Modulated Fragmentation of Proapoptotic Peptide Nanoparticles Regulates Cytotoxicity

Article abstract of DOI:10.1021/jacs.6b11302

Peptides perform a diverse range of physiologically important functions. The formulation of nanoparticles directly from functional peptides would therefore offer a versatile and robust platform to produce highly functional therapeutics. Herein, we engineered proapoptotic peptide nanoparticles from mitochondria-disrupting KLAK peptides using a template-assisted approach. The nanoparticles were designed to disassemble into free native peptides via the traceless cleavage of disulfide-based cross-linkers. Furthermore, the cytotoxicity of the nanoparticles was tuned by controlling the kinetics of disulfide bond cleavage, and the rate of regeneration of the native peptide from the precursor species. In addition, a small molecule drug (i.e., doxorubicin hydrochloride) was loaded into the nanoparticles to confer synergistic cytotoxic activity, further highlighting the potential application of KLAK particles in therapeutic delivery.

Full text of DOI:10.1021/jacs.6b11302

Article
pubs.acs.org/JACS
Modulated Fragmentation of Proapoptotic Peptide Nanoparticles
Regulates Cytotoxicity
Tomoya Suma,† Jiwei Cui,† Markus Mullner,‡ Shiwei Fu,† Jenny Tran,† Ka Fung Noi,† Yi Ju,†
̈
and Frank Caruso*,†
†ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and the Department of Chemical and Biomolecular
Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia
‡Key Centre for Polymers and Colloids, School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
S
* Supporting Information
ABSTRACT: Peptides perform a diverse range of physiologi-
cally important functions. The formulation of nanoparticles
directly from functional peptides would therefore offer a versatile
and robust platform to produce highly functional therapeutics.
Herein, we engineered proapoptotic peptide nanoparticles from
mitochondria-disrupting KLAK peptides using a template-
assisted approach. The nanoparticles were designed to
disassemble into free native peptides via the traceless cleavage
of disulfide-based cross-linkers. Furthermore, the cytotoxicity of
the nanoparticles was tuned by controlling the kinetics of
disulfide bond cleavage, and the rate of regeneration of the native
peptide from the precursor species. In addition, a small molecule
drug (i.e., doxorubicin hydrochloride) was loaded into the nanoparticles to confer synergistic cytotoxic activity, further
highlighting the potential application of KLAK particles in therapeutic delivery.
other constituent materials is reduced, thus lowering the risk of
adverse side effects. Furthermore, the biodegradability of
peptides ultimately reduces cumulative toxicity. Recently,
peptide assemblies have been obtained by conjugation of
amino acid sequences, hydrophobic chains, or polymers,16−19
improving delivery efficiency of pharmaceutical agents.
However, engineering self-assembled peptide nanoparticles of
a specific size that are stable under normal physiological
conditions, but can disassemble under certain biological
triggers, remains a challenge.16 Furthermore, irreversible
coupling reactions reduce the bioactivity of peptides,20−23
highlighting the need for better disassembly strategies that yield
native-state peptides. Combining peptide delivery with other
therapeutics is often desired but requires additional function-
alization or loading steps, which may hamper the activity of
peptides and their self-assembly processes.16,17
Herein, we introduce a strategy to engineer functional
peptide nanoparticles, using mesoporous silica nanoparticles
(MSNs) as sacrificial templates (Scheme 1), and reversible
chemistries engineered into the peptide network (Scheme 2).
Our approach has distinct advantages in that (i) template
assembly allows the facile engineering of peptide nanoparticles
with defined sizes;24−26 (ii) small molecule drugs can be loaded
without hampering the assembly process; (iii) the disassembly
and drug release properties can be tuned through the cross-
INTRODUCTION
■
Peptides have highly sophisticated and diverse physiological
functions that remain challenging to mimic using synthetic
molecules. The roles of peptides range from signaling for
regulation of immunity, reproduction, and homeostasis1,2 to
self-defense against pathogens or predators.3 Advancements in
genetic engineering, computational design, and chemical
synthesis have allowed the creation of diverse artificial peptide
variants that have superior functionality and stability when
compared with naturally derived peptides.4−6 The number of
approved peptide-based drugs is increasing, with applications in
areas including gastrointestinal disorders, anemia, and cancer.1,5
Nevertheless, the biomedical application of peptides is still
limited because they generally exhibit poor membrane
permeability, are prone to enzymatic degradation and renal
clearance, and have limited accumulation at target sites.
