Poly(sarcosine)-Based Nano-Objects with Multi-Protease Resistance by Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA)

Article abstract of DOI:10.1021/acs.biomac.8b01326

Poly(sarcosine) (PSar) is a non-ionic hydrophilic polypeptoid with numerous biologically relevant properties, making it an appealing candidate for the development of amphiphilic block copolymer nanostructures. In this work, the fabrication of poly(sarcosine)-based diblock copolymer nano-objects with various morphologies via aqueous reversible addition-fragmentation chain-transfer (RAFT)-mediated photoinitiated polymerization-induced self-assembly (photo-PISA) is reported. Poly(sarcosine) was first synthesized via ring-opening polymerization (ROP) of sarcosine N-carboxyanhydride, using high-vacuum techniques. A small molecule chain transfer agent (CTA) was then coupled to the active ω-amino chain end of the telechelic polymer for the synthesis of a poly(sarcosine)-based macro-CTA. Controlled chain-extensions of a commercially available water-miscible methacrylate monomer (2-hydroxypropyl methacrylate) were achieved via photo-PISA under mild reaction conditions, using PSar macro-CTA. Upon varying the degree of polymerization and concentration of the core-forming monomer, morphologies evolving from spherical micelles to worm-like micelles and vesicles were accessed, as determined by dynamic light scattering and transmission electron microscopy, resulting in the construction of a detailed phase diagram. The resistance of both colloidally stable empty vesicles and enzyme-loaded nanoreactors against degradation by a series of proteases was finally assessed. Overall, our findings underline the potential of poly(sarcosine) as an alternative corona-forming polymer to poly(ethylene glycol)-based analogues of PISA assemblies for use in various pharmaceutical and biomedical applications.

Full text of DOI:10.1021/acs.biomac.8b01326

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Article
Poly(Sarcosine)-Based Nano-Objects with Multi-Protease Resistance by
Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA)
Spyridon Varlas, Panagiotis G. Georgiou, Panayiotis Bilalis,
Joseph R. Jones, Nikos Hadjichristidis, and Rachel K. O'Reilly
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01326 • Publication Date (Web): 23 Oct 2018
Downloaded from http://pubs.acs.org on October 27, 2018
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Poly(Sarcosine)-Based Nano-Objects with Multi-
Protease Resistance by Aqueous Photoinitiated
Polymerization-Induced Self-Assembly (Photo-
PISA)
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Spyridon Varlas,a Panagiotis G. Georgiou,a,b Panayiotis Bilalis,c Joseph R. Jones,a Nikos
Hadjichristidis,c* and Rachel K. O’Reillya*
a School of Chemistry, University of Birmingham, B15 2TT, Birmingham, UK
b Department of Chemistry, University of Warwick, Gibbet Hill Road, CV4 7AL, Coventry, UK
c
Physical Sciences and Engineering Division, KAUST Catalysis Center, Polymer Synthesis
Laboratory, King Abdullah University of Science and Technology (KAUST), 23955, Thuwal,
Saudi Arabia
*Corresponding Authors: nikolaos.hadjichristidis@kaust.edu.sa (N.H.) and
r.oreilly@bham.ac.uk (R.K.O.R.)
KEYWORDS
polymerization-induced self-assembly (PISA), ring-opening polymerization (ROP), reversible
addition-fragmentation chain-transfer (RAFT) polymerization, photoinitiated polymerization,
poly(sarcosine), protease resistance.
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ABSTRACT
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Poly(sarcosine) (PSar) is a non-ionic hydrophilic poly(peptoid) with numerous biologically
relevant properties, making it an appealing candidate for the development of amphiphilic block
copolymer nanostructures. In this work, the fabrication of poly(sarcosine)-based diblock
copolymer nano-objects with various morphologies via aqueous reversible addition-fragmentation
chain-transfer (RAFT)-mediated photoinitiated polymerization-induced self-assembly (photo-
PISA) is reported. Poly(sarcosine) was first synthesized via ring-opening polymerization (ROP)
of sarcosine N-carboxyanhydride, using high-vacuum techniques. A small molecule chain transfer
agent (CTA) was then coupled to the active ω-amino chain end of the telechelic polymer for the
synthesis of a poly(sarcosine)-based macro-CTA. Controlled chain-extensions of a commercially
available water-miscible methacrylate monomer (2-hydroxypropyl methacrylate) were achieved
via photo-PISA under mild reaction conditions, using PSar macro-CTA. Upon varying the degree
of polymerization and concentration of the core-forming monomer, morphologies evolving from
spherical micelles to worm-like micelles and vesicles were accessed, as determined by dynamic
light scattering and transmission electron microscopy, resulting in the construction of a detailed
phase diagram. The resistance of both colloidally stable empty vesicles and enzyme-loaded
nanoreactors against degradation by a series of proteases was finally assessed. Overall, our
findings underline the potential of poly(sarcosine) as an alternative corona-forming polymer to
poly(ethylene glycol)-based analogues of PISA assemblies for use in various pharmaceutical and
biomedical applications.
