) D

h and PdI = average ± standard deviation (the approximate magnitude of errors of
the particles’ hydrodynamic diameter were automatically generated by the Zetasizer
Nano instrument based on three independent scans).

referred to as the low-ligand density (LLD) polymersomes consisting of 5 ± 80 nm (major axes) particles and 5 ± 400 nm (majoraxes) particles with varying peptide ligand densities. The remaining ﬁve particles are the high-ligand density (HLD) ± 100 nm(major axes) particles. The perylene moiety provides the necessary hydrophobicity and liquid crystallinity for the polymer
to self-assemble into its anisotropic shape while serving as ahighly stable ﬂuorescent component that is convenient for invitro imaging.[21] These polymersomes were functionalized witha heptapeptide FSRPAFL, which we discovered through a peptide phage-display library as having selectivity and high binding

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www.advhealthmat.deaﬃnity against human medulloblastoma (DAOY) cells.[22] The
stealth property of these ellipsoidal polymersomes was determined through their association and clearance by human phagocytes and compared against a known “stealthy” PEG-based particle by incubating the particles with whole human blood.[23]We hypothesized that the higher peptide ligand density will
give greater selectivity to these polymersomes and that the peptide will not alter their-like stealth property. Our results showedthat indeed the higher ligand density particles were more selective. More interestingly, this increase was greatly enhanced whena certain ligand density threshold was reached. Further, the addition of the medulloblastoma cell-targeting peptide also markedly
improved the stealth property of these particles.

2. Results and Discussions
2.1. Polymer Synthesis and Characterization
Poly(ethylene
glycol)-block-poly(N-isopropylacrylamide-coperylene diester monoimide) (PEG43 -b-P(NIPAM21 -co-PDMI9 ))polymer, which was derived from PEG43 -b-P(NIPAM21 -co-tBocAEA9 ) was synthesized according to our previously reported
protocol.[20,24] The ﬁrst step in the synthesis of PEG43 -bP(NIPAM21 -co-t-BocAEA9 ) involved the copolymerization
reaction between two monomers: 1) N-isopropylacrylamide(NIPAM) and 2) tert-butyloxycarbonyl-aminoethyl acrylate(t-BocAEA), using a reversible addition-fragmentation chaintransfer (RAFT) polymerization technique in the presenceof PEG43 -modiﬁed trithiocarbonate macro-RAFT agent. The
use of t-Boc-aminoethyl acrylate (t-BocAEA) was a convenient
way to pre-install a protected primary amine functionality asa handle for the perylene moiety, which acts both as a ﬂuorescent probe and to direct the self-assembly of the polymers
toward non-spherical polymersomes. The perylene was added
to the polymer block in the form of an N-(pentaﬂuorophenyl
butyl ester)-perylene-3,4,9,10-tetracarboxylic monoimide dibutyl
ester (PDMI-PFP).[20] Here, the t-Boc protecting group wasused to avoid cleavage of the RAFT trithiocarbonate moiety by
the primary amine during RAFT polymerization (Scheme S1,
Supporting Information).
To facilitate the peptide ligand attachment, a diblock
copolymer,
namelypoly(ethyleneglycol)-block-poly(Nisopropylacrylamide-co-N-(tert-butoxylcarbonyl)
aminoethyl
acrylate-co-pentaﬂuorophenyl
ester
acrylate)(PEG43 -bP(NIPAM21 -co-t-BocAEA9 -co-PFP4 )) was synthesized via RAFT
polymerization following modiﬁcations to previously reported
procedures (Scheme S1, Supporting Information).[20,25] The
design of this polymer was intended to provide an orthogonal
handle for the incorporation of two distinct functionalities.
The ﬁrst component was the previously mentioned perylene
moiety, which was reacted with the amino group of the block
copolymer, and the second moiety was a heptapeptide (as atargeting ligand) which was attached through a reaction withthe pentaﬂuorophenyl ester group via the N-terminus. The
success of these syntheses and the degree of polymerization was
conﬁrmed and determined using 1 H NMR spectroscopy anddimethylacetamide (DMAc) gel permeation chromatography(Figure S1, Supporting Information).