Nanoparticles have shown promise as delivery carriers of
various therapeutics such as small molecule drugs, proteins,
nucleic acids, and peptides.7,8 Various peptide delivery
formulations have been developed, including liposomes,
nanogels, polyplex particles, and inorganic nanoparticles.9−11
However, challenges still remain regarding loading, stability and
controlled release of peptides from carriers.12,13
In this context, it would be advantageous to assemble
functional peptides into nanoscale carriers.14−16 In addition to
the high peptide loading, the resulting peptide nanoparticles
would offer a range of potential benefits. For example, as the
particles are assembled from bioactive peptides the amount of
Received: October 31, 2016
© XXXX American Chemical Society
A
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a
Scheme 1. Preparation of PPNs Using Template Assembly
a
KLAK peptides modified with three different disulfide-introducing reagents (i.e., 4-nitrophenyl 4-(2-pyridyldithio)benzyl carbonate (NDBC), 4-
nitrophenyl 2-(2-pyridyldithio)ethyl carbonate (NDEC), or succinimidyl 3-(2-pyridyldithio)propionate (SPDP)) were immobilized onto
mesoporous silica nanoparticles (MSNs), followed by cross-linking with 8-arm-PEG-SH and template removal, resulting in proapoptotic peptide
nanoparticles (i.e., B-PPN, E-PPN, or S-PPN, respectively).
Scheme 2. Proposed Mechanism of Disulfide Cleavage and
Fragmentation of (a) 4-Dithiobenzyl Urethane of B-PPN,
(b) 2-Dithioethyl Urethane of E-PPN, and (c) 3-
Dithiopropyl Amide of S-PPN
or dithiobis(succinimidyl propionate), are used,31 molecular
pendant groups remain on the peptides even after cleavage of
the disulfide bond (Scheme 2c). Furthermore, the presence of
such pendant groups reduces the bioactivity of the
peptides.21−23,32 In this regard, reversible disulfide chemistry,
such as 4-dithiobenzyl urethane (4-DBU)22,33,34 or 2-
dithioethyl urethane (2-DEU),31,35 undergo complete removal
from peptides after the cleavage of disulfide bonds, ultimately
regenerating unmodified amino groups (Scheme 2a and b).
Such reversibility in disulfide bond cleavage has not been
studied for nanoparticles in mammalian cells, to our knowledge.
Herein, we study the effect of disulfide bond cleavage on the
disassembly and drug release properties of peptide nano-
particles, leading to the development of new nanoparticles with
modulated functionalities. These studies will provide useful
information on the design of environment-sensitive function-
alities for future biomedical applications, such as prodrug,36
PEGylation of proteins,22,23,32 cross-linking protein par-
ticles,31,37 latent fluorophores,35 reversible activation of endo-
somolytic agents,38,39 and signal amplification for ultrasensitive
detection.40
In this study, functional peptide particles mainly composed
of KLAK peptides, referred to herein as proapoptotic peptide
nanoparticles (PPNs), were prepared (Scheme 1). A cationic
amphipathic peptide, (KLAKLAK)2 (referred to herein as
KLAK) is of interest for application in cancer therapy, because
of the selective disruption of mitochondrial membranes, and
thereby apoptosis of mammalian cells.41,42 To improve the
stability and internalization into cancer cells, KLAK peptides
have been encapsulated into liposomes,43 conjugated onto iron
oxide nanoparticles,44 chemically modified and assembled into
nanoparticles,19,45 or complexed with polyelectrolytes.46
However, engineering of the nanoparticles with tunable
geometry, disassembly, and therefore cytotoxicity has been a
challenge. Herein, we present KLAK particles with apoptosis-
promoting capabilities programmed by tunable fragmentation
linking chemistry; and (iv) the reversible functionality allows
regeneration of pristine peptides and small molecule drugs by a
biological trigger, endowing peptide nanoparticles with tunable
bioactivity.