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INTRODUCTION
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In modern polymer and materials science, poly(ethylene glycol) (PEG) and ethylene
glycol-based polymers mainly from meth(acrylate) monomers are considered the gold
standard for the design of nanostructures and materials with “stealth-like” properties.1-3
PEG is widely utilized in various biotechnological applications such as drug/protein
delivery and targeted diagnostics,4, 5 surface coatings,6 household and personal care
products,7, 8 and cell cryopreservation.9 This is mainly attributed to the high flexibility and
hydrophilicity, low cellular toxicity and biocompatible character of PEG that make it an
efficient material for such applications.10 However, PEG is known to be a non-bio-based
and non-biodegradable polymer with limited functionality, while studies have also shown
oxidative activity of PEG in physiological cellular environments, immune response of
patients and accelerated blood clearance phenomena caused by anti-PEG antibodies.11-14
Consequently, in recent years there is a growing demand for finding alternatives to
overcome these limitations of PEG.15
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A broad variety of hydrophilic polymers including poly(glycerols),16 poly(oxazolines),17
poly(peptides)18 and poly(peptoids)19 with comparable physicochemical properties to PEG have
been currently explored and proposed as promising alternatives. Among them, poly(peptoids), an
important family of biomaterials, only recently have attracted the attention of scientific
community.19, 20 Poly(sarcosine) (PSar), also referred as poly(N-methylated glycine), is the
simplest member of the poly(peptoid) family that displays PEG-like properties and is based on an
achiral endogenous but non-proteinogenic amino acid, sarcosine, mainly found in muscle
tissues.21, 22 N-Substitution of amino acid residues in poly(peptoids) is a common synthetic
procedure to promote the random coil conformation over other secondary structures (e.g. α-helices
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and β-sheets) and to confer substantial flexibility to the polymer chains thus enhancing the
resistance of the derived materials toward enzymatic degradation.20, 23 PSar is a non-ionic,
hydrophilic, highly biocompatible and potentially biodegradable polymer that exhibits low cellular
toxicity, limited interactions with blood components and “stealth-like” non-immunogenic
character.23, 24 In addition to aqueous solubility, PSar is also highly soluble in common polar
organic solvents. Moreover, PSar can be synthesized via living ring-opening polymerization
(ROP) of the corresponding N-carboxyanhydride (NCA) to produce well-defined telechelic homo-
and copolymers with low dispersities.25, 26 Further post-polymerization modification of the living
amino-terminal chain end allows for the insertion of various functionalities and the combination
of PSar with other synthetic polymers. Overall, the unique features of PSar suggest that it can be
effectively utilized as a PEG-alternative corona-forming block imparting higher colloidal stability
(i.e. prevention of protein-induced aggregation), minimal systemic toxicity and longer circulation
times in vivo to amphiphilic block copolymer assemblies.23
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Over the last years, studies on the synthesis and self-assembly behavior of PSar-based block
copolymers for the development of nanostructures of biotechnological interest have been
presented, although they involve the use of conventional self-assembly procedures in dilute
aqueous solutions.13, 23, 27-32 Recently, polymerization-induced self-assembly (PISA) has
been established as an efficient methodology for facile one-pot fabrication of polymeric
nano-objects with predictable morphologies at high solids concentrations (10-50% w/w).33-
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Existing limitations of traditional self-assembly strategies (i.e. direct dissolution, thin-
film hydration, and solvent-switch) such as low polymer concentrations (≤1% w/w) and
laborious post-polymerization processing steps for targeting certain morphologies are
proven to be overcome by PISA. Only selected monomers have the ability to undergo PISA
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since a solubility transition of the gradually growing core-forming block from solvent-
soluble to solvent-immiscible is required.34 In principle, PISA can be achieved by using
any known polymerization technique, although the vast majority of PISA studies to date
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involve implementation of reversible-deactivation radical polymerization (RDRP)
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techniques, including atom transfer radical polymerization (ATRP),37,
nitroxide-
mediated polymerization (NMP),39, 40 and reversible addition-fragmentation chain-transfer
(RAFT) polymerization,41-44 under either dispersion or emulsion polymerization
conditions, owing to their versatility and broad applicability. Ring-opening metathesis
polymerization (ROMP) has been also introduced lately as a non-radical approach for PISA
in both aqueous and organic media.45-47
In particular, there has been rapidly growing research interest in aqueous RAFT-mediated
photoinitiated PISA (photo-PISA) via either the well-described “photoiniferter”
mechanism of chain transfer agents (CTAs) or by using special photoinitiators and
photoredox catalysts under ultraviolet or visible-light irradiation.42, 48-54 This is mainly
attributed to the ambient temperatures and mild reaction conditions of photo-PISA that are
not harmful to sensitive biomolecules (i.e. drugs, enzymes, membrane proteins) enabling
their direct encapsulation into well-defined polymeric vesicles for the one-pot development
of cargo-loaded delivery vehicles and nanoreactors of further bio-related interest.55-59
Herein, the preparation of diblock copolymer nano-objects with different morphologies bearing
a PSar hydrophilic corona via a combination of NCA ROP and RAFT-mediated photo-PISA is
reported. First, sarcosine NCA (Sar NCA) was synthesized from the corresponding N-Boc-
protected amino acid. Living ROP of Sar NCA using high-vacuum techniques and an amino
initiator yielded an ω-telechelic PSar homopolymer with monomodal molecular weight
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distribution and low dispersity (ĐM). Due to the absence of side chain functional groups
found in poly(peptides), quantitative coupling between an acid-functionalized small
molecule CTA and the sterically accessible N-terminus of PSar was achieved, resulting in
the formation of a water-soluble PSar macromolecular CTA (macro-CTA). As a next step,
PSar macro-CTA was used to carry out aqueous photo-PISA reactions of 2-hydroxypropyl
methacrylate (HPMA) under dispersion polymerization conditions by varying PHPMA
degrees of polymerization and total solids concentration (Scheme 1). The obtained PSar-b-
PHPMA diblock copolymer assemblies were characterized by dynamic light scattering
(DLS), zeta-potential analysis and transmission electron microscopy (TEM) imaging, resulting in
the construction of a morphologies diagram. To further explore the robust nature and PEG-like
characteristics of PSar-based nano-objects, the colloidal stability of PSar-b-PHPMA unilamellar
vesicles in physiological media and the effect of a series of typical proteolytic enzymes (i.e. a-
chymotrypsin, trypsin and pepsin) on empty and protein-loaded vesicles were evaluated
revealing the great potential of such materials for biomedical applications.
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Scheme 1. Schematic illustration of the synthetic route followed for the synthesis of PSar59-
b-PHPMAn (n = 100, 200, 300, and 400) diblock copolymer nano-objects at [solids] = 10, 15,
and 20 wt% via aqueous RAFT-mediated photo-PISA, using a PSar59-based macro-CTA.
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EXPERIMENTAL SECTION
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Materials and methods
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Materials and characterization techniques used are given in detail in the Supporting Information
(SI).
Synthetic Procedures
Synthesis of sarcosine N-carboxyanhydride (Sar NCA). Boc-N-methylglycine (Boc-Sar-OH)
(7.0 g, 0.037 mol) was added into a three-neck round-bottom flask and degassed under vacuum
overnight. Then, 300 mL of dry DCM was distilled and the solution was left under stirring for 30
min at 0 °C. Subsequently, phosphorous trichloride (PCl3) (4.0 mL, 0.046 mol) was diluted into
30 mL of dry DCM, added dropwise to the flask via a dropping funnel and the reaction was left to
proceed for 4 h at 0 °C under nitrogen atmosphere. The solvent and the volatiles were removed
under reduced pressure, yielding a yellowish oil as the crude reaction product. The crude product
was then sublimated at 70 °C under high vacuum (10−5 mbar) resulting in the formation of Sar
NCA crystals (3.1 g, 0.027 mol, 73%, m. p.: 103–104 °C (lit.: 102−105 °C)).60 1H-NMR (400
MHz, DMSO-d6): δ (ppm) 4.23 (s, 2H, CH2), 2.87 (s, 3H, CH3). FT-IR (neat): ν (cm-1) 1848, 1761
(C=O).