Adv. Healthcare Mater. 2020, 2000261

Heptapeptide FSRPAFL, which was previously described tohave selectivity toward our target medulloblastoma cell line
(DAOY),[22] was conjugated onto the PEG43 -b-P(NIPAM21 -co-tBocAEA9 -co-PFP4 ) polymer backbone using a diisopropylethylamine base in N,N-dimethylformamide for 48 h at 40 °C. To synthesize the LLD polymer, only 1.5 molar equivalent of this peptide
was reacted onto the polymer backbone, which contains four repeating units of pentaﬂuorophenol—as indicated by the 1 H NMR(Scheme S2, Supporting Information). Six molar equivalents of
the heptapeptide was reacted with the polymer in a similar manner to create a polymer with HLD. This synthesis strategy allows
a ﬁne-tuning of the peptide units on the polymer backbone (with0, 0.375, 0.75, 1, 1.125, 1.5, 2, 3, or 4 equivalent peptide units
per polymersome). The progress of the conjugation reaction wasmonitored qualitatively using 19 F NMR, where the gradual decrease in the broad polymeric pentaﬂuorophenyl signal was indicative of the extent of conjugation (Figure 1).
The 19 F NMR spectra of the crude reaction mixture for the LLD
polymer at 24 h post reaction showed signals from the ﬂuorine ofthe pentaﬂuorophenol group. The broad singlet peaks at 𝛿 −152,−157, and −162 ppm (Figure 1, signals a, b, and c), suggested
that the polymer-bound pentaﬂuorophenyl was still present, andhence incomplete conjugation. Sharp signals at 𝛿 −168, −171,
and −185 ppm were indicative of free pentaﬂuorophenol, indicating that some conjugation had occurred (Figure 1, signals d,e, and f). Thus, the reaction was left for another 24 h. At 48 h,
the 19 F NMR spectra suggested that the proportion of polymerbound pentaﬂuorophenyl had been reduced, but not completely(Figure 1).
It was important to ensure that no free pentaﬂuorophenylgroups were left on the polymer backbone, as cross-linking withthe free amine on the amino-ethyl ester amine would impede
subsequent attachment of the PDMI-PFP. Hence, in order tofully substitute the pentaﬂuorophenol group, propargylamine
was added into the reaction mixture as a less-hindered primaryamine and stirred for a further 24 h. The disappearance of thebroad singlet peaks (labeled a–c in Figure 1) suggested that there
were no polymer-bound pentaﬂuorophenyl moieties left on the
LLD polymer backbone (Figure 1C). The absence of this ﬂuorine
signal indicated that the reaction between the pentaﬂuorophenyl
ester and the free amine of the linear peptide did occur. Hence,
the targeting peptides were successfully conjugated to the polymer backbone.
The HLD polymer, on the other hand, did not require the additional propargylamine capping step described above. After 72 hreaction with the six molar equivalents of peptide, the 19 F NMR
spectra showed no unreacted pentaﬂuorophenol groups left on
the HLD polymer backbone (Figure S2, Supporting Information). The reaction products were dialyzed to remove any unreacted peptides and free pentaﬂuorophenol. This procedure wasfollowed by the deprotection of the tert-butylcarbonyl (t-Boc) moiety to produce free amines, which upon conjugation with the N(pentaﬂuorophenyl butyl ester)-perylene-3,4,9,10-tetracarboxylic
monoimide dibutyl ester (PDMI-PFP) yielded the ﬁnal polymer
products. Successful attachment of the perylene moiety onto thepolymer backbones was conﬁrmed using 1 H–13 C 2D NMR (Figure S3, Supporting Information). However, signals corresponding to protons on the peptide moiety could not be assigned due
to poor resolution (Figure S3, Supporting Information).

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www.advhealthmat.deFigure 1. 19 F NMR spectra of the progression of low density peptide functionalization and propargylamine capping onto PEG43 -b-P(NIPAM21 -co-tBocAEA9 -co-PFP4 ) in CDCl3 . 19 F NMR trace: A) after 24 h reaction with FSRPAFL peptide; B) after 48 h reaction with peptide; C) after 24 h capping withpropargylamine, showing the disappearance of a–c peaks; and D) after dialysis against dichloromethane and ethanol.

2.2. Polymersome Formation and Characterization

2.4. Tuning the Size of Polymersomes

Ellipsoidal polymersomes in this study were formed through asolvent-switch method with tetrahydrofuran as the good solvent
and water as the antisolvent (Figure 2A).[16,20,26,27] The solubilityof non-peptide bearing block copolymer PEG43 -b-P(NIPAM21 co-PDMI9 ) was diﬀerent from that of its peptide-bearing counterparts. While block copolymer PEG43 -b-P(NIPAM21 -co-PDMI9 )dissolved reasonably well in tetrahydrofuran, block copolymer, PEG43 -b-P(NIPAM21 -co-PDMI9 -co-Peptide1.5(LLD) or 4(HLD) ),did not. Therefore, in order to create a homogeneous solution, the peptide-bearing polymers were dissolved in N,Ndimethylformamide (10% of the ﬁnal volume), prior to dilution
with tetrahydrofuran. Water was then added to the polymer
solution at a constant volume (in this case, 500 µL), triggering
aggregation and self-assembly. The tetrahydrofuran solvent
was evaporated slowly over 16 to 20 h yielding ellipsoidal polymersomes in water (Figure 2A). The N,N-dimethylformamide
solvent leftovers in the peptide-bearing polymersomes wasremoved by dialysis against water.

Previous work in the group has found that polymersome sizesof this speciﬁc system can be tuned by adjusting the volumeof tetrahydrofuran during formation.[20] Two diﬀerent volumes
were chosen in this study (i.e., 500 and 800 µL) to yield polymersomes with hydrodynamic diameters of ≈70 nm and ≈200 nm,
as conﬁrmed by dynamic light scattering (DLS) (Figure S4,
Supporting Information; Table 1). Due to the ellipsoidal natureof the polymersomes, DLS can only provide a rough estimate of
the hydrodynamic diameter (Dh ) of these polymersomes. This
discrepancy is due to the spherical geometry (hard spheres) thatis assumed in the measurement process, hence discounting thepossible rotational diﬀusion (tumbling motion) of these particles
in solution.[28] A more accurate measurement (including the
major/minor axes and aspect ratio) of these polymersomes wasdetermined by taking transmission electron microscopy (TEM)
images and using ImageJ analysis (Figures S5–S7, Supporting
Information).
Here the TEM images of the particles showed that the addition of the peptide units onto the polymer backbone did not alter
the shape of the resulting polymersomes. This observation suggests that the peptide did not disrupt the liquid crystallinity ofthe particle. Note that the DLS measurements of the polymersomes sizes did not correlate to the TEM image analyses (using
ImageJ) in relation to large polymersomes (Figure S6, Supporting Information) compared to the small polymersomes (Figure
S5, Supporting Information). The major axes of the large polymersomes as determined from the from transmission electron
microscopy (TEM) images indicated that they were 404 ± 64 nm
in size (as opposed to 194 ± 2.1 nm as indicated by DLS). On theother hand, the hydrodynamic size of the small polymersomes
according to the DLS measurement (70 ± 0.6 nm) was a closer
match to the major axes (80 ± 12 nm) indicated by the TEM images (Figure 2B,C).