To achieve the triggered disassembly and subsequent peptide
release, we chose disulfide-based chemistry to stabilize peptide
nanoparticles. Disulfide-based chemistry has been extensively
studied for controlled drug release within intracellular27,28 and
tumor environments29 that have an elevated chemical reducing
potential. However, when conventional conjugation reagents,
such as succinimidyl 3-(2-pyridyldithio)propionate (SPDP)30
B
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1
alcohol was obtained as a pale yellow oil (yield: 183.2 mg, 74%). H
kinetics, which are potentially useful for anticancer drug
delivery. To tune the triggered disassembly of PPNs and the
release of native bioactive peptides, three disulfide-based
reversible cross-linking strategies were compared (Scheme 2).
Interestingly, a marked difference in the cytotoxicity of PPNs
from the three different reversible disulfide linkages was
observed. To investigate the underlying mechanism, cellular
association, particle disassembly, and disulfide cleavage
mechanisms were studied with a focus on the different disulfide
bonds. Furthermore, the potential of PPNs in therapeutic
delivery applications was examined using a model anticancer
drug, doxorubicin hydrochloride (DOX). Additionally, the
effect of conjugation chemistry on the cytotoxic potency of the
DOX-loaded PPNs was evaluated. The present study provides a
framework to exploit reversible chemistry to tune the
therapeutic potency of PPNs and cargo and furthermore
demonstrates that PPNs may be a promising platform for future
application in nanomedicine.
NMR (400 MHz, CDCl3, 25 °C): 8.45 (d, 1H), 7.61 (m, 2H), 7.50 (d,
2H), 7.30 (d, 2H), 7.08 (m, 1H), 4.65 (d, 2H) (Supporting
Information, Figure S3). 13C NMR (100 MHz, CDCl3, 25 °C, δ):
159.4, 148.6, 138.1, 130.4, 129.7, 129.6, 127.9, 121.0, 120.0, 64.6
(Supporting Information, Figure S4).
4-(2-Pyridyldithio)benzyl alcohol (170 mg, 0.7 mmol) was
dissolved in anhydrous CH2Cl2 (5 mL), which was added to a
solution containing 4-nitrophenyl chloroformate (282 mg, 1.4 mmol),
pyridine (111 mg, 1.4 mmol), and a catalytic amount of DMAP in
anhydrous CH2Cl2 (5 mL). After overnight stirring, the reaction
mixture was washed with 2 N HCl (aqueous) and brine, and dried
over MgSO4. After removal of excess solvent by evaporation, the
product was purified by silica gel chromatography (ethyl acetate/
hexane = 1/4 (v/v), Rf = 0.23) to obtain 4-nitrophenyl 4-(2-
pyridyldithio)benzyl carbonate as a pale yellow solid (yield: 229.3 mg,
1
81%). H NMR (400 MHz, CDCl3, 25 °C, δ): 8.49 (d, 1H), 8.26 (d,
2H), 7.61 (d, 2H), 7.55 (d, 2H), 7.40−7.32 (m, 4H), 7.20 (m, 1H),
5.22 (s, 2H) (Supporting Information, Figure S5). 13C NMR (100
MHz, CDCl3, 25 °C, δ): 159.0, 155.4, 152.3, 148.9, 145.4, 138.0,
137.1, 133.4, 129.5, 127.5, 125.2, 121.7, 121.2, 120.0, 70.2 (Supporting
Information, Figure S6).