Synthesis of 4-cyano-4-[(ethylsulfanylthiocarbonyl)sulfanyl] pentanoic acid (CEPA). A
previously described process was followed for the synthesis of 4-cyano-4-
[(ethylsulfanylthiocarbonyl)sulfanyl] pentanoic acid chain transfer agent (CEPA CTA).61 In
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particular, sodium ethanethiolate (10.0 g, 0.119 mol, 1 eq) was suspended in 500 mL of dry diethyl
ether at 0 °C. Carbon disulfide (7.74 mL, 0.131 mol, 1.1 eq) was subsequently added dropwise
over 10 min, resulting to the formation of a thick yellow precipitate of sodium S-ethyl
trithiocarbonate. After 2 h of stirring at room temperature, solid iodine (15.1 g, 0.059 mol, 0.5 eq)
was added to the reaction medium. After 2 h, the solution was washed three times with aqueous
sodium thiosulfate (1 M), water and finally saturated NaCl solution. The organic layer was
thoroughly dried over MgSO4 and the crude bis-(ethylsulfanylthiocarbonyl) disulfide was then
isolated by rotary evaporation (16.2 g, 0.059 mol).
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A solution of bis-(ethylsulfanylthiocarbonyl) disulfide (16.2 g, 0.059 mol, 1 eq) and 4,4′-azobis(4-
cyanopentanoic acid) (ACVA) (24.8 g, 0.0885 mol, 1.5 eq) in 500 mL ethyl acetate was heated at
reflux for 18 h under N2(g) atmosphere. Following rotary evaporation of the solvent, the crude
CEPA CTA was isolated by flash column chromatography using silica gel as the stationary phase
and 75:25 DCM-petroleum ether as the eluent. The isolated product was precipitated out of
solution using hexane, leaving a yellow-light orange solid. The final product was collected and
dried under reduced pressure to afford pure CEPA CTA (21.36 g, 0.081 mol, 69%). 1H-NMR (400
MHz, CDCl3): δ (ppm) 3.35 (q, 2H, S-CH2-CH3), 2.38-2.71 (m, 4H, CH2-CH2), 1.89 (s, 3H,
C(CN)-CH3), 1.36 (t, 3H, S-CH2-CH3). 13C-NMR (100 MHz, CDCl3): δ (ppm) 217.0, 177.2,
119.2, 46.5, 33.5, 31.7, 29.5, 25.0, 12.9. FT-IR (neat): ν (cm-1) 2235 (CN), 1709 (C=O), 1073
(C=S). HRMS: m/z [C9H13NO2S3+Na]+ calc. 286.0001 g mol-1, exp. 286.0001 g mol-1.
Synthesis of poly(sarcosine)59 (PSar59) via ring-opening polymerization (ROP) of Sar NCA.
In a flame-dried round bottom flask, n-hexylamine (0.048 mL, 3.6×10-5 mol) was added, followed
by distillation of 30 mL highly pure DMF. The flask was transferred to the glove box and a 10 mL
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solution of Sar NCA (2.9 g, 0.0252 mol) in DMF was added and the solution was vigorously stirred
at room temperature. Periodically, the solution was pumped to remove the CO2 (g) produced
during polymerization. The consumption of Sar NCA was monitored by FT-IR spectroscopy
through removal of an aliquot of the solution in the glove box. Upon completion of the
polymerization reaction (4 h), the final PSar homopolymer was precipitated in diethyl ether and
dried under vacuum overnight (1.5 g, 83%). 1H-NMR (400 MHz, D2O): δ (ppm) 4.49-4.05 (br m,
CH2 of PSar backbone), 3.21 (m, 2H, CH2-CH2-NH), 3.10-2.81 (br m, CH3 of PSar side chain),
1.50 (s, 2H, (CH2)3-CH2-CH2-NH), 1.29 (s, 6H, CH3-(CH2)3-CH2), 0.86 (s, 3H, CH3-(CH2)3-CH2).
Mn, NMR = 4,200 g mol−1 (DPPSar, NMR = 59). FT-IR (neat): ν (cm-1) 1641 (C=O of amide). SEC (5
mM NH4BF4 in DMF) Mn, SEC RI = 7,700 g mol−1, ĐM, SEC RI = 1.08. Mw, SLS = 5,500 g mol-1.
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Synthesis of poly(sarcosine)59-CEPA macro-CTA (PSar59-CEPA macro-CTA). PSar59-CEPA
macro-CTA was synthesized by dicyclohexylcarbodiimide (DCC) coupling between PSar59 and
CEPA CTA according to previously reported methods.42, 48, 59 Poly(sarcosine) homopolymer (Mn,
NMR = 4,200 g mol-1, PSar59) (1 g, 2.4×10-4 mol, 1 eq) was dissolved in 20 mL of dry DCM. The
resulting solution was then purged with N2 (g) for 30 min. After complete dissolution, CEPA CTA
(0.253 g, 9.6×10-4 mol, 4 eq), DCC (99 mg, 4.8×10-4 mol, 2 eq) and DMAP (5.9 mg, 4.8×10-5 mol,
0.2 eq) were added to the reaction mixture. The amide formation reaction proceeded with stirring
at room temperature for 18 h under continuous N2 (g) flow. After this period, DCC (99 mg, 4.8×10-
4 mol, 2 eq) and DMAP (5.9 mg, 4.8×10-5 mol, 0.2 eq) were added for a second time to the reaction
mixture and then stirred at room temperature for an additional period of 6 h under continuous N2
(g) flow. The solution was then filtered to remove unreacted DCC and DMAP. Following rotary
evaporation of DCM, the resulted PSar59-CEPA macro-CTA was collected by precipitation into
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250 mL of cold diethyl ether, redissolved in deionized water (DI) and dialyzed against DI water
for 48 h (dialysis membrane MWCO = 1,000 Da). The purified PSar59-CEPA macro-CTA was
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then lyophilized to give a yellowish solid as the final product (0.82 g, 1.8×10-4 mol, 76%). H-
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NMR (400 MHz, CDCl3): δ (ppm) 4.35-3.85 (br m, CH2 of PSar backbone), 3.35 (q, 2H, CH3-
CH2-S-(C=S)), 3.21 (m, 2H, CH2-CH2-NH), 3.10-2.85 (br m, CH3 of PSar side chain), 2.69 (m,
2H, CH2-(C=O)-N-CH3), 2.52-2.35 (m, 2H, C(CN)-CH2), 1.91 (s, 3H, CH3-C-(CN)), 1.49 (s, 2H,
(CH2)3-CH2-CH2-NH), 1.36 (t, 3H, CH3-CH2-S-(C=S)), 1.25 (d, 6H, CH3-(CH2)3-CH2), 0.88 (s,
3H, CH3-(CH2)3-CH2). SEC (5 mM NH4BF4 in DMF) Mn, SEC RI = 8,300 g mol−1, ĐM, SEC RI = 1.09.
