Mapping the supramolecular assembly space of poly(sarcosine)-: B-poly(propylene sulfide) using a combinatorial copolymer library

Article abstract of DOI:10.1039/d0cc00925c

A combinatorial copolymer library was created to rapidly screen the landscape of self-assembled nanostructure morphologies formed by block copolymers composed of hydrophilic peptoid polysarcosine (PSarc) and hydrophobic poly(propylene sulfide) (PPS) block

Full text of DOI:10.1039/d0cc00925c

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Mapping the supramolecular assembly space of poly(sarcosine)-b-
poly(propylene sulfide) using a combinatorial copolymer library
Molly Frey,a Mike Vincent,b Sharan Bobbala,b Rajan Burt,b and Evan Scotta,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A combinatorial copolymer library was created to rapidly screen polymer chemistry to overcome downstream difficulties that often
the landscape of self-assembed nanostructure morphologies arise at the blood-polymer interface, principally protein adsorption
formed by block copolymers composed of hydrophilic peptoid and rapid clearance by the immune system.4 Here, we developed and
polysarcosine (PSarc) and hydrophobic poly(propylene sulfide) employed such a pipeline to evaluate and optimize the self-assembly
(PPS) blocks. By probing rationally selected hydrophilic/ of a novel poly(sarcosine)-b-poly(propylene sulfide) (PSarc-b-PPS)
hydrophobic copolymer weight fractions, the rapid and diblock copolymer as a new class of NCA-b-PPS polymer for
reproducible fabrication of micellar and vesicular nanostructures nanostructures desirable for applications in controlled drug delivery.
was optimized.
Poly(propylene sulfide) (PPS) is a non-toxic and highly versatile
hydrophobic polymer that forms stable lyotropic mesophases in
One major challenge in the development of novel self-assembled aqueous solution.5 It is often selected as the hydrophobic block for
bionanomaterials is evaluating whether block copolymer constructs self-assembled nanocarriers due to the sensitivity of its repeated
are capable of stably forming a useful nanocarrier morphology in sulfide units to oxidation that generates hydrophilic sulfoxide and
aqueous environments. The assembled morphology is highly sulfone moieties.6 These hydrophilic derivatives result in rapid
dependent on the ratio of hydrophilic to hydrophobic blocks, as well disassembly of the aggregate structure for both on-demand drug
as the specific chemistry and functionalization.1 The impact of these delivery as well as clearance of the material through the kidneys,
variables on the self-assembly process can be estimated through which is an advantage over alternative hydrophobic blocks like
thermodynamic modeling and molecular dynamics simulations. polystyrene that can instead remaining present within the circulation
However, these computational approaches are rarely capable of or tissues.5,7 Nanostructures assembled from copolymers containing
predicting the true final assembled aggregate morphology and its PPS hydrophobic blocks are able to trigger intracellular release after
associated supramolecular properties.2,3 Alternatively, an cellular uptake into oxidizing endo-lysosomes.8 Additional evidence
experimental screening pipeline could facilitate the development suggests that the PPS block can also temporarily permeabilize the
and testing of novel polymer prototypes for minimal time, cost, and lysosomal membrane and allow for cytosolic release of therapeutic
quantity. This evaluation process must be (1) comprehensive to cargo.8 Furthermore, the low glass transition temperature of PPS
minimize false negatives, and (2) rapid to quickly halt further permits rapid reorganization of hydrophobic domains during self-
development of dysfunctional prototypes that fail to meet the assembly, enabling the stable formation of diverse nanostructures
defined self-assembly criteria. Importantly, this pipeline must using scalable self-assembly techniques like flash nanoprecipitation
achieve these development goals without sacrificing the rigorous (FNP).9,10 PPS can be quickly and inexpensively synthesized through
characterization of the material properties being screened. It is with the initiation of commercially available propylene sulfide by a small-
this motivation that we sought to create a robust pipeline that molecule thiolate ion. Materials incorporating PPS polymer have
utilizes a combinatorial approach to rapidly and efficiently probe been utilized successfully in drug delivery systems and are non-toxic
nanoparticle characteristics across an otherwise extensive range of in nonhuman primates, making this hydrophobic block a promising
copolymer prototypes that varied in their hydrophilic block weight option for a novel self-assembling PSarc-based material.11
fractions. Furthermore, this approach allows for the customization of
N-carboxyanhydride (NCA) polymers are especially attractive
options for the development of biomimetic materials, since the
resulting peptide backbone formed between amino acid monomer
units closely resembles endogenous peptides in a similar capacity to
solid phase peptide synthesis. Additionally, the ring-opening
polymerization (ROP) of NCA monomers with a primary amine is
conducted under mild, easily controlled conditions to yield polymer
populations of reliable dispersity.12 The monomers themselves offer
a. Interdisciplinary Biological Sciences, Northwestern University, Evanston, IL
b. Department of Biomedical Engineering, Northwestern University, Evanston, IL
† Corresponding Author: Evan Scott, PhD, Assistant Professor of Biomedical
Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208,
evan.scott@northwestern.edu
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Fig. 1 Schematic representation for the rapid screening of PSarc-b-PPS polymer. Polymer blocks of PSarc (blue) and PPS (orange) are synthesized with an
phenylthiosulfate-cysteine linker (green) and paired across a combinatorial library (a) to cover the full range of relevant fPSarc (b). The diblock copolymers
are subjected to self-assembly thin film rehydration or flash nanoprecipitation (c). The stabilized nanostructures are characterized to determine the
morphological properties of each population (d).