2.3. Tuning the Ligand Density of the Polymersomes
Ligand density of the peptide on the surface of polymersomes
was tuned by blending the non-peptide and peptide-bearing polymers during polymersome formation (step 1, Figure 2A). The total mass of both polymers (i.e., 0.5 mg) was kept constant acrossall samples. For ease of discussion, these polymersomes are referred according to their sizes (small or large), and the composition of peptide-bearing block copolymers PEG43 -b-P(NIPAM21 co-PDMI9 -co-peptide1.5(LLD) or 4(HLD) ) beginning from 0, 25, 50, 75,
to 100%. In the case of 0%, the polymersome was formed with
only the non-peptide polymer, while the 100% samples weremade out of only the peptide-bearing polymer (≈4 equivalent orunits of peptides per polymer).

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www.advhealthmat.deFigure 2. Polymersome formation using a solvent-switching method. A) PEG43 -b-P(NIPAM21 -co-PDMI9 polymer (1) and PEG43 -b-P(NIPAM21 -co-PDMI9 co-peptide1.5 (LLD) or 4 (HLD) polymer (2) were dissolved in THF (in red). Addition of water (in blue) induces self-assembly of the polymer. Diﬀerent ratios of
PEG43 -b-P(NIPAM21 -co-PDMI9 and PEG43 -b-P(NIPAM21 -co-PDMI9 -co-peptide1.5/4 polymer mixture in “Step 1” are key in tuning the ligand density of thepolymersome, while the increasing volume of THF corresponds to an increase in resulting polymersome sizes. B,C) TEM images of the two polymersome
populations (small and large low-ligand density (LLD) polymersomes) of varying peptide ligand-density (expressed in percentage). Peptide ligand density
on polymersomes is indicated by the percent value: 0% for polymersomes made of non-peptide PEG43 -b-P(NIPAM21 -co-PDMI9 ) polymer only, and 100%for polymersomes made of PEG43 -b-P(NIPAM21 -co-PDMI9 -co-peptide1.5 ). Scale bars: 200 nm.

These diﬀerences in sizes from the two techniques indicatedthat the sizes of the larger polymersomes were underestimatedby the DLS. Despite this inconsistency, DLS is still a useful toolfor checking particle dispersity in solution (expressed in termsof a polydispersity index, PdI) as an indication of their size
distributions.[28] Using these two polymers and by adjusting thetetrahydrofuran volumes, two populations of ellipsoidal polymersomes according to their sizes, having varying ligand densities
were successfully generated. The stability of these polymersomesin the cell culture media and at pH ranging from 3–8 was conﬁrmed (Figure S16, Supporting Information).

2.5. Active Transport of Polymersomes into Medulloblastoma
Cells
The eﬀect of varying the peptide ligand density and particle
sizes on the uptake of the polymersomes inside the medulloblastoma cells (DAOY) was investigated by ﬂow cytometry. We previously reported a detailed characterization of the targeting peptide (FSPRPAFL) used for functionalizing these polymersomes
and showed that the peptide is capable of mediating a ligandmediated endocytosis.[22] Herein we conﬁrm that such metabolically active mechanism was also responsible for the transport ofthe polymersomes into the target cells. In brief, DAOY cells wereseeded onto tissue culture plates and incubated with 0.025 mg

Adv. Healthcare Mater. 2020, 2000261

mL−1 of low-ligand density polymersomes at 37 and 4 °C for 2 h(concentration was chosen according to the maximum tolerated
concentration tested, see Figure S8, Supporting Information).
Note that 0.025 mg mL−1 concentration of the HLD polymersomes was toxic for the cells at 72 h. However, this concentration is well tolerated by the cells within 2 h post-incubation asdemonstrated by cytotoxicity assays (see Figure S8C, Supporting Information). Hence, the cellular uptake study proceededwith this concentration. After removal of the polymersomes, thecells were washed with phosphate saline buﬀer, trypsinized, andharvested for ﬂow cytometry analysis using methods previously
described.[22] The ﬂuorescent intensities were corrected for thediﬀerences observed in each sample (Figure S9, Supporting Information). Single cell populations were sorted using ﬂow cytometry. The median ﬂuorescence intensity of the polymersomeincubated cells was indicative of the concentration of polymersomes that were internalized by the cells as demonstrated by confocal microscopy (Figure S10, Supporting Information).
It was observed that at 4 °C (at which cells remained viable but metabolically inactive),[29,30] minimal cellular uptake wasachieved as indicated by the baseline ﬂuorescence intensity ofthe single cells across the ten diﬀerent polymersome samples(small and large, 0% to 100% peptide ligand density). This was
not the case at a physiological temperature of 37 °C, where the increase in peptide density was associated to an increase in cellular
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www.advhealthmat.deintensity. The cellular internalization of the polymersomes wasinhibited at 4 °C (according to a paired t-test comparing the samples at both temperatures, p (two-tailed) = 0.000005. This suggested that these polymersomes were taken up by the cells via
a metabolically energy-dependent process rather than passive
membrane diﬀusion (Figure S11, Supporting Information).