Synthesis of PSar59-b-PHPMAn diblock copolymer nano-objects by aqueous RAFT-mediated
photoinitiated polymerization-induced self-assembly (photo-PISA). All photo-PISA reactions
were performed in a custom-built photoreactor setup (see the Supporting Information for details).
This ensured the polymerization solutions were only exposed to the light from the 400−410 nm
LED source placed underneath the vials (radiant flux of 800 mW@400 mA). A typical synthetic
procedure to achieve PSar59-b-PHPMA200 diblock copolymer nano-objects at 15 wt% solids
content by aqueous RAFT-mediated photo-PISA is described.42, 59 PSar59-CEPA mCTA (20 mg,
4.4×10-6 mol, 1 eq) and HPMA (128 mg, 8.9×10-4 mol, 200 eq) were dissolved in deionized water
(0.84 mL) in a sealed 15 mL scintillation vial bearing a magnetic stirrer bar. The resulting
polymerization solution was degassed by purging with N2 (g) for 15 min. The sealed vial was
incubated at 37 °C with magnetic stirring under 405 nm light irradiation for 90 min to ensure full
monomer conversion. After this period, the reaction mixture was exposed to air and allowed to
cool to room temperature. The resulting solution of particles was then diluted ten-fold in DI water
1
and purified by three centrifugation/resuspension cycles in DI water at 14000 rpm. H-NMR in
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methanol-d4 and DMF SEC analyses of the pure copolymers were performed after lyophilization
of an aliquot of particles. TEM, DLS and zeta potential analyses were performed on samples after
dilution to an appropriate analysis concentration. 1H-NMR (400 MHz, CD3OD): δ (ppm) 5.60 (br
s, OH), 4.47-4.10 (br m, CH2 of PSar backbone), 4.03 and 3.88 (br s, CH and CH2 of PHPMA
side chain), 3.63 (br s, CH of PHPMA side chain, isomer peak), 3.10-2.89 (br m, CH3 of PSar side
chain), 2.23-1.75 (br m, CH2 of PHPMA backbone), 1.50-0.75 (br m, CH3 of PHPMA backbone
and CH3 of PHPMA side chain).
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Encapsulation of HRP into PSar59-b-PHPMA400 vesicles by one-pot aqueous RAFT photo-
PISA. For the preparation of HRP-loaded PSar59-b-PHPMA400 vesicles by aqueous photo-PISA at
10 wt% solids content, a typical synthetic protocol was followed.57 PSar59-CEPA mCTA (10 mg,
2.2×10-6 mol, 1 eq) and HPMA (128 mg, 8.9×10-4 mol, 400 eq) were dissolved in deionized water
(1.14 mL) in a sealed 15 mL scintillation vial bearing a magnetic stirrer bar. Once homogeneous,
0.1 mL of a 200 U mL-1 HRP solution in DI water was added. The resulting polymerization
solution was degassed by purging with N2 (g) for 15 min. The sealed vial was incubated at 37 °C
with magnetic stirring under 405 nm light irradiation for 90 min to ensure full monomer
conversion. After this period, the reaction mixture was exposed to air and allowed to cool to room
temperature. The resulting solution of particles was then diluted ten-fold in 100 mM PB (pH = 7.0)
and purified by five centrifugation/resuspension cycles in 100 mM PB (pH = 7.0) at 14000 rpm
for the removal of unreacted monomer and free HRP enzyme.
Incubation of empty and HRP-loaded PSar59-b-PHPMA400 vesicles with different proteolytic
enzymes (proteases). Empty PSar59-b-PHPMA400 vesicles purified in 100 mM phosphate buffer
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(PB) (pH = 7.0 for a-chymotrypsin and trypsin or pH = 1.8 for pepsin) at 10× dilution (1 wt%
solids content) (2 mL) were incubated with either 25 mg mL-1 a-CT (pH = 7.0), 25 mg mL-1 trypsin
(pH = 7.0) or 25 mg mL-1 pepsin (pH = 1.8) solutions (0.2 mL) at 37 oC. Aliquots were taken over
a period of 72 h and samples were analyzed by DLS, DMF SEC and dry-state TEM imaging to
determine the effect of different proteases on the characteristics of particles.
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HRP-loaded PSar59-b-PHPMA400 vesicles purified in 100 mM PB (pH = 7.0 for a-CT and trypsin)
at 10× dilution (1 wt% solids content) (2 mL) were incubated with either 100 mM PB (pH = 7.0),
25 mg mL-1 a-CT or trypsin (pH = 7.0) solutions (0.2 mL) at 37 oC for a period of 72 h. Aliquots
were taken at 18 h and 72 h and the relative activities of particles were assessed by kinetic
colorimetric analysis using a plate reader. For the free HRP, 2 U mL-1 solutions in 100 mM PB
(pH = 7.0) (2 mL) were incubated with either 100 mM PB (pH = 7.0), 25 mg mL-1 a-CT or trypsin
(pH = 7.0) solutions (0.2 mL) at 37 oC for a period of 72 h. Aliquots were taken at 18 h and 72 h
and the relative activities of free HRP solutions were assessed in an identical manner. Pepsin was
not used in case of HRP-loaded vesicles due to significant difference in optimum pH range of the
two enzymes.
Kinetic colorimetric analyses for determination of activity of HRP-loaded PSar59-b-
PHPMA400 vesicles in presence of different proteases. Purified HRP-loaded PSar59-b-
PHPMA400 vesicles incubated with either PB, a-CT or trypsin at 20× dilution (0.5 wt% solids
content) in 100 mM PB (pH = 7.0) (120 μL) were diluted with 100 mM PB (pH = 7.0) (40 μL) in
a 96-well plate microwell. O-dianisidine (4 mM, 20 μL) was then added. Finally, a 35 wt% aqueous
solution of hydrogen peroxide (20 μL) was added and the change in absorbance at λ = 492 nm was
recorded every minute for a period of 60 min using a plate reader. In a similar process, the free
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HRP solutions incubated with either PB, a-CT or trypsin at 1 U mL-1 in 100 mM PB (pH = 7.0)
(20 μL) were diluted with 100 mM PB (pH = 7.0) (140 μL) in a 96-well plate microwell. O-
dianisidine (4 mM, 20 μL) was then added. Finally, a 35 wt% aqueous solution of hydrogen
peroxide (20 μL) was added and the change in absorbance was monitored in an identical manner.