a vast range of modular functionalization as each heterocyclic ring
A critical component of this strategy is the selection of a
can accommodate a unique side chain without significantly altering protected linker between the blocks that enables unrestricted
the polymerization chemistry. This allows for the incorporation of pairing of distinct chain lengths of PSarc and PPS. This will maximize
functional groups found in the 20 natural amino acids as well as a the utility of each synthesized polymer and minimize the mass of
virtually unlimited number of non-canonical derivatives.13 Finally, material needed to conduct the screen. We began exploring
polymers formed from NCA monomers maintains a primary amine potential heterobifunctional linkers beginning with small molecules
terminal end, which can be utilized for any of the existing conjugation that could potentially initiate one or both of these chains.
strategies to biological substrates or further chemical modifications. Unfortunately, a large number of these candidates failed to
Sarcosine, i.e. N-methyl glycine, is the simplest NCA-derived adequately isolate the nucleophilic requirements of amines and
monomer and conserves the broad chemical properties of peptide thiolates and limit ROP to only one end of the polymer at a time
backbones while minimizing side chain complexities. Polymerization (Table S1). As a result, the majority of our initial trials yielded
of poly(sarcosine) (PSarc) has previously been shown to resist protein homopolymers extending off both sides of the heterobifunctional
adsorption similarly to poly(ethylene glycol) (PEG)14 and effectively initiator. The inability to avoid this cross-reactivity resulted in
shield charges on nanocarriers like liposomes.15 To the best of our polymers that did not maintain initiation capability on the opposite
knowledge, diblock copolymers formed from PSarc and PPS have not end, eliminating any possible use as a linker for future steps.
been previously synthesized or employed for nanomaterial
We therefore selected a cysteine initiator with side chain
fabrication. Thus the optimal hydrophilic/hydrophobic weight protection from the phenylthiosulfate moiety, previously shown to
fractions required to reproducibly assemble useful nanostructure be selectively susceptible to thiolate ions over amines.19 Collectively,
morphologies from such a copolymer are unknown. Furthermore, this initiator offers both a primary amine on the amino end of the
previous work with self-assembling PSarc copolymers demonstrated cysteine for NCA initiation as well as a disulfide bond to end cap the
vesicle formation at hydrophilic weight fractions (fPSarc) of 0.18 thiolate of PPS chains. These three components were incorporated
[P(TPE-NAG)25-b-PSar46],16 0.54 (GA-PSar43-PMLG18),17 and 0.86 as shown in Scheme S1. The selected blocks suit a maximally efficient
(PLLA25-PSarc150).18 This formation of a single morphology from such approach given the undesirable use of phosgene to synthesize the
a wide range of fPSarc shows that distinct PSarc copolymers can have monomers and the added complexities of N-methylation on a ROP
drastically different tendencies for nanostructure self-assembly. mechanism with a secondary instead of a primary amine.20.
Using PSarc as a minimal NCA placeholder, we therefore employed a Hydrophilic PSarc chains of lengths 22 and 29 were synthesized for
pipeline to systematically establish baseline fPSarc ranges for self- this library and validated using NMR. These PSarc lengths were
assembly of distinct nanostructures from NCA-b-PPS copolymers.