2.6. Comparing Polymersome Cellular Association in Non-Target
Cells
A key function we aimed to demonstrate with these polymersomes was selectivity toward their target cancer cell throughthe addition of a medulloblastoma cell-targeting peptide (in thiscase, peptide FSRPAFL). The selectivity of the polymersomes toward the target medulloblastoma cell line, DAOY, was assessedby comparing their cellular associations for non-target cells(HEK293). A cellular association study using ﬂow cytometry was
performed comparing the ﬂuorescence intensity of cells treatedwith the same concentration of the polymersomes solution incell culture media. The HEK293 cell line, which is a human embryonic kidney cell line with similar characteristics to neuronalcells was used as the control non-target cell line.[31]Interestingly, according to the ﬂow cytometry measurement on
the LLD polymersomes the non-liganded polymersomes associated more to the target cells, DAOY, compared to the non-target
cells, HEK293 (0% peptide polymersome samples, both small
and large, p = 0.000014 for large polymersome and p = 0.000045
for small polymersomes) (Figure 3). The addition of the peptide
onto the polymersomes led to an incremental nanoparticle uptake into DAOY cells. Notably, the same eﬀect was not observedin the HEK293 cells, where the presence of the peptide ligand
on both the small and the large polymersomes did not alter thecellular uptake of these particles. This ﬁnding indicates that theincrease in peptide-ligand density on the polymersomes surface
led to the increased selectivity of these polymersomes toward thetarget tumor cells compared to the control non-tumor cells.
The cellular association pattern of the small LLD polymersomes was very similar to the HLD polymersomes at 37 °C except for the 100% small polymersomes where a signiﬁcant difference was observed (Figure 4). The degree of association of the
100% peptide-bearing polymersome, as indicated by the median
ﬂuorescence intensity of the particles, was almost three times
higher for the small HLD polymersomes compared to the small
LLD polymersomes. This stark increase in cellular associationsuggests that an enhanced interaction was achieved once a highpeptide ligand density threshold (in this case, 4 peptide units
per polymersomes) was reached.[32] Interestingly, the same phenomenon was not observed in two diﬀerent non-target cell lines(HEK293 and HBEC5i) (Figure 4A,B), which demonstrated thatthe multivalent binding was a cell-speciﬁc event.
The observed enhancement in binding and selectivity toward
the DAOY cells over the non-target non-tumor cells can be explained based on the latest understanding of multivalent targeting of cell-receptors.[32] In particular the work of Frenkel
and co-workers on the concept of selectivity in multivalency
helps explains the results here.[33,34] In short, while overall binding strengths might be expected to increase somewhat linearly with the number of interacting ligands (here the pep-

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Figure 3. Representative graph of the ﬂow cytometry measurements forcomparing the cellular association of A) large versus B) small LLD polymersomes in DAOY and HEK293 cells. The diﬀerence in the median ﬂuorescence intensity (MFI) of DAOY and HEK293 (i.e., MFIDAOY –MFIHEK293 )is represented by the dashed lines (—). Two-way ANOVA, Tukey’s multiple
comparison tests, were calculated for the diﬀerence in the MFI of thesetwo cell lines, for a, p = 0.000014; b, p = 0.000003; c, p = 0.000001; d,p = 0.00000008; e, p = 0.00000004; f, p = 0.000045; g, p = 0.000022; h, p
= 0.0000027; i, p = 0.0000003; and j, p = 0.00000006. Error bars represent
standard error of the mean (n = 3).

tide) on the nanoparticles, the selectivity between cells withmany target receptors (here DAOY) and those with fewer receptors (the non-targeting, HEK293 and HBEC5i) will be highly
non-linear. This is consistent with there being a thresholdconcentration of ligands, above which the interaction of thenanoparticle becomes much stronger for the cell lines with manytarget receptors than those cell lines with fewer receptors (compare the uptake at 100% ligand density in Figure 4A with Figure 4B). Multivalent systems that show such selectivity are also
usually characterized by very steep (sigmoidal) non-linear increase in the bound fraction of ligands toward their target cellline once a certain threshold concentration of ligands has been

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2.7. Stealth Characteristics of Peptide-Bearing Polymersomes in
Human Blood

Figure 4. Representative graphs of the ﬂow cytometry measurements forcomparing the uptake of small HLD and LLD polymersomes in DAOY,HEK293, and HBEC5i cells (a human brain microvascular endothelial cellline). A) Low ligand density (LLD) polymersomes with the 0–1.5 equivalent peptide units corresponding to 0 to 100% ligand density, B) high ligand density (HLD) polymersomes with the 0–4 equivalent peptide units
corresponding to 0 to 100% ligand density. Each data point indicates themedian ﬂuorescence intensity (MFI) of the detected polymersomes signal.
Error bars represent standard error of the mean (n = 3).

reached. Here it appears that this threshold concentration, which
resulted in a more optimal interaction with the target cell, corresponds to the 100% HLD ligand concentration (equivalent to 4
peptides per polymersome).
Overall, the selectivity of the polymersome was more pronounced in polymersomes with higher peptide ligand density.
This was especially true for the 100% high ligand density small
polymersomes where a threefold increase in association was observed (as referred to in Figure 4). Comparisons of polymersomes
cellular uptake in DAOY, HEK293, and HBEC5i cells suggested
that the polymersomes in general associate more with the target
cell (DAOY). We also found that the size of the polymersomes
was, in this case, less of a factor in the selectivity of these polymersomes (as indicated in Figure 3).