Synthesis of 4-Nitrophenyl 2-(2-Pyridyldithio)ethyl Carbo-
nate (NDEC). 2,2′-Dithiodipyridine (880 mg, 4.0 mmol) was
dissolved in CH2Cl2, and 2-mercaptoethanol (313 mg, 4.0 mmol)
was added. After 2 h of stirring, the excess solvent was removed,
followed by purification with silica gel chromatography (ethyl acetate/
hexane = 1/3 (v/v), Rf = 0.4) to obtain 2-(2-pyridyldithio)ethyl
EXPERIMENTAL SECTION
■
Materials. 2-Mercaptoethanol, 3-mercaptopropionic acid, 4-
mercaptobenzoic acid, lithium aluminum hydride (LiAlH4), N-
hydroxysuccinimide (NHS), 4-nitrophenyl chloroformate, N,N′-
dicyclohexylcarbodiimide (DCC), reduced L-glutathione (GSH),
tetraethyl orthosilicate (TEOS), cetyltrimethylammonium tosylate
(CTAT), triethanolamine, ammonia solution (28−30%), hydrofluoric
acid (HF), ammonium fluoride (NH4F), triethylamine (TEA), 4-
(dimethylamino)pyridine (DMAP), α-cyano-4-hydroxycinnamic acid,
3-(N-morpholino)propanesulfonic acid (MOPS), anhydrous dimethyl
sulfoxide (DMSO), anhydrous pyridine, and anhydrous dichloro-
methane (CH2Cl2) were purchased from Sigma-Aldrich (MO, USA).
2,2′-Dithiodipyridine and anhydrous tetrahydrofuran (THF) were
obtained from Alfa Aesar (MA, USA). 8-Arm-poly(ethylene glycol)-
thiol (8-arm-PEG-SH) (Mw: 10 kDa) was purchased from JenKem
Technology (Beijing, China). Alexa Fluor 488 succinimidyl ester (AF-
488-NHS), Alexa Fluor 568 succinimidyl ester (AF-568-NHS), an
Alexa Fluor 488 annexin V/dead cell apoptosis kit, Hoechst 33342,
and 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-car-
boxyanilide inner salt (XTT) were purchased from Life Technologies
(CA, USA). KLAK peptide (HOOC-KLAKLAKKLAKLAK-NH2) was
obtained from GL Biochem (Shanghai, China). DOX was purchased
from OChem Inc. (IL, USA). All chemicals were used as received
without further purification. High-purity water with a resistivity of
greater than 18.2 MΩ cm was obtained from a three-stage Millipore
Milli-Q plus 185 purification system (Millipore Corporation, MA,
USA).
1
alcohol as a pale yellow liquid (yield: 400 mg, 53%). H NMR (400
MHz, CDCl3, 25 °C, δ): 8.49 (d, 1H), 7.56 (t, 1H), 7.38 (d, 1H),
7.15−7.11 (m, 1H), 5.70 (s, 1H), 3.78 (s, 2H), 2.94 (t, 2H)
(Supporting Information, Figure S7). 13C NMR (100 MHz, CDCl3, 25
°C, δ): 159.1, 149.8, 136.8, 121.9, 121.5, 58.2, 42.7 (Supporting
Information, Figure S8).
2-(2-Pyridyldithio)ethyl alcohol (187 mg, 1.0 mmol) was dissolved
in anhydrous CH2Cl2 (10 mL), to which anhydrous pyridine (158 mg,
2 mmol) and a catalytic amount of DMAP were added. Then, 4-
nitrophenyl chloroformate (403 mg, 2.0 mmol) was added, and the
reaction mixture was stirred overnight. The mixture was washed with 2
N HCl (aqueous) and brine, followed by drying over MgSO4. After
excess solvent was removed by evaporation, silica gel chromatography
(hexane/ethyl acetate = 1/3 (v/v), Rf = 0.24) was conducted to obtain
4-nitrophenyl 2-(2-pyridyldithio)ethyl carbonate as a pale yellow oil
1
(yield: 193.5 mg, 55%). H NMR (400 MHz, CDCl3, 25 °C, δ): 8.53
(d, 1H), 8.27 (d, 2H), 7.72 (d, 2H), 7.36 (d, 2H), 7.21−7.16 (m, 1H),
4.55 (t, 2H), 3.17 (t, 2H) (Supporting Information, Figure S9). 13C
NMR (100 MHz, CDCl3, 25 °C, δ): 158.7, 155.3, 152.2, 148.4, 145.5,
138.6, 125.3, 121.7, 121.5, 121.1, 66.5, 37.0 (Supporting Information,
Figure S10).