Different lengths of PSarc and PPS blocks were combinatorially achieve a copolymer library with an fPSarc ranging from 0.25 to 0.52.
paired (Fig. 1a) to produce five copolymers spanning a range of fPSarc Rational selection of these weight fractions was based on our
matched with hydrophobic PPS unit lengths of 25, 35, and 62 to
using minimal material (Fig. 1b). The copolymers were subjected to prior work with the self-assembly of PEG-b-PPS copolymers.7,11 These
thin film rehydration (TF) or FNP for self-assembly (Fig. 1c). The specific ranges have known morphological significance when
resulting formulations were then characterized by dynamic light subjected to self-assembly in aqueous conditions via TF or FNP. For
scattering (DLS) to measure nanostructure diameter and example, PEG-b-PPS vesicular morphologies reliably formed via FNP
polydispersity, electrophoretic light scattering (ELS) to measure zeta around the fPEG 0.21 to 0.31 range while filamentous and spherical
potential, transmission electron microscopy (TEM) for structural micellar morphologies formed at 0.38 and higher.10 This is also
visualization, and small-angle x-ray scattering (SAXS) for an extensive consistent with previous studies showing triblock combinations of
investigation of morphological characteristics (Fig. 1d). Finally, we PEG-b-PPS polymer to form vesicles via TF at fPEG 0.20 to 0.30,
examined cytotoxicity of the formed nanostructures to ensure that cylindrical morphologies from fPEG 0.30 to 0.42, and micelles at fPEG
the baseline chemical character of PSarc-b-PPS nanomaterials do not 0.43 and above.21 In this work, we synthesized PSarc-b-PPS polymers
induce unintended cell death.
with a fPSarc between 0.25 and 0.52 for distinguishing between
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micellar, vesicular, and potentially worm-like nanostructures (Table
1, Tables S2-S6). PPS lengths were maintained between 20-75 units
to ensure retention of oxidation-responsive characteristics that we
have validated in our prior work for controlled delivery
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DOI: 10.1039/D0CC00925C
applications.22-25
2
Nanocarrier morphology is highly dependent on the method of
assembly, as the local interactions between material components
and external conditions dictate supramolecular organization. For
example, delicate filomicelle assemblies are more amenable to
gentle TF strategies while the cubic internal architecture of
bicontinuous nanospheres have been observed following high
energy regimes including FNP and sonication.21,24,26 Utilizing multiple
assembly methods allowed us to better evaluate which
nanostructure morphologies are attainable with the specified
polymer under a broa d set of formation conditions. In this way, the
pipeline provides a more complete understanding of polymer self-
assembly capability, preventing early termination of copolymer
prototypes that fail to form nanostructures under one condition but
not another. Here, all five copolymer preparations aggregated
following TF, but formed stable suspensions after FNP using the same
THF:Milli-Q water solvent system (Fig. S5 & S6, Table S6 & S7).
The FNP formulations were characterized by DLS and zeta
potential, showing a distinct trend in the development of a second
independent population as the fPSarc decreases (Fig. 2). The
formations of higher fPSarc copolymers (0.52 and 0.44) have a single,
monodisperse population from 32 to 43 nm, a range consistent with
a micellar morphology. As the hydrophilic weight fraction decreases
and the packing of polymer chains shifts, a second population around
91 to 105 nm begins to appear, indicating a second independent
morphology from the smaller peak. For all five polymer samples, the
slightly negative zeta potential of -3.82 to -8.37 mV is expected of the
PSarc-b-PPS chemistry and indicates overall structural stability.
TEM of the negatively stained soft structures shows the
existence of populations consistent with the hydrodynamic
diameters indicated by DLS (Fig. 2b-f). The micrographs further
support unique morphological characteristics between the larger
and smaller populations based on the appearance of spherical
structures with aqueous lumens, where collapse is often readily
visible. In the larger fPSarc of 0.52 and 0.44, the monodisperse smaller
diameter structures show spherical centers absent of stain,
suggestive of micelles. In the smaller fPSarc of 0.38 to 0.25, the
Fig 2 Graphical correlation between the hydrophilic weight fraction and the
resulting micelle (MC, red line) or polymersome (PS, blue line) morphology
(a). Representative TEM micrographs are shown for each copolymer sample
(b-f) with the corresponding number-weighted DLS trace (inset). SAXS
scattering profiles were overlayed with the best fitting scattering model (g-k)
along with calculated 2 values and radii (inset). These orthogonal techniques
were used to analyze polymers PSarc29-b-PPS25 (fPSarc = 0.52; b, g*), PSarc29-b-
PPS35 (fPSarc = 0.44; c, h), PSarc22-b-PPS35 (fPSarc = 0.38; d, i), PSarc29-b-PPS62 (fPSarc
= 0.31; e, j), and PSarc22-b-PPS62 (fPSarc = 0.25; f, k). Due to difficulty in staining
of fPSarc 0.52, MC are identified with red squares for improved visualization.
additional larger diameter structures among the micelles have dark
spherical centers consistent with
a
vesicular morphology
(polymersomes) having an aqueous lumen capable of collapsing and
pooling the dark uranyl formate stain during sample preparation.