Adv. Healthcare Mater. 2020, 2000261

Next, we evaluated the eﬀect of the tumor cell-targeting peptide attached to the small HLD polymersomes and its potential association with human blood immune cells by ﬂow cytometry (Figure 5A). We studied the small HLD polymersomes incomparison to an alternate PEG-based particle we have previously developed.[23] We found that the non-peptide bearing polymersomes had a similar and low level of association with blood
phagocytes (monocytes and granulocytes) as the PEG particles,
although a higher level of association with B cells was observed(Figure 5A). The association of particles with B cells is an eﬀectcommonly driven by association of the particles with complement in plasma and complement receptors found on the surfaceof B cells.[35]We were concerned that the addition of the peptide ligand to
the PEG based polymersomes could interfere with the hydrophobicity of the particles and reduce the stealth-like properties of theparticle, as previously observed with antibody labeled particles.[36]However, we observed the opposite, with the highest ligand density polymersomes having reduced association with neutrophils(granulocytes), monocytes, and B cells which we speculate wasdue to the protective nature of the cancer cell-targeting peptide
against binding to human blood plasma proteins (Figure 5B).
The reduced association of the peptide-targeted polymersomes
and B cell association observed in the whole human blood assay
suggested the possibility that interactions between plasma proteins in blood and the particles (the formation of a biomolecular corona) were mediating some aspects of the particle association with blood immune cells. We therefore performed a“washed blood” assay where all plasma proteins were removed
from blood prior to incubation with the polymersomes, as previously described.[37] We found that particle association with blood
immune cells was essentially absent when plasma was removed(Figure 5C), demonstrating a dominant eﬀect of plasma proteins on mediating association of polymersomes with blood immune cells. In contrast, the comparator PEG based particles[36]
had low but detectable association with blood immune cells inboth the presence and absence of plasma illustrating that the lowfouling properties of that system are not inﬂuenced as much byplasma proteins. Consistent to our previous observation on different sized polymersome particle associations with cancer cells
above (Figure 3), the size of the particles (80 nm versus 400 nm,
major axes) did not contribute to any diﬀerence in association toward the diﬀerent types of blood cells (Figure S14, Supporting
Information). At the same time, this also suggests that the targeting capacity of the ligands on the polymersome particles were
not negated by the presence of PEG units.

3. Conclusion
The concept of a “stealthy” nanoparticle to evade immune cellrecognition is a popular avenue for improving the bioavailability of drug carriers. However, these particles tend to lacktargeting capabilities. Here in our work, we reported the ﬁrst
synthesis of a selective and multivalent polymersome using two
block co-polymers. Using this system, we studied the eﬀect of

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www.advhealthmat.deFigure 5. Percentage cell association of small non-peptide bearing and 100% HLD polymersomes to blood cells compared to PEG-based particles. A)
Schematic illustration of the experimental setup where the particles were incubated with either a whole blood containing plasma proteins or washedblood with no proteins. B) Graphical representation of the particle association to the whole blood samples. C) Graphical representation of the particle
association to washed blood samples. Error bars represent standard error of the mean (n = 3).

targeting ligand density on nanoparticle selectivity and stealthiness. We developed an ellipsoidal particle system that has aunique tunable size and shape property with versatile surfacefunctionality. These particles were functionalized with our inhouse peptide phage-display cancer cell-targeting peptide, which
we showed to eﬀectively target a medulloblastoma cells in vitro.
We demonstrated that the particle associated with medulloblastoma cell was modulated by tuning the peptide ligand density on
the particles. More importantly, we showed that the eﬀect was,in fact, non-linear. A threshold of ligand density was required to
unlock the targeting potential of these particles. Further, thesepolymersomes also had low levels of association with primary
human blood phagocytes. The stealth properties of the polymersomes were improved by including the peptide targeting moiety,an eﬀect that was likely driven by the protective nature of the peptide against binding to blood plasma proteins. Overall, this ellipsoidal polymersome system not only provides valuable insights
into ligand design but also is itself a highly promising platform
for further development of a selective theranostics for in vivo
applications.

4. Experimental Section
Materials: All chemicals were purchased from commercial sources
and were used as received unless otherwise stated. Deuterated chloroform
was ﬁltered over anhydrous potassium carbonate and neutral aluminumoxide prior to use. Cell lines (DAOY, HEK293, and HBEC5i) used in thisstudy were obtained from American Type Culture Collection (ATCC). Cells
were grown in a standard T75 cell culture ﬂask (Corning, USA) and maintained at 37 °C and 4% CO2 and grown until ≈80–90% conﬂuent. Cells
were kept only for 3 months (or before reaches passage 30). For every routine passaging, cells were rinsed with a Dulbecco’s phosphate buﬀeredsaline (PBS) and harvested by adding Trypsin. Cells were regularly (≈every
3 months) tested for mycoplasma and were negative.

Adv. Healthcare Mater. 2020, 2000261

Polymer Synthesis: Synthesis of the Polymer through RAFT Copolymerization: Synthesis of PEG43 -b-P(NIPAM21 -co-t-BocAEA9 -co-PFP4 ) by
RAFT copolymerization followed the reported procedure by Wong andco-workers with slight modiﬁcation.[20] NIPAM (438 mg, 3.87 mmol),t-BocAEA (300 mg, 1.39 mmol), poly(ethylene glycol) methyl ether
2-(dodecylthiocarbonothioylthio)-2-methyl propionate (PEG43 -DDMAT,
351 mg, 0.15 mmol), pentaﬂurorophenol anhydride (128 µL, 0.77 mmol),and 2,2′-azobis(2-methylpropionitrile) (0.2 m in toluene) (155 µL,
0.31 mmol) were dissolved in N,N-dimethylformamide (1 mL), and degassed by purging with nitrogen for 25 min. The degassed mixture wasthen placed in a preheated oil bath at 65 °C. After 170 min, the reaction vessel was exposed to air and quenched in an ice bath. The reaction was monitored using 19 F NMR, by observing the disappearance of ﬂuorine signal,
which indicated the complete exchange of pentaﬂuorophenol group on thepolymer backbone. The crude reaction mixture was then diluted in minimal amounts of tetrahydrofuran and precipitated into cold hexane:diethyl
ether (2:1, v/v). The resulting precipitate was collected by centrifugation,
redissolved in minimal amounts of tetrahydrofuran and precipitated into
cold hexane:diethyl ether (2:1, v/v) again. The puriﬁcation process was repeated four times in total. The puriﬁed product was collected and dried under high vacuum to yield PEG43 -b-P(NIPAM21 -co-t-BocAEA9 -co-PFP4 ) as alight yellow solid (943.9 mg). The number of NIPAM and t-BocAEA repeating units in the diblock terpolymer were determined using NMR (CDCl3 )(see Figure S1, Supporting Information). Mn, GPC = 13 800 g mol−1 ; Mn ,−1
NMR = 9336 g mol ; Ð = 1.06.
Polymer Synthesis—Peptide Conjugation and Capping Step (LLD Polymer): PEG43 -b-P(NIPAM21 -co-t-BocAEA9 -co-PFP4 ) (300 mg, 1 equiv.),
FSRPAFL peptide[22] (36 mg, 1.5 equiv.), and diisopropylethylamine(100 µL, excess) were dissolved in peptide-grade N,N-dimethylformamide(4 mL) and stirred for 2 days at 40 ˚C under nitrogen ﬂow. The reaction mixture was then dialyzed in a mixture of dichloromethane/ethanol(1:1). Solvents were replaced three times after stirring for at least 2 h. The
product was recovered after dialysis (3500 MWCO) and dried in vacuo
to aﬀord dry yellow oil (281.1 mg). Since the 19 F NMR spectra (see Figure 1) indicated that there were still some unreacted polymer, crude product was redissolved in N,N-dimethylformamide (4 mL), along with more
peptide (31 mg), and diisopropylethylamine (1 mL), and stirred at 40 °C
under nitrogen ﬂow for another 24 h. A reduction in the ﬂuorine signal

