Straightforward access to biocompatible poly(2-oxazoline)-coated nanomaterials by polymerization-induced self-assembly

Article abstract of DOI:10.1039/c9cc00407f

We report the synthesis of poly(2-ethyl-2-oxazoline)-based (PEtOx) nanoobjects by polymerization-induced self-assembly (PISA). First, well-defined PEtOx macromolecular chain transfer agents were synthesized by cationic ring-opening polymerization and clic

Full text of DOI:10.1039/c9cc00407f

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DOI: 10.1039/C9CC00407F
Poly(2-ethyl-2-oxazoline) chain transfer agents are employed in photoinitiated RAFTPISA, providing
access to biocompatible core-shell polymeric nanostructures with various morphologies.

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Straightforward access to biocompatible poly(2-oxazoline)-coated
nanomaterials by polymerization-induced self-assembly
Dao Le,‡ Friederike Wagner, Masanari Takamiya, I-Lun Hsiao, Gabriela Gil Alvaradejo, Uwe
ab
‡ab
a
ac
ab
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
ab
Strähle, Carsten Weiss, Guillaume Delaittre *
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report the synthesis of poly(2-ethyl-2-oxazoline)-based (PEtOx)
Polymerization-induced self-assembly (PISA), developed in
nanoobjects by polymerization-induced self-assembly (PISA). First, the last decade, has become an important approach for the
well-defined PEtOx macromolecular chain transfer agents were fabrication of ABCP NPs with elaborate and precise
synthesized by cationic ring-opening polymerization and click macromolecular architecture, colloidal morphology, and
chemistry. The photoinitiated PISA of 2-hydroxypropyl functionality.17–23 In a nutshell, PISA is based on two events: (i)
methacrylate mediated by these PEtOx produced nanoobjects chain extension of a solvophilic macromolecular precursor with
spanning the full range of core-shell morphologies. The a monomer forming a solvophobic block and (ii) synchronous
nanoparticles exhibited high biocompatibility and stealth property self-assembly of the so-formed amphiphilic block copolymer
in vitro or in vivo, as well as thermoresponsive behavior.
leading to core-shell NPs. As opposed to the classic
nanoprecipitation–solvent exchange/evaporation method, PISA
can be performed at high polymer concentration (up to 50 vs.
Poly(2-alkyl/aryl-2-oxazoline)s (PAOx), synthesized by ring-
opening cationic polymerization (CROP), are valuable polymers
for the biomedical field thanks to their biocompatibility, stealth
behavior, and their chemical and physical versatility.1–4
Coinciding with the recently renewed interest in PAOx, the first
poly(2-ethyl-2-oxazoline) (PEtOx) conjugate already entered
Phase II clinical trials for treatment of Parkinson´s disease.5
Several studies focused on the synthesis of functional PAOx to
1
–2 wt%) and gives in situ access to morphologies as diverse as
plain spheres, rods/fibers, and hollow vesicles. Reversible-
deactivation radical polymerization, mostly reversible addition-
fragmentation transfer (RAFT) polymerization, is the major
underlying polymerization mechanism for PISA, occurring under
either dispersion or emulsion polymerization conditions with
styrene, (meth)acrylate, or (meth)acrylamide monomers. Other
polymerization mechanisms are slowly making an appearance
6
–8
9
achieve functional hydrogels, surface coatings, and polymer-
10,11
peptide/protein/drug conjugates.
Besides, the interest in
in the PISA realm, such as ring-opening metathesis
PAOx-based complex structures is also raising.12 PEtOx brush-
arm star and comb-shaped polymers incorporating nitroxide
radicals and perfluorinated segments, respectively, are being
24–27
polymerization,
in order to expand the chemistry and
28
functionality of NPs. However, because of the interest in
greener solvent and of the general requirement for polar
solvents to produce biocompatible nanoparticles (i.e., alcohols
and water), employment of ionic polymerization is still
1
3,14
reported for magnetic resonance imaging.