SAXS with synchrotron radiation was performed on these
samples to definitively elucidate their morphological characteristics.
The calculated radii for each of the five polymer samples were again
consistent with the values found through DLS and TEM (Table 1). The
scattering profiles of samples with fPSarc of 0.52, 0.44, and 0.38 fit the
core-shell sphere model (Fig. 2g-i), presenting strong correlating
evidence for a micelle-type structure of these particles. Additionally,
the scattering profiles of samples with fPSarc of 0.31 and 0.25 fit the
vesicle model (Fig. 2j-k), suggesting the majority presence of vesicles
as the larger diameter population observed by DLS and TEM. SAXS
was particularly important for the vesicles of 0.31 weight fraction
(Fig. 2e), since the appearance of these structures deviated from the
appearance more commonly observed for negatively stained vesicles
Table 1 Parameters and characterization of PSarc-b-PPS polymers.
b
aMn was calculated from 1H-NMR. Dpeak is to describe more clearly the DLS
c
samples with multiple populations and high PDI. TEM visual identification as
micelle (MC) or polymersome (PS). dSAXS scattering for majority population; χ2
refers to best fitting model as core-shell (cs) or vesicle (v). Labels: d = diameter
(nm); PDI = polydispersity index; ZP = zeta potential (mV); dpeak = diameter of
population peaks (nm); rcore = core radius (nm); rshell = shell radius (nm).
This journal is © The Royal Society of Chemistry 20xx
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NCA-b-PPS materials but can be easily modified to investigate the
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DOI: 10.1039/D0CC00925C
supramolecular assembly space of other polymer systems. Overall,
this work significantly advances our ability to develop novel self-
assembling NCA-b-PPS block copolymers, which will help increase
the diversity of nanocarrier platforms available for meeting unique
drug delivery challenges.
Fig 3 Cytotoxicity of the representative copolymer formulations in
RAW 264.7 macrophages dosed from 0.125 mg/mL to 0.500
mg/mL. The dashed line highlights the 75% viability threshold.
Conflicts of interest
There are no conflicts to declare.
reported in this study. SAXS modeling clarified this morphological
ambiguity, with a good fit between the obtained scattering profile
and a spherical vesicle model (2 < 1; Fig. 2j).
References
Together, these orthogonal analyses converge on a cohesive
morphological description of the PSarc-b-PPS copolymer samples.
Prototype polymer formulations with a fPSarc above 0.38 resulted in a
single monodisperse population of micelles, while formulations with
a fPSarc below 0.38 result in a second majority population of
polymersomes (Fig. 2a). The polymers of fPSarc = 0.38 appeared to
exist at the border of the two morphologies, as the DLS and TEM
profiles matched those of the polymersome samples, but the SAXS
scattering profile best fit the core-shell model of the micelle samples.
Overall, this work highlights the innate differences across the
analytical methods as DLS and SAXS average across the population,
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morphological characteristics of even minor subpopulations. These
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ethyl eosin and DiI, which confirms the presence of a defined
hydrophobic space to stably retain the cargo (Table S7). As confirmed
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capable of forming diverse morphologies in aqueous media with
definitive organic and aqueous lumens, which may accommodate
the encapsulation of hydrophobic and hydrophilic therapeutics as
observed for PEG-b-PPS.
To assess the toxicity of the assembled nanostructures to
mammalian cells, we performed an MTT assay using RAW 264.7
macrophages after treatment with copolymer concentrations (0.125
- 0.50 mg/mL). These structures were largely non-toxic, as observed
by the cell viability near or above 75% in each case (Fig. 3). Future
use of these materials will require cell-specific toxicity studies
relevant to the application of interest. However, the absence of
toxicity in murine-derived macrophages, capable of extensive
nanoparticle endocytosis,27-29 gives reason to be optimistic on
cellular interactions and new directions for biomedical applications.
In summary, we developed and optimized a screening workflow
that utilizes a combinatorial library of polymer variants to minimize
the material required to comprehensively assess the self-assembling
properties of the new NCA-b-PPS polymer system. As a pilot
example, we have shown that PSarc-b-PPS is a novel self-assembling
copolymer capable of stably forming both micellar and vesicular
morphologies in aqueous media. Our extensive characterization
efforts suggest PSarc-b-PPS to potentially be a versatile diblock
copolymer platform for nanocarrier fabrication, as it is amenable to
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the assembly of diverse morphologies via
a scalable FNP
methodology. Our pipeline is particularly well-suited for evaluating
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