Synthesis of 4-Nitrophenyl 4-(2-Pyridyldithio)benzyl Carbo-
nate (NDBC). NDBC was synthesized following a reported method
(Supporting Information, Scheme S1).36 To a suspension of LiAlH4
(0.74 g, 19.5 mmol) in anhydrous THF (10 mL), 4-mercaptobenzoic
acid (1.0 g, 6.5 mmol) in anhydrous THF (10 mL) was slowly added
at 0 °C. After overnight stirring, the reaction was quenched by the
addition of water (500 μL). The pH of the mixture was adjusted to 2
by the addition of 2 N HCl (aqueous; ∼ 30 mL), followed by
extraction with ethyl acetate. The mixture was washed with water and
brine, and dried over MgSO4. The product was purified by silica gel
chromatography (ethyl acetate/hexane = 1/2 (v/v), Rf = 0.30) to
obtain 4-mercaptobenzyl alcohol as a white powder (yield: 559 mg,
Synthesis of Succinimidyl 3-(2-Pyridyldithio) Propionate
(SPDP). SPDP was synthesized following a reported method
(Supporting Information, Scheme S1).30 2,2′-Dithiodipyridine (2.2 g,
9.96 mmol) was dissolved in ethanol (16 mL), to which acetic acid
(0.3 mL) and 3-mercaptopropionic acid (0.528 g, 4.98 mmol) were
added. After 2 h of stirring, ethanol and acetic acid were removed by
evaporation. Then, the resultant pale yellow oil was purified through a
basic Al2O3 column. First, CH2Cl2/ethanol = 3/2 (v/v) was flushed
through the oil-containing Al2O3 column until a colorless filtrate was
obtained. Then, CH2Cl2/ethanol/acetic acid = 60/40/4 (v/v/v) was
flushed to elute the desired product. The solvents were removed by
evaporation to obtain 3-(2-pyridyldithio)propionic acid as a pale
yellow oil (yield: 745.8 mg, 70%). 1H NMR (400 MHz, CDCl3, 25 °C,
δ): 8.50 (d, 1H), 7.75−7.66 (m, 2H), 7.20 (t, 1H), 3.06 (t, 2H), 2.79
(t, 2H) (Supporting Information, Figure S11). 13C NMR (100 MHz,
CDCl3, 25 °C, δ): 175.1, 159.0, 148.6, 138.2, 121.5, 121.1, 34.1 (2C)
(Supporting Information, Figure S12).
1
62%). H NMR (400 MHz, D2O, 25 °C, δ): 7.22 (d, 2H), 7.15 (d,
2H), 4.42 (s, 2H) (Supporting Information, Figure S1). 13C NMR
(100 MHz, CDCl3, 25 °C, δ): 138.4, 130.2, 130.0, 129.6, 127.8, 127.5,
64.8 (Supporting Information, Figure S2).
4-Mercaptobenzyl alcohol (140 mg, 1 mmol) was dissolved in
CH2Cl2 (0.75 mL) and mixed with a solution of 2,2′-dithiodipyridine
(220 mg, 2 mmol) in CH2Cl2 (0.75 mL). After overnight stirring, the
excess solvent was removed by evaporation, followed by purification
by silica gel chromatography (ethyl acetate/hexane = 1/1 (v/v), Rf =
0.35). After drying under reduced pressure, 4-(2-pyridyldithio)benzyl
3-(2-Pyridyldithio)propionic acid (690 mg, 3.2 mmol) was
dissolved in anhydrous CH2Cl2, and then NHS (440 mg, 3.8 mmol)
and DCC (784 mg, 3.8 mmol) were added. After 3 h of stirring, the
precipitate was removed by filtration, and the solvent was removed by
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evaporation. After reconstitution in CH2Cl2, the undissolved portion
was removed by filtration. The excess solvent was removed, and the
product was crystallized in ethanol at −20 °C. The obtained crystals
were dissolved in CH2Cl2, followed by crystallization in isopropanol at
−20 °C to obtain SPDP as a white powder (yield: 649.5 mg, 65%). 1H
NMR (400 MHz, CDCl3, 25 °C, δ): 8.51 (d, 1H), 7.74 (d, 2H), 7.17
(m, 1H), 3.14 (t, 2H), 3.06 (t, 2H), 2.82 (s, 4H) (Supporting
Information, Figure S13). 13C NMR (100 MHz, CDCl3, 25 °C, δ):
168.8, 166.9, 159.0, 148.6, 138.5, 121.3, 120.6, 33.0, 30.9, 25.6
(Supporting Information, Figure S14).