Patterned PEtOx
bottle-brush copolymer brushes on diamond were reported for
15
biosensor design.
Notably, PAOx-based core-shell
29
limited. Therefore, many interesting biocompatible polymers
nanoparticles (NPs) are promising nanocarriers for
such as PAOx have not yet been included in PISA nanoobjects.
In this study, we describe the first combination of CROP and
RAFTPISA to obtain PAOx-based nanoobjects spanning a wide
range of morphologies. First, dithiobenzoate-functionalized
PAOx were prepared by CROP of a 2-oxazoline monomer in
aprotic solvent, followed by capping to introduce the chain
transfer agent (CTA) moiety necessary for RAFT polymerization.
The functional PAOx were then used as macromolecular CTA
1
6
biomedicine. Until now, these have been exclusively prepared
by classic self-assembly of pre-synthesized amphiphilic block
copolymer (ABCP).
aInstitute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology (KIT),
Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
E-mail: guillaume.delaittre@kit.edu
b
Institute for Chemical Technology and Polymer Chemistry (ITCP), Karlsruhe Institute
of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany.
c
School of Food Safety, College of Nutrition, Taipei Medical University, No.250,
(macroCTA)
in
photoinitiated
aqueous
RAFTPISA
Wuxing St., Taipei 11031, Taiwan.
(photoRAFTPISA). The influence of several parameters on the
‡
These authors contributed equally.
Electronic Supplementary Information (ESI) available: Materials, characterization morphologies was investigated, i.e., mode of synthesis of the
methods, experimental procedures, additional data, in-depth discussion of biological
data (PDF). See DOI: 10.1039/x0xx00000x
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Scheme 1. Synthetic routes towards PEtOx-stabilized NPs by merging CROP and photoRAFTPISA. SPTP
=
Sodium phenyl-2,4,6-
trimethylbenzoylphosphinate.
macroCTAs, chain length ratio between the solvophilic and Nevertheless, as a living character is not mandatory to obtain
3
4
solvophobic blocks, and concentration. Besides, we nanoparticles by PISA, we further employed all four
demonstrate that the obtained NPs are biocompatible in vitro macroCTAs.
and in vivo, and hence are suitable for potential bioapplications.
2-Hydroxypropyl methacrylate (HPMA) was used as core-
In particular, PEtOx was used in this study for its great forming monomer in water. Since PEtOx exhibits a lower critical
3
5
hydrophilicity and its potential as alternative to the established solution temperature in water at approx. 60 °C,
30
polyethylene glycol (PEG). PEtOx was synthesized by CROP photopolymerization at 25 °C was applied to maintain good
using methyl tosylate as initiator. As shown in Scheme 1, the solubility and ability for steric stabilization. Firstly, PISA
CTA end group could be incorporated either by direct capping experiments with the short PEtOx-CTA M1 were performed
with a deprotonated carboxylic acid-functionalized CTA (Route with varying targeted degrees of polymerization of HPMA
31,32
A)
or by a two-step sequence consisting of a nucleophilic (DPHPMA) and total solids content (Table S3). All PISA
substitution with sodium azide and a subsequent copper- experiments were conducted for 3 h and led to full HPMA
1
catalyzed azide–alkyne cycloaddition (CuAAC) (Route B). Both conversion, as verified by H NMR spectroscopy (Figure S16). As
routes showed similar kinetic features and fulfilled elementary observed by TEM, only pure spherical NPs were obtained in the
criteria for a controlled/living polymerization, i.e., linear DPHPMA = 30–100 range at 5 wt% solids (Figure S18, left). At 10
increase of pseudo-first order plot, linear increase of number- wt%, a mixture of spheres and nanofibers was obtained for
average molar mass with conversion, and clear shift of size- DPHPMA = 50 (Figure S18, right). Higher DPHPMA or solids