All measurements were performed in quadruplicate.
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Colloidal stability studies of PSar59-b-PHPMA400 vesicles in physiological media. To assess
the colloidal stability of empty PSar59-b-PHPMA400 vesicles (prepared at [solids] = 10 wt%) and
their interaction with complex physiological media, a typical protocol was followed.62 FBS and
cell growth medium were first incubated at 37 oC for 15 min. Purified PSar59-b-PHPMA400 vesicles
were suspended in DI water at a final concentration of 1 wt% prior to mixing with FBS or cell
growth medium. Then, 80 μL of 1 wt% vesicles were dispersed in either 10 mL DI water or cell
growth medium or 10 mL of 1:9 FBS:DI H2O solution, with gentle agitation. The resulting particle
solutions were incubated at 37 oC and Dh changes of vesicles were monitored by DLS over a period
of 72 h.
RESULTS AND DISCUSSION
Synthesis and characterization of poly(sarcosine)59 (PSar59) homopolymer. The first
step for the preparation of PSar homopolymer involved the synthesis of the corresponding
N-carboxyanhydride of sarcosine (Sar NCA). This was achieved by cyclization reaction of
N-Boc-protected sarcosine using PCl3 as chlorinating agent for the formation of Sar NCA
ring molecule (Scheme 1). The reaction was completed in 4 h as evidenced by FT-IR
spectroscopy (Figure S2, SI). The appearance of two characteristic peaks at 1761 and 1848
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cm-1 are indicative of the v(C=O) vibrations of the Sar NCA ring. The crude product was
purified by sublimation in vacuuo resulting in the formation of Sar NCA crystals. The
successful synthesis and purification of Sar NCA was confirmed by 1H-NMR spectroscopy
in DMSO-d6 (Figure S1A, SI). Although the “Fuchs-Farthing” method is considered the
standard approach for the synthesis of amino acid NCAs (i.e. use of phosgene or derivatives
at elevated temperatures), the alternative mild synthetic procedure followed herein resulted
in highly pure final product of Sar NCA at high yield.
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Next, ω-telechelic PSar homopolymer was synthesized via living ring-opening
polymerization (ROP) of Sar NCA. The polymerization reaction proceeded under high
vacuum at room temperature for 4 h using n-hexylamine as the initiator and DMF as the
solvent, and was terminated by precipitation in diethylether (Scheme 1). Monitoring of the
ROP kinetics was carried out via FT-IR spectroscopy as evidenced by the disappearance of
the NCA peaks (1761 and 1848 cm-1) and the gradual appearance of a sharp peak at 1641
cm-1 attributed to the v(C=O) vibration of the formed poly(peptoid) amide bonds (Figure
S2, SI). SEC analysis in DMF with 5 mM NH4BF4 was carried out to get a rough molecular
weight estimate of the homopolymer as PMMA can be poor standard for PSar, while it
revealed a narrow monomodal molecular weight distribution peak of low dispersity (Mn,
SEC RI = 7,700 g mol−1, ĐM RI = 1.08) (Figure 1B).60 1H-NMR spectroscopy in D2O was used
for the determination of the average degree of polymerization (DP) of the final purified
PSar by comparing the integral ratio of the peak corresponding to –CH3 group of
hexylamine at 0.86 ppm (I0.86 ppm = 3.00) to the peak of –CH3 groups of PSar backbone at
2.81-3.10 ppm (I2.81-3.10 ppm = 177.25) (ca. DPPSar = 59, Mn, NMR = 4,200 g mol−1) (Figure S1B,
SI). Analysis of static and dynamic light scattering over a range of scattering lengths (10.7
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≤ q ≤ 23.0 μm-1) was used to estimate the weight-average molecular weight and intensity-
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weighted
hydrodynamic
radius
of
PSar59
in
DI
water
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3
〈 〉
(Figure S3, SI). The mutual
(
푀푤 = (5.50 ± 0.09) × 10 Da; 푅ℎ 푍 = (1.95 ± 0.03) nm
)
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consistency of these two measurements was then verified by reference to an empirical
formula published previously by Weber et al.60
Figure 1. (A) 1H-NMR spectrum of PSar59-CEPA macro-CTA in CDCl3. (B) Normalized SEC RI
molecular weight distributions for PSar59 homopolymer (black trace) and PSar59-CEPA macro-
CTA (red trace), along with the corresponding Mn and ĐM values. Mn and ĐM values were
calculated from PMMA standards using 5 mM NH4BF4 in DMF as the eluent.
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Aqueous RAFT-mediated photoinitiated polymerization-induced self-assembly (photo-
PISA) of PSar59-b-PHPMAn diblock copolymer nano-objects. In order to conduct aqueous
RAFT-mediated photo-PISA reactions using PSar59 as the hydrophilic steric stabilizer, a small
molecule CTA (CEPA CTA) suitable for methacrylate monomers was introduced to the PSar
chain ends to afford a PSar-based macro-CTA. This was achieved through amide bond
formation between the acid-functionalized CEPA CTA and the sterically accessible amino
end group of PSar by DCC coupling chemistry under dry conditions (Scheme 1). Quantitative
amidation efficiency (≥98%) was calculated from 1H-NMR spectroscopy in chloroform-d by
comparing the integral ratio of the peak corresponding to –CH3 group of hexylamine at 0.88
ppm (I0.88 ppm = 1.00) to the peak of –CH3 group of CTA at 1.91 ppm (I1.91 ppm = 0.98) (ca.
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1
amidation efficiency (%) = (I1.91 ppm/I0.88 ppm)×100, Figure 1A). Based on H-NMR analysis,
there is approximately 2% of non-fuctionalized PSar59 homopolymer that is not separated
from PSar59-CEPA macro-CTA as they both precipitate from diethyl ether. SEC analysis of
the purified and lyophilized PSar59 macro-CTA in 5 mM NH4BF4 in DMF revealed the
successful attachment of CEPA, as judged by the small increase of molecular weight
compared to PSar59 and the characteristic absorbance of the trithiocarbonate group of the
macro-CTA peak at λ = 309 nm (Mn, SEC RI = 8,300 g mol−1, ĐM RI = 1.09) (Figure 1B).