Preparation of MSNs. MSNs were prepared following a
previously published method.47 Briefly, CTAT (960 mg) and
triethanolamine (174 mg) were dissolved in water (50 mL) at 80
°C. Then, TEOS (7.8 mL) was quickly added to the above solution.
The mixture was stirred at 80 °C for 2 h. The synthesized MSNs were
washed with water and ethanol, dried at 80 °C, and finally calcined at
550 °C for 6 h.
Preparation of PPNs. PPNs from NDBC chemistry (B-PPNs)
were prepared as follows. The MSNs were suspended in MOPS buffer
(50 mM, pH 8.0) at a concentration of 10 mg mL−1. KLAK peptide
was dissolved in anhydrous DMSO with 1% TEA at a concentration of
10 mg mL−1. To the solution containing KLAK peptide, a solution of
NDBC (100 mg mL−1 in anhydrous DMSO) was added at a NDBC-
to-KLAK molar ratio of 4:1. After 2 h of incubation, the solution
containing the NDBC-modified KLAK peptide (60 μL) was mixed
with the MSN suspension (150 μL), followed by the addition of
MOPS buffer (700 μL, 50 mM, pH 8.0). The suspension was
incubated for 3 min and centrifuged at 1000g for 3 min, followed by
removal of the supernatant and the addition of MOPS buffer (700 μL,
50 mM, pH 8.0). This process was repeated twice. Then, 8-arm-PEG-
SH in MOPS buffer (50 μL, 50 mM, pH 8.0) was added at a SH-to-
NDBC molar ratio of 1.1:1. After 1 h of stirring, the suspension was
centrifuged at 1000g for 3 min, followed by removal of the supernatant
and the addition of MOPS buffer (700 μL, 50 mM, pH 8.0). This
process was repeated once. Finally, the MSN templates were removed
using buffered HF solution (5 M HF/13.3 M NH4F/H2O = 1/6/3,
pH ∼6.5) (Caution! Hydrofluoric acid is highly toxic. Extreme care
should be taken when handling HF solution, and only small quantities
should be prepared.), followed by three washing cycles with water. The
obtained particles were dispersed by sonication and any aggregated
particles were removed by centrifugation at 1000g for 5 min. The
process was repeated twice to obtain dispersed particles. The S-PPNs
and E-PPNs were prepared following a similar procedure, using SPDP
and NDEC, respectively, instead of NDBC.
Determination of Peptide Concentration. The peptide
concentration was determined by Lowry’s method with a slight
modification.48,49 Briefly, a solution containing an unknown
concentration of PPNs (10 μL) was mixed with a solution (100 μL)
of 0.2 M NaOH supplemented with 4% Na2CO3, 2% CuSO4, and 4%
sodium tartrate at a volume ratio of 100:1:1, followed by 10 min of
incubation. Then, 2 M Folin−Ciocalteu’s phenol reagent (10 μL) was
added, followed by 20 min of incubation. The absorbance at 750 nm
was measured using an Infinite M200 microplate reader (Tecan,
Switzerland). The absorbance at 750 nm in the absence of CuSO4 was
used as background (disulfides and sulfhydryls are known to interfere
with Lowry’s method).49 The concentration of peptide was calculated
from a calibration curve obtained using peptide solutions with known
concentrations.
solvent). For the ζ-potential measurements, a particle dispersion in
phosphate buffer (pH 7.4, 10 mM) (800 μL) was placed into a folded
capillary cell (DTS1070, Malvern Instruments). The ζ-potential was
obtained from the electrophoretic mobility and by using the
Smoluchowski equation: ζ = 4πην/ε (η: viscosity of the solvent, ν:
electrophoretic mobility, ε: dielectric constant of the solvent).