exclusion chromatography (SEC) traces (Figure S2). However, concentration led to precipitation. Similar results were achieved
the nature of the capping reaction impacted the dispersity of for the CuAAC-based PEtOx-CTA M3 with mainly plain spherical
the PEtOx (Đ): Đ values were systematically slightly higher when morphology but also large, possibly multicompartmented
capping with the carboxyl CTA, as compared with NaN
3
,
particles (Table S5 and Figure S22). While both short PEtOx-
concomitant with the presence of a significant shoulder at CTAs seemed to provide insufficient stabilization and led to
higher molar masses on the SEC traces (Figure S2C). In Route B, aggregation at high DPHPMA and high concentration, PISA with
reaction conditions to introduce the CTA moiety by CuAAC longer analogs allowed the formation of nanoobjects spanning
(time, solvent) also influenced the shape of the molar mass the full range of morphology. Pure spheres, worm-like
distribution (MMD), and Đ (Figure S7). Performing CuAAC for 4 nanofibers, vesicles as well as morphology mixture were
hours in dichloromethane (DCM) led to the smallest broadening obtained with M2 (Table S4 and Figure S20). Higher-order
of the MMD. This shouldering phenomenon may be – at least to morphologies evolved with increasing DPHPMA and solids
some extent – due to partial decomposition of the CTA moiety contents. However, a significant raise in dispersity was also
to a thiol, which can be readily oxidized to disulfides and observed, which is most probably due to non-reinitiating PEtOx-
produce high molar mass coupled species (Figure S5, B and C).33 CTA rather than to a loss of control. Indeed, SEC traces of PISA
Indeed, CuAAC of PEtOx-N
conditions led to a significant reduction of MMD broadening species consistent with non-functionalized PEtOx and
Figure S8). Overall, Route B required one more step but unreacted POx-CTA (Figure S19). By using the better-defined
3
with propargyl alcohol in similar samples showed important amounts of low-molecular-mass
(
provided well-defined polymers with Ɖ ≤ 1.2 (Figure S9). For PEtOx-CTA M4, strongly improved MMDs were obtained with a
each route, two batches of PEtOx-CTAs were synthesized for total consumption of POx-CTA (Figure S23). Concomitantly, PISA
–
1
PISA experiments, with M
n
of approx. 5500 and 9500 g mol . NPs displayed well-defined morphologies, as well as further
1
The characteristics of these polymers are listed in Table S1. H more intricate shapes such as donut- and jellyfish-like (Figure
NMR and SEC coupled to electrospray ionization mass 1). At low solids content (5 wt%) and with DPHPMA up to 200, only
spectrometry (SEC–ESI-MS) data ascertained high RAFT spheres or donuts were observed, as for higher solids content
functionality in all polymers (Figures S3, S4, S10, S11, and S12). with DPHPMA ≤ 100. However, a range of nanoobjects from
Chain extension experiments with benzyl methacrylate in spheres to vesicles was achieved when targeting higher DPHPMA
.
solution confirmed the previous trends: While macroCTAs from Particularly, conditions for the preparation of pure phases were
Route A underwent only partial chain extension (Figure S13), established, even for jellyfish-like objects, an intermediate
Route B led to clean block copolymers (Figures S14 and S15). stage of the nanofiber-to-vesicle morphological transformation.
2
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Figure 2. Temperature-induced morphological transformation of (A) original
nanofibers (M4-b-PHPMA125, 20 wt% at 25 °C) and vesicles (M4-b-PHPMA175
5 wt% at 25 °C) in as-synthesized dispersions, and (B) vesicles (M4-b-
,
1
PHPMA125, 0.1 wt% at 40 °C) at low concentration. Representative solutions
and TEM images in (A) from the original nanofiber sample.
were obtained (Table S7 and Figure S27) and expected maxima
absorption and emission for fluorescein and rhodamine B
(Figure S28).