Subsequently, the prepared water-soluble PSar59 macro-CTA was chain-extended under
RAFT dispersion PISA conditions using the well-documented water-miscible monomer 2-
hydroxypropyl methacrylate (HPMA) (mixture of 2-hydroxypropyl methacrylate - 75 mol %
and 2-hydroxyisopropyl methacrylate – 25 mol%) for the formation of the water-insoluble
core-forming block. Aqueous RAFT-mediated photo-PISA reactions of HPMA for the
fabrication of PSar59-b-PHPMAn diblock copolymer nano-objects were carried out under 405
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nm visible-light irradiation (radiant flux of 800 mW@400 mA) at 37 °C (N2 atmosphere) in
the absence of a photoinitiator or catalyst (Scheme 1). As an initial step, the required photo-
PISA reaction time to ensure complete monomer conversions was determined via kinetic
study of a PSar59-b-PHPMA400 system at 10 wt% total solids content. Aliquots were
withdrawn from the polymerization mixture every 5 min and samples were analysed by 1H-
NMR spectroscopy in methanol-d4 for monomer conversion calculation and SEC analysis
using 5 mM NH4BF4 in DMF as the eluent. As shown in Figure 2A, the photo-PISA reaction
followed pseudo-first-order kinetics separated into two regimes with quantitative monomer
conversion (>99%) achieved after 90 min of irradiation time. Based on the semilogarithmic
plot, the first regime from 0 to 25 min corresponds to growing solvent-soluble PSar59-b-
PHPMAn chains, while for the second regime a significant increase in polymerization rate
typically occurring in a PISA process was observed after approximately 25 min ascribed to a
monomer conversion of 36.5% that is attributed to the onset of particle micellization resulting
in a relatively high local HPMA concentration.33, 63 SEC monitoring during the kinetic study
revealed the linear evolution of Mn values with conversion and verified the controlled
character of the photo-PISA process, while ĐM values remained relatively low with
progression of conversion (ĐM max = 1.50), given that a high DP of PHPMA was targeted in
this case (Figure 2B).
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Figure 2. (A) Polymerization kinetics for aqueous RAFT-mediated photo-PISA of HPMA
using PSar59-CEPA as the macro-CTA at [solids] = 10 wt% (target DPPHPMA = 400) (inset:
ln([M]0/[M]) versus irradiation time kinetic plot). (B) Evolution of number-average molecular
weight (Mn) and molar mass distribution (ĐM) values with monomer conversion for aqueous
RAFT-mediated photo-PISA of HPMA using PSar59-CEPA as the macro-CTA at [solids] =
10 wt% (target DPPHPMA = 400). (C) Normalized SEC RI molecular weight distributions for
PSar59-CEPA macro-CTA (black trace) and PSar59-b-PHPMAn diblock copolymers (n = 100
- red trace, 200 - blue trace, 300 - green trace, and 400 - purple trace) at [solids] = 10 wt%,
along with their corresponding Mn and ĐM values. (D) Evolution of Mn (filled circles) and ĐM
(empty circles) values calculated from SEC RI analysis with increasing target DP of PHPMA
for PSar59-b-PHPMAn diblock copolymers prepared via aqueous RAFT-mediated photo-
PISA at [solids] = 10, 15, and 20 wt%. In all cases Mn and ĐM values were calculated from
PMMA standards using 5 mM NH4BF4 in DMF as the eluent.
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Next, a series of aqueous photo-PISA reactions under the same mild polymerization
conditions were carried out for 90 min for the development of PSar59-b-PHPMAn diblock
copolymer nano-objects of different morphologies by targeting various DPs of the core-
forming PHPMA block (DPPHPMA = 100, 200, 300, and 400) and total solids concentrations
([solids] = 10, 15, and 20 wt%). In all cases complete monomer conversion (≥98%) was
achieved in 90 min of irradiation time, as determined by 1H-NMR spectroscopic analysis in
methanol-d4 of the crude copolymer samples (Table S1, SI). The PSar59-b-PHPMAn nano-
object samples were purified by consecutive centrifugation-resuspension cycles in DI water
for the removal of unreacted monomer (Figure S4, SI). Based on SEC analysis of lyophilized
samples using 5 mM NH4BF4 in DMF as the eluent, the well-controlled character of photo-
PISA reactions at different solids content was revealed. Specifically, in all cases symmetrical
monomodal molecular weight distributions were observed shifting linearly toward higher
molecular weight (Mn) values upon increasing the DP of PHPMA with no apparent trace of
bimolecular termination (Figures 2C and S5, SI). Based on SEC RI chromatograms of PSar59-
b-PHPMAn diblock copolymers, a low molecular weight peak that corresponds to non-
separated and non-functionalized PSar59 homopolymer (~4–5% of PSar59-CEPA macro-CTA
trace in all cases, ca. 2% from 1H-NMR analysis) is observed, but since it doesn’t contribute
to RAFT-mediated chain-extensions of PHPMA or affect the overall nano-object
characteristics it was not taken into consideration for the calculation of Mn and ĐM values of
the main PSar59-b-PHPMAn diblock copolymer peak in each case (Figure S6, SI).
Importantly, for a series of samples with specified block copolymer composition (i.e. same
target DP of PHPMA) at different total solids concentration ranging from 10-20 wt%,
comparable Mn values were measured throughout. Low dispersity values were calculated
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when targeting shorter PHPMA blocks with increasing ĐM upon gradually increasing either
the DP of the core-forming block or the total solids content (Figure 2D and Table S1). This
behavior of Mn and ĐM values’ progression is typical for dispersion PISA systems.