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was observed. Propargylamine (15 µL, 6 equiv.) was then added to the reaction mixture to react all unreacted pentaﬂuorophenyl groups for 24 h.
The disappearance in polymer-bound ﬂuorine signal (broad, Figure 1) indicated that the polymer-bound pentaﬂuorophenyl group has been fully
substituted. The reaction was then stopped and precipitated onto hexane/ether (1:1), centrifuged, and dried in vacuo. The product was dialyzed in dichloromethane/ethanol (1:1; v/v). Yellow pale solid (292 mg)
PEG43 -b-P(NIPAM21 -co-t-BocAEA9 -co-peptide1.5 ) was obtained after drying in vacuo.
Polymer
Synthesis—Tert-Butyloxycarbonyl(t-Boc)-Deprotection
and PDMI-PFP Conjugation (LLD polymer): PEG43 -b-P(NIPAM21 co-t-BocAEA9 -co-FSRPAFL1.5 )
(100
mg)
was
dissolved
indichloromethane/triﬂuoroacetic acid (1:1, v/v, 12 mL) was added tothe solution drop wise on an ice bath. Stirring then continued at room
temperature for 3 h. The complete deprotection of t-Boc was conﬁrmed by
1 H NMR. After 3 h, the reaction mixture was dried and washed azeotropically using dichloromethane (5 × 6 mL) to remove the residual acid.
After ﬁnal drying in vacuo, PEG43 -b-P(NIPAM21 -co-AEA9 -co-peptide1.5 )was obtained as a pale yellow solid (128 mg). PEG43 -b-P(NIPAM21 -coAEA9 -co-peptide1.5 ) (50 mg) and PDMI-PFP (43 mg) were suspended in
peptide-grade N,N-dimethylformamide (5 mL). Diisopropylethylamine(1 mL) was then added to the suspension and stirred at 40 °C under
nitrogen ﬂow. After 2 days, the crude reaction mixture was puriﬁed using
size exclusion chromatography with N,N-dimethylformamide as theeluent. The product was then dialyzed in dichloromethane/ethanol (1:1,v/v). The product was then concentrated in vacuo to aﬀord red solid(12.3 mg). 1 H–13 C HSQC NMR spectrum of PEG43 -b-P(NIPAM21 -coPDMI9 -co-peptide1.5 ) was obtained using dimethylformamide-d6 (seeFigure S3, Supporting Information). Mn, GPC = 18 300 g mol−1 ; Mn , NMR= 12 680 g mol−1 ; Ð = 1.16.
Polymer Synthesis—Peptide Conjugation (HLD Polymer): PEG43 -bP(NIPAM21 -co-t-BocAEA9 -co-PFP4 ) (125 mg, 1 equiv.), FSRPAFL peptide(114 mg, 6 equiv.), and diisopropylethylamine (100 µL, excess) were dissolved in peptide-grade N,N-dimethylformamide (5 mL) and stirred for
3 days at 40 °C under nitrogen ﬂow. The reaction mixture was then dialyzed in a mixture of dichloromethane/ethanol (1:1). Solvents were replaced three times after stirring for at least 2 h. The product was recovered after dialysis and dried in vacuo to aﬀord PEG43 -b-P(NIPAM21 -co-tBocAEA9 -co-peptide4 ) as a yellow oil (201 mg). Mn, GPC = 17 800 g mol−1 ;Mn, NMR = 9336 g mol−1 ; Ð = 1.01.
Polymer Synthesis—Tert-butyloxycarbonyl (t-Boc)-Deprotection and
PDMI-PFP
Conjugation(HLD
Polymer): PEG43 -b-P(NIPAM21 co-t-BocAEA9 -co-FSRPAFL4 )
(201
mg)
was
dissolved
indichloromethane/triﬂuoroacetic acid (1:1, v/v, 12 mL) was added tothe solution dropwise on an ice bath. Stirring then continues at room
temperature for 3 h. The complete deprotection of t-Boc was conﬁrmed by
1 H NMR. After 3 h, the reaction mixture was dried and washed azeotropically using dichloromethane (5 × 6 mL) to remove the residual acid.
After ﬁnal drying in vacuo, PEG43 -b-P(NIPAM21 -co-AEA9 -co-peptide4 )was obtained as a pale yellow solid (185 mg). PEG43 -b-P(NIPAM21 -coAEA9 -co-peptide4 ) (97 mg) and PDMI-PFP (71 mg) were suspended in
peptide-grade N,N-dimethylformamide (10 mL). Diisopropylethylamine(0.1 mL) was then added to the suspension and stirred at 40 °C under
nitrogen ﬂow. After 2 days, the crude reaction mixture was puriﬁed using
size exclusion chromatography with N,N-dimethylformamide as theeluent. The product was then dialyzed in dichloromethane/ethanol (1:1,v/v). The PEG43 -b-P(NIPAM21 -co-PDMI9 -co-peptide4 ) product was thenconcentrated in vacuo to aﬀord red solid (200 mg). Mn , GPC = 18 920 g
mol−1 ; Mn , NMR = 12 678 g mol−1 ; Ð = 1.02.
Polymersome Formation: In a typical experiment, a 4 mL sample tube
equipped with a magnetic stir bar (2.5 mm length × 5 mm width) was
charged with 0.5 mg of PEG43 -b-P(NIPAM21 -co-PDMI9 ) and PEG43 -bP(NIPAM21 -co-t-BocAEA9 -co-peptide1.5/4 ).[20] Stock solution of PEG43 -bP(NIPAM21 -co-PDMI9 ) was prepared using neat tetrahydrofuran, while
polymer was ﬁrst dissolved in N,N-dimethylformamide (10% (v/v) of ﬁnal
volume) before addition of tetrahydrofuran. Small polymersomes populations were formed by using 500 µL of total tetrahydrofuran (or 10% (v/v)
N,N-dimethylformamide in tetrahydrofuran), while large polymersomes