Transmission Electron Microscopy (TEM). A suspension
containing the PPNs or MSNs was casted on Formvar-coated copper
grids, followed by overnight air-drying. The grids were then washed
with pure water to remove salt and allowed to dry in air. TEM imaging
was conducted on a CM120 BioTWIN instrument (Philips, Germany)
operating at an acceleration voltage of 120 kV.
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight
Mass Spectrometry (MALDI-ToF MS). The PPNs were incubated
in phosphate buffer (pH 7.4, 10 mM) containing 5 mM GSH. After
incubation for either 2 or 24 h, the dispersions were centrifuged
(8000g, 3 min). The supernatant was collected and mixed with a
solution of α-cyano-4-hydroxycinnamic acid (10 mg mL−1) in a 1/1
(v/v) water/acetonitrile mixture containing 0.1% trifluoroacetic acid.
MALDI-ToF MS measurements were subsequently performed
(Autoflex, Bruker, MA, USA).
KLAK Peptide Release Study. The PPNs were prepared from
KLAK peptides labeled with AF-568. The PPNs (250 μL, 1000 μg
mL−1 of KLAK) were dialyzed against phosphate buffer (10 mL, pH
7.4, 20 mM) containing either 5 mM or 10 μM GSH using a dialysis
membrane with a molecular weight cutoff of 7000 Da. At each time
point, the solution outside the dialysis membrane (500 μL) was
withdrawn and replaced with the same volume of fresh buffer
containing GSH. The KLAK peptide concentration in the solution was
determined based on the fluorescence of AF-568, which was measured
using an Infinite M200 microplate reader.
Preparation of DOX-Loaded PPNs. The DOX-loaded PPNs
were prepared following a similar procedure. The DOX was dissolved
in anhydrous DMSO with 1% TEA at a concentration of 10 mg mL−1.
To this solution, NDBC (100 mg mL−1 in anhydrous DMSO) was
added at a NDBC-to-DOX molar ratio of 1.1:1, followed by 2 h of
incubation. A desired amount of this solution was then mixed with a
NDBC-modified KLAK peptide solution prior to immobilization into
MSNs. Following immobilization, the particles were washed twice with
MOPS buffer (50 mM, pH 8.0). Then, 8-arm-PEG-SH in MOPS
buffer (50 mM, pH 8.0) was added at a SH-to-NDBC molar ratio of
1.1:1. The MSN templates were then removed following the same
procedure as that described for preparing PPNs in the absence of
DOX loading. To determine the composition, the PPNs were
prepared from AF-633-labeled KLAK peptides. The KLAK peptides
and DOX concentration were determined by fluorescence spectropho-
tometry and UV−visible spectrophotometry (NanoDrop 2000,
Thermo Fisher Scientific, MA, USA).
DOX Release Studies. The DOX-loaded PPNs (300 μL, 250 μg
mL−1 of DOX) were dialyzed against phosphate buffer (10 mL, pH
7.4, 20 mM) containing either 5 mM or 10 μM GSH using a dialysis
membrane with a molecular weight cutoff of 7000 Da. At each time
point, the solution outside the dialysis membrane (500 μL) was
withdrawn and replaced with the same volume of fresh buffer
containing GSH. The DOX concentration in the solution was
determined based on fluorescence from DOX, which was obtained
using an Infinite M200 microplate reader.
Cell Viability Assays. HeLa cells were plated on a 96-well plate
(Costar 3596, Corning, MA, USA) at a cell density of 5000 cells per
well in cell culture media (100 μL), followed by incubation for 24 h.
The PPNs, free KLAK peptides, DOX-loaded PPNs, and free DOX
were added at varying concentrations of KLAK peptides or DOX.
After 48 h of incubation, the cell viability was evaluated by an XTT
assay. The absorbances at 475 and 650 nm, used as references, were
recorded using an Infinite M200 microplate reader. The cell viability
was calculated as a ratio against the absorbance obtained from
nontreated cells.