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Exhaustive dry-state stained transmission electron microscopy (TEM) imaging along with
dynamic light scattering (DLS) and zeta-potential analyses were used for the characterization
of PSar59-b-PHPMAn block copolymer nano-objects in solution (Figures S7-S18 and Table
S2, SI). Morphologies evolving from spherical micelles (S - spheres) to worm-like micelles
(W - worms) and vesicular nanostructures (V - vesicles) along with intermediate mixed
morphologies were observed upon targeting higher DPs of the core-forming PHPMA block
and solids concentration. In particular, a mixture of spheres and short worms was obtained in
the case of PSar59-b-PHPMA100 diblock copolymer system at [solids] = 10 and 15 wt%, while
a pure phase of worms could be accessed for the same block copolymer composition at 20
wt%, as sphere-sphere fusion is more favorable at higher solids content. This was evident
macroscopically by the formation of a clear free-standing gel in the reaction vial after photo-
PISA. In all three cases, low hydrodynamic diameter (Dh) and polydispersity (PD) values were
measured ranging from 29 - 47 nm and 0.06 - 0.16, respectively, accompanied by narrow
particle size distributions. Aqueous photo-PISA reaction targeting DPPHPMA = 200 at [solids]
= 10 wt% lead to the formation of a mixed phase of all three morphologies (S+W+V), due to
coexistence of two particle populations initially indicated by DLS analysis and shown by
TEM imaging. A mixture of worm-like micelles and spherical vesicles was formed for the
same DP of PHPMA at [solids] = 15 and 20 wt%. In these cases Dh and PD values were found
to be relatively higher (Dh = 200 - 500 nm and PD = 0.13 - 0.36) compared to the ones
corresponding to PSar59-b-PHPMA100 due to existence of mixed morphologies of small and
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larger nano-objects. For DPPHPMA = 300 at [solids] = 10 wt%, an intermediate morphology
between worms and vesicles was observed, while pure vesicular morphologies were formed
at higher solids concentrations for the same target DP of PHPMA. Interestingly, a pure phase
of long tubular vesicles was detected for PSar59-b-PHPMA300 at [solids] = 15 wt% of average
Dh = 1360 nm and PD = 0.22 showing great promise for nanocarrier design applications as it
is proven that non-spherical particles exhibit longer circulation times in vivo and could be
more easily uptaken by cells.64 In the case of 20 wt% total solids content for DPPHPMA = 300,
micron-sized oligolamellar vesicles of relatively low PD were detected by dry-state TEM
imaging. More importantly, pure spherical and elongated unilamellar vesicles with Dh = 321
nm and narrow particles’ size distribution were obtained for PSar59-b-PHPMA400 formed at
10 wt% that could potentially be utilized for nanoreactor development. Finally, a mixture of
elongated tubular and multilamellar vesicles was formed in case of DPPHPMA = 400 at [solids]
= 15 wt%, while an intriguing morphology of large perforated vesicles with Dh = 1515 nm
and PD = 0.22 was observed at 20 wt% solids content. The unusual higher-order vesicular
morphologies observed at exceedingly high DPs and wt% of PHPMA are mainly attributed
to the development of significant hydrophobic interactions between the PHPMA cores and
the short hydrophobic segment of hexylamine located in front of the hydrophilic stabilizer
block of PSar that could promote further entanglement of the polymer chains and formation
of loops potentially aiding the fabrication of flower-like nanostructures.
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Based on the obtained results, a detailed phase diagram was constructed to summarize the
observed nano-object morphologies of different PSar59-b-PHPMAn formulations developed
upon varying DPPHPMA and [solids] (wt%) and to allow for the facile reproducibility of our
findings (Figure 3). Importantly, in all PSar59-b-PHPMAn nano-object formulations, zeta-
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potential values of around 0 mV (zeta-potential = -0.15 – +0.07 mV) were measured from
microelectrophoretic analysis at neutral pH that were independent of pH variations from
acidic (pH = 4.0) to basic (pH = 9.0) values (Table S2, SI), revealing the absence of net
charges on the outer surface of particles and their promising “stealth” character as they can
prevent the activation of the immune system upon insertion to the body.
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Figure 3. Detailed morphologies diagram for PSar59-b-PHPMAn diblock copolymer nano-
objects prepared via aqueous RAFT-mediated photo-PISA of HPMA by varying the total
solids content and DP of PHPMA, along with representative dry-state TEM images of
different formulations, stained with 1 wt% uranyl acetate (UA) solution. Key: S – spherical
micelles (blue), W – worm-like micelles (yellow), V – vesicles (red).
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Colloidal stability of PSar59-b-PHPMA400 unilamellar vesicles and resistance against
degradation by proteolytic enzymes. Based on the constructed morphologies diagram, the
developed PSar59-b-PHPMA400 unilamellar vesicles formed at 10 wt% solids as a pure
morphology were isolated and their potential use for nanoreactor fabrication and future in
vitro and in vivo studies was explored. Additionally, the resistance of both empty and enzyme-
loaded vesicles against a series of different proteases was also assessed.
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First, the colloidal stability of empty PSar59-b-PHPMA400 vesicles in a range of complex
media (i.e. DI water, fetal bovine serum (FBS) and cell culture medium) was evaluated, by
monitoring the Dh changes over time using DLS upon incubation at 37 °C for a total time
period of 72 h (Figure S19, SI). Not surprisingly, the average size of vesicles in DI water
didn’t change significantly over extended incubation periods ranging from 340 to 380 nm. In
the case of vesicles incubated in aqueous FBS solution, a negligible Dh increase to 405 nm
was observed after 24 h of incubation time which was more evident after 72 h (Dh = 448 nm),
indicating slow time-dependent agglomeration of particles with blood components such as
serum proteins (e.g. albumins and globulins).62 On the contrary, for empty vesicles incubated
in cell growth medium, a minor Dh decrease was observed after 24 h of incubation time to
220 nm while their size remained constant for the rest of the study. Overall, the obtained
results revealed the good colloidal properties of PSar59-b-PHPMA400 vesicles in physiological
media for prolonged time periods.
Additionally, to further assess the effect of common proteolytic enzymes on the PSar
poly(peptoid) corona of PSar59-b-PHPMA400 vesicles and the potential ability of the formed
nanostructures to act as protective cages of delicate enzymes for development of vesicular
nanoreactors, particle solutions at 10-fold dilution from original concentration were incubated
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with a series of proteases (i.e. a-chymotrypsin, trypsin and pepsin) at 37 °C and appropriate
pH for a period of 72 h (Figure 4). Structural and molecular characteristics’ changes of empty
PSar59-b-PHPMA400 were monitored by DLS and SEC analyses and dry-state TEM imaging
for determination of the ability of hydrophilic and non-ionic PSar59 stabilizer block to resist
proteolysis. Size variations of the vesicle solutions incubated with either a-chymotrypsin (a-
CT) or trypsin at pH = 7.0 and pepsin at pH = 1.8 were measured by DLS analysis (Figure
4A). In case of a-CT and trypsin, the overall dimensions of particles remained constant in the
range of 305-335 nm for the total incubation period of 72 h, while for pepsin a slight size
increase to 380 nm was monitored after 24 h mainly attributed to the exceedingly low pH
level of the solution affecting the measurements upon extended incubation time periods. Near-
identical SEC molecular weight distributions were recorded for lyophilized samples in all
three cases with Mn and ĐM values being similar to those of empty PSar59-b-PHPMA400
diblock copolymers formed by aqueous photo-PISA at 10 wt% (Figure 4B), showing no
apparent peptoid bond hydrolysis taking place. Dry-state TEM imaging of empty vesicles
after 72 h of incubation time with each protease proved that no changes in shape and size of
nano-objects occurred showing their excellent stability toward biodegradation from various
proteolytic enzymes (Figures 4C and S20, SI).