Adv. Healthcare Mater. 2020, 2000261

were formed using 800 µL of the organic solvents. Gentle shaking was applied to homogenize the solution before water (500 µL) was added directly
into the polymer solution in a single portion. The mixture was then stirred
at room temperature in the fume hood (100 rpm) to allow the evaporationof tetrahydrofuran (≈16–20 h). Samples containing polymer were dialyzed
against water to remove N,N-dimethylformamide from the polymersome
solutions.
Synthesis of PEG Particles: PEG particles were prepared based ona mesoporous silica (MS) templating method according to a previous
protocol.[23,36,38] MS templates with an average size of 400 nm were synthesized using polyelectrolyte-surfactant complexes as templates according to a previously reported method.[39] For ﬂuorescence labeling, PEG
particles were labeled with AF647. The number of particles were counted
by ﬂow cytometry (Apogee A50-Micro ﬂow cytometer, UK) before adding
to cells.
Transmission Electron Microscopy: TEM measurements were carriedout on an FEI Tecnai G2 20 TEM operating at an accelerating voltage of
200 kV. Images were acquired using a BM Eagle 2K CCD Camera. Samples were prepared by dropping 10 µL of polymersome solution (0.2 mg
mL−1 ) onto a formvar-coated carbon grid. The drop was immediately blotted with a pre-cut ﬁlter paper to leave behind a thin ﬁlm of sample before
being dried using a gentle stream of nitrogen. No staining was used.
Cytotoxicity Studies: In order to assess the biocompatibility of thesepolymersomes, cytotoxicity proﬁles of the particles were determined using
AlamarBlue Assay. DAOY cells in high-glucose Dulbecco’s modiﬁed Eagle medium (DMEM) containing 10% fetal bovine serum and l-glutamine(1000 cells per 100 µL) were seeded into 96-multiwell tissue culture plates
and allowed to adhere overnight. The following day, polymersomes of
0.025 mg mL−1 , 2.5 µg mL−1 , 0.25 µg mL−1 , 25 ng mL−1 , and 2.5 ng
mL−1 concentrations were prepared by dissolving the stock solution (1 mg
mL−1 ) in DMEM. After removal of cell media, the polymersome solutions(100 µL) were pipetted onto the wells in triplicate. A solution of cell culture media devoid of any polymersomes serves as a control. The cells
were incubated at 37 °C with 5% CO2 . 72 h post-treatment the polymersomes were removed and replaced with fresh alamarBlue reagent (10%in DMEM cell culture media). The cells were incubated for another 6 h at
37 °C with 5% CO2 before subjected to ﬂuorometric measurements using a
microplate spectrophotometer at 570 nm (reference wavelength 595 nm).
IC50 values were calculated using GraphPad Prism 7 (see Figure S8, Supporting Information). Since the highest tested concentration for the HLDpolymersomes was toxic for the cells at 72 h, the test was repeated with a
2 h incubation. Results from this test indicated that the cells were viable
at this time point (see Figure S8C, Supporting Information).
Flow Cytometry: Cellular uptake of the polymersomes was assessedusing a ﬂow cytometry as previously described.[22] Cells in DMEM media(DAOY 200 000 cells per mL) were seeded onto a 6-well tissue culture
plates and allowed to adhere overnight. The following day, the cell culture media was replaced with polymersomes solutions (0.05 mg mL−1 )that were prepared by diluting the stock solutions (1 mg mL−1 ) with cell
culture media. The cells were incubated at either 37 °C with 5% CO2 or
4 °C in a cold room. After 2 h, the treatment solutions were removed
and the cells were washed with PBS (1 mL). After aspiration of PBS, cells
were treated with Trypsin (2 mL) for 2 min, neutralized with PBS (4 mL),and centrifuged for 1 min (1200 rpm). After removal of the supernatant,cells were re-suspended into PBS containing 2 mm of ethylenediaminetetraacetic acid (EDTA) and 2% fetal bovine serum (350 µL). Cell uptake wasmeasured using BD FACSCanto benchtop cell analyzer. Percentage cellular
uptake was calculated and graphed using GraphPad Prism 7 (see Figure
S11, Supporting Information).
Confocal Microscopy: Cellular uptake of the polymersomes in medulloblastoma cells was investigated using live cell imaging microscopy
technique. Confocal microscope analyses were performed on a Zeiss 880,
equipped with a 63.0 × 1.40 NA oil immersion objective. Fluorescence
images were recorded using a 488 laser with a detector wavelength rangeof 493–674 nm. Image processing was performed with FIJI, an open
source image processing package based on ImageJ. Cells in culture media(DAOY 20 000 cells per mL) were seeded onto a 6-well glass-bottom
tissue culture plate (20 mm) and allowed to adhere overnight. Cells were