To assess the biocompatibility and ability of FMA-labeled
macroRAFT agent and (bottom) TEM images of representative samples PEtOx NPs to evade clearance by the reticuloendothelial system
Figure 1. (Top) Phase diagram for RAFTPISA of HPMA using PEtOx-CTA M4 as
(empty symbols from above). Larger TEM images are provided as Figure S25. (RES), murine macrophages (RAW 264.7), human umbilical vein
endothelial cells (HUVEC), and human epithelial lung cancer
PISA with higher-molar-mass PEtOx-CTAs therefore gave better
results in terms of colloidal stability and morphology control.
There is however no clear influence of the degree of livingness
of the macromolecular CTAs on the morphological control.
cells (A549) were incubated for 24 h, using fluorescein-labeled
carboxylated polystyrene (PS-COOH) NPs as reference
38
material. RVB-labeled NPs were administered to zebrafish
embryos and their biocompatibility, biodistribution, and
clearance were examined for 24 h post-injection (hpi). As
controls, PS-COOH NPs as well as rhodamine-labeled dextran
were used in parallel. Both in vitro and in vivo studies are
reported in more detail in the Supporting Information.
3
6
In analogy to systems reported by Armes and co-workers,
phase transition of the final dispersions could be triggered by
changing temperature. At high concentration, the vesicle-to-
nanofiber-to-sphere transitions were reversible, irrespective of
the initial state, e.g., nanofibers and vesicles for M4-b-
PHPMA125 and M4-b-PHPMA175 at 25 °C, respectively (Figure
Briefly, as expected for NPs covered with stealth polymers,
PEtOx NPs show none-to-very low uptake in all cell types and
2
A). Such order-order transitions in a narrow temperature
–
1
did not reduce cell viability up to 100 μg mL (Figures S31–S33).
In the zebrafish embryos, no noticeable morphological or
behavioral change was detected after intravenous injection of
a 1 mg mL– dispersion of RVB-labeled NPs (Figure 3). In
contrast to dextran, which was rapidly cleared and only slightly
accumulated in endothelial cells in the caudal vein (CV) after 24
h (C, F, C´, and F´), RVB-PEtOx NPs mainly circulated in the
bloodstream (B, E and B´, E´). For comparison, PS-COOH NPs
were not detected in the circulation but were, as expected,
entrapped in the RES (macrophages and endothelial cells) in the
CV (D, and D´). However, contrary to the more simplistic in vitro
experiments employing murine macrophages, a small fraction
of RVB-PEtOx NPs could be detected in macrophages in vivo (E,
and E´). In summary, our PEtOx NPs are biocompatible and
display enhanced retention in the blood circulation.
Furthermore, to accurately monitor clearance by the RES, the
range are due to not only the weakly hydrophobic nature of the
3
7
core-forming PHPMA block but also the temperature-
dependent solubility of PEtOx. However, at low concentration,
e.g., 0.1 wt%, the transitions became irreversible (Figure 2B)
because the fusion of spheres or fibers is less likely under these
conditions. For instance, vesicles of M4-b-PHPMA125 obtained
by heating a concentrated solution at 40 °C and carefully diluted
at the same temperature irreversibly led to nanofibers, then
spheres by gradual cooling.
Since PEtOx is considered as an attractive alternative to PEG
for in vivo applications, we sought to preliminary assess the
biocompatibility and blood circulation behavior of the present
PEtOx-coated NPs. For imaging purposes, fluorescent NPs were
thus produced by simply adding a small amount of fluorescein
methacrylate (FMA) or rhodamine 4-vinylbenzyl ester (RVB)
comonomer to a PISA recipe (Scheme S2). Spherical NPs with
intensity-average hydrodynamic diameters of 40 and 60 nm
1
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are acknowledged for the synthesis of RVB and SPTP,
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respectively.
Conflicts of interest
There are no conflicts to declare.
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Figure 3. (A) Schematic of a 3 day-post-fertilization zebrafish embryo
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