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Based on these findings, the ability of PSar59-b-PHPMA400 vesicles to protect other sensitive
hydrophilic enzymes from proteolysis by encapsulation in their inner aqueous lumen compared to
free enzymes was further investigated. Horseradish peroxidase (HRP) was selected as a model
enzyme for encapsulation into PSar59-b-PHPMA400 vesicles via a one-pot photo-PISA
methodology previously described by our group.57-59 In these studies, it was shown that such
enzymes could tolerate photo-PISA reaction conditions, retain activity and communicate with the
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external aqueous environment by passive diffusion of small molecules through the semipermeable
and relatively hydrated PHPMA membrane of vesicles providing a read-out of permeability.
Indeed, control experiments of either purging a HRP solution with N2 (g) for 15 min or exposure
to 405 nm irradiation for 90 min after N2 (g) bubbling showed no loss of enzyme activity as
compared to the untreated enzyme (Figure S21, SI). Purified HRP-loaded vesicular nanoreactors
and free HRP were incubated with either a-CT or trypsin at pH = 7.0 for 72 h, and their relative
activities were determined by colorimetric assays at λ = 492 nm over time and were normalized
against control experiments of HRP-loaded vesicles and free enzyme incubated solely with
phosphate buffer at pH = 7.0 in absence of proteases. It should be noted that pepsin was not used
for incubation of HRP-loaded vesicles as the optimum pH ranges of the two enzymes differ
significantly. As shown in Figure 4D, quantitative retention of activity was achieved in case of
HRP-loaded vesicles after 18 and 72 h of incubation time with either a-CT (96%) or trypsin (92%).
On the contrary, a significant loss of activity of 29.2% for a-CT and 41% for trypsin was noticed
in case of free HRP attributed to gradual enzyme degradation from the different proteases after 18
h. An additional activity decrease to 54.9% for a-CT and 32.9% for trypsin was measured for the
free enzyme solution after 72 h, clearly showing the robust nature and protective character of PSar-
based vesicles toward other encapsulated biomolecules.
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Importantly, when performing the described studies to determine the resistance against proteolytic
degradation of purified empty and HRP-loaded PEG113-b-PHPMA400 unilamellar vesicles of
similar size and overall characteristics previously developed by our group,42 an near-identical
retention of enzyme activity was observed in the case of HRP-loaded vesicles after 18 and 72 h of
incubation time with either a-CT or trypsin. However, a significant Dh and PD increase was
monitored in the case of empty PEG113-b-PHPMA400 unilamellar vesicles after 18 h of incubation
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time with different proteases (a-CT, trypsin or pepsin), as judged by DLS analysis, mainly
attributed to the enhanced protein-induced particle aggregation that occurs in this case after a
certain incubation period (Figure S22, SI).
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Figure 4. (A) Monitoring of hydrodynamic diameter (Dh) changes of empty PSar59-b-
PHPMA400 vesicles in 100 mM PB upon incubation with a-CT (pH = 7.0), trypsin (pH = 7.0)
or pepsin (pH = 1.8) at 37 oC for 72 h (the error bars show the standard deviation from five
repeat measurements). (B) Normalized SEC RI molecular weight distributions for PSar59-b-
PHPMAn diblock copolymers after incubation with a-CT (pH = 7.0), trypsin (pH = 7.0) or
pepsin (pH = 1.8) at 37 oC for 72 h. Mn and ĐM values were calculated from PMMA standards
using 5 mM NH4BF4 in DMF as the eluent. (C) Representative dry-state TEM images of
empty PSar59-b-PHPMA400 vesicles after incubation with a-CT (pH = 7.0), trypsin (pH = 7.0)
or pepsin (pH = 1.8) for 72 h, stained with 1 wt% UA. (D) Normalized relative activities of
HRP-loaded PSar59-b-PHPMA400 vesicles and free HRP after incubation with a-CT or trypsin
at 37 oC for 18 h (red) and 72 h (blue) (the error shows the standard deviation from four repeat
measurements). The normalized relative activities are defined as the ratio between the
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absorbance of the samples and the absorbance of untreated HRP-loaded vesicles or free HRP,
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CONCLUSIONS
To conclude, we demonstrate an efficient methodology for the fabrication of poly(peptoid)-
based block copolymer nano-objects with predictable morphologies at high concentrations
by combining living NCA ROP and aqueous RAFT-mediated photo-PISA. In particular,
poly(sarcosine) was utilized as a novel hydrophilic stabilizer block for controlled RAFT
chain-extensions of a methacrylate monomer able to undergo PISA under aqueous
dispersion polymerization conditions targeting different DPs of the core-forming block and
total solids concentrations. A diverse set of nano-object morphologies including higher-
order structures was obtained, as evidenced by the construction of a phase diagram. The
colloidal stability of single phase vesicles and their ability to encapsulate hydrophilic
enzymes protecting them from proteolysis were thoroughly assessed and compared to their
PEG-based counterparts, showing great promise for use of the developed materials in
various biomedical applications. Our findings circumvent the current limitations of
conventional block copolymer self-assembly techniques, such as dilute conditions and
multiple laborious post-polymerization processing and purification steps for targeting
certain morphologies, underlining the potential of poly(sarcosine) as an alternative corona-
forming polymer to PEG-derived polymers for fabrication of PISA nano-objects with bio-
relevant character.
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ASSOCIATED CONTENT
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Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI:
10.1021/acs.bio-mac.xbxxxxx.
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Materials and characterization methods, additional NMR, FT-IR, SEC and DLS data,
additional dry-state TEM images, HRP control experiments and colloidal stability results.
AUTHOR INFORMATION
Corresponding Authors
*Email: nikolaos.hadjichristidis@kaust.edu.sa
*Email: r.oreilly@bham.ac.uk
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
This work was supported by the ERC (grant number 615142), EPSRC and King Abdullah
University of Science and Technology (KAUST).
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