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www.advhealthmat.deincubated with 0.025 mg mL−1 of polymersome samples (a concentrationthat was conﬁrmed to be non-toxic toward the cells, (see Figure S10,
Supporting Information) for 2 h and imaged on a confocal microscope. An
increase in particle uptake was observed after a longer incubation periodof 4- and 8-h (data not shown). For the purpose of this study, 2 h period
was selected as both a convenient time point as well as to ensure thatthe measured cellular uptake was not a result of particle oversaturation.
The images in Figure S10, Supporting Information, showed that all of
the polymersome samples were taken up by the cell and internalized
into the cytosolic compartment (red). Minimal localization inside thenucleus was observed as indicated by the hollow appearance in thecenter of the cells (Figure S10, Supporting Information). A similar cellular
uptake proﬁle was observed across all the samples regardless of peptide
functionalization.
Human Blood Preparation: Fresh whole blood was collected from ahealthy human donor after obtaining informed consent in accordancewith The University of Melbourne Human Research Ethics approval(No. 1 443 420) and the Australian National Health and Medical ResearchCouncil Statement on Ethical Conduct in Human Research. Blood was
drawn by venepuncture into Vacuette collection tubes containing sodium
heparin (Greiner Bio-One) and gently inverted ﬁve times. Cell counts
were obtained using a CELL-DYN Emerald analyzer (Abbott). To prepare
washed blood, 3 mL whole blood was topped up with 47 mL PBS andcentrifuged at 950 × g for 10 min, slow brake. The supernatant was removed and this wash step was repeated ﬁve times. The absence of plasma
proteins was conﬁrmed by the lack of absorbance of the supernatant at
280 nm (Nanodrop 2000, ThermoFisher Scientiﬁc, Australia). Blood cells
were resuspended in serum-free RPMI 1640 media (ThermoFisher) at thesame cell concentration as whole blood.
Blood Association Assay: Whole or washed blood (100 µL) was introduced into FACS tubes and incubated at 37 °C for 10 min according topreviously reported method.[37] Small polymersomes (non-peptide bearing and 100% HLD) were added directly at 1 µg mL−1 concentration andincubated for 1 h at 37 °C, before placing all tubes on ice. Erythrocytes werelysed with 1X Pharm Lyse buﬀer (BD Biosciences). WBCs were pelleted(500 g, 7 min.) and washed twice with PBS. Cells were phenotyped on ice(1 h) using optimized concentrations of ﬂuorescently tagged antibodies to
identify the following white cell populations: T cells (antibody CD3- AF700,
BD Biosciences, clone SP34-2), monocytes (antibody CD14-APC-H7 BD
Biosciences, clone MϕP9), neutrophils (antibody CD66b-BV421, BD Biosciences, clone G10F5), B cells (antibody CD19-BUV395, BD Biosciences,clone SJ25C1), NK cells (antibody CD56-PE, BD Biosciences, clone B159),
and a cocktail of antibodies to identify dendritic cells (lineage-1 FITC antibodies BD Biosciences and HLA-DR PE-CF594 antibody, BD Biosciences,clone G46-6). The stained cells were washed twice in cold (4 °C) PBS/2 mm
EDTA/0.5% BSA, and ﬁxed (1% v/v formaldehyde in PBS). The subpopulations of white blood cells associating with the perylene-labeled polymersomes was measured by ﬂow cytometry (LSRFortessa, BD Biosciences)
and analyzed using FlowJo V10 with cell types identiﬁed using the gating
strategy shown in Supporting Information (see Figure S15, Supporting Information).
Statistical Analysis: The statistical two-way ANOVA (repeated measures with both factors) analysis reported in this manuscript was generated using GraphPad Prism version 7 for Windows, GraphPad Software,
San Diego California USA (www.graphpad.com). This statistical analysis
was performed for all the ﬂow cytometry cellular association experimentsthat utilized a Tukey’s multiple comparison test. Error bars presented in
the ﬁgures were reﬂective of the standard error of the mean based on three
independent measurements collected for each individual parameter. Signiﬁcance was presented in terms of p values, which denoted the conﬁdence
interval of the diﬀerences between two means.

Supporting Information
Supporting Information is available from the Wiley Online Library or fromthe author.

Adv. Healthcare Mater. 2020, 2000261

Acknowledgements
M.K., F.C., S.J.K, and P.T. were in part supported by the Australian ResearchCouncil Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036) which also provided a PhD scholarship to K.T. This work was supported by grants from the Ross Trust Foundation (J.A.M.) and Steadfast Foundation (J.A.M.). J.A.M. was in part supported by funding from the Olivia Lambert Foundation. F.C. acknowledges
the award of a National Health and Medical Research Council Senior Principal Research Fellowship (GNT1135806). The authors thank the staﬀ ofMark Wainwright Analytical Centre at UNSW.

Conﬂict of Interest
The authors declare no conﬂict of interest.

Keywords
cancer drug delivery, multivalency, nanomedicines, peptides, polymericnanoparticles, targeting ligands
Received: February 17, 2020
Revised: April 20, 2020
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