Zetasizer Measurements. The hydrodynamic diameter, scatter-
ing light intensity (SLI), and zeta(ζ)-potential of the particles were
measured using a Zetasizer Nano ZS instrument (Malvern Instru-
ments, Malvern, UK) equipped with a He−Ne ion laser (λ = 633 nm)
as an incident beam. For the dynamic light scattering (DLS)
measurements, a particle dispersion in phosphate buffer (pH 7.4, 10
mM) (60 μL) was placed in a micro cuvette (ZEN0040, Malvern
Instruments). The photon correlation function was analyzed by the
cumulant method to derive the diffusion coefficients (DC) of the
particles. The obtained DC was converted into the hydrodynamic
diameter (DH) using the Stokes−Einstein equation: DH = kBT/3πηDC
(kB: Boltzmann constant, T: absolute temperature, η: viscosity of the
Annexin V/Propidium Iodide (PI) Assays. HeLa cells were
plated on Nunc Lab-Tek II 8-well-chambered coverglass slides at a cell
density of 10 000 cells per well in cell culture media (200 μL). After 24
D
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h of incubation, the PPNs were added at a KLAK concentration of 20
μM. After 24 h of incubation, the cells were stained with a dead cell
apoptosis kit with annexin V-AF-488 and PI (Thermo Fisher Scientific,
MA, USA) following the manufacturer’s protocol. Fluorescence
microscopy images were taken using an Olympus IX71 inverted
fluorescence microscope (Olympus, Japan) equipped with a 40×
objective.
Table 1. Physicochemical Properties of MSNs and PPNs
Prepared from Three Different Disulfide Linkages
ab
,
a
a
chemistry size (nm)
PDI
ζ-potential (mV)
MSN
−
135
181 15
183
190 15
6
0.14 0.03
0.16 0.04
0.17 0.02
0.14 0.03
−34
2
B-PPN
E-PPN
S-PPN
NDBC
NDEC
SPDP
2
3
2
1
1
1
7
Flow Cytometry. HeLa cells were plated on a 96-well plate at a
cell density of 5000 cells per well in cell culture media (100 μL). After
24 h of incubation, the PPNs were added at a KLAK peptide
concentration of 10 μM. At various time points, the medium was
removed and the cells were washed with phosphate-buffered saline
(PBS). After treatment with trypsin, the cells were suspended in ice-
cold PBS, and flow cytometry analysis of the cells was conducted using
an A50 Micro Flow Cytometer (Apogee Flow Systems, UK).
Deconvolution Microscopy. HeLa cells were plated on Nunc
Lab-Tek II 8-well-chambered coverglass slides at a cell density of
10 000 cells per well in cell culture media (200 μL), followed by 24 h
of incubation. For the cellular internalization study, the PPNs were
added at a concentration of 10 μM. After 10 h of incubation,
deconvolution microscopy images were taken using a Delta Vision
(Applied Precision) equipped with a 60× 1.42NA oil objective with a
standard FITC/TRITC/Cy5 filter set. For analysis of the DOX-loaded
PPNs, the particles were added at a DOX concentration of 2 μM. After
24 h of incubation, deconvolution microscopy images were taken.
a
Determined in phosphate buffer (pH 7.4, 10 mM). The results are
expressed as the mean standard deviation obtained from four
b
samples. Size denotes hydrodynamic diameter measured by DLS.
potentials of the PPNs were close to neutral, with negligible
variation across the three types of cross-linked particles. This
result suggests that the PPNs made of KLAK peptides and PEG
have comparable compositions (Table 1). Stability under
physiological salt conditions is important for the biological
application of peptide nanoparticles. As peptides are weakly
charged polyelectrolytes of low molecular weight, the presence
of high concentrations of salt may induce dissociation of the
peptides from the nanoparticles unless they are covalently
attached. Furthermore, electrostatic repulsion between nano-
particles is screened in high salt concentrations, which may
result in unexpected nanoparticle aggregation. The DH and
polydispersity index (PDI) of the PPNs remained constant for
at least 6 h of incubation (Supporting Information, Figure S15),
thus demonstrating the stability of the PPNs under
physiological salt conditions, probably because of the presence
of PEG in the structure.
Cytotoxicity of PPNs against Cancer Cells. The
cytotoxicity of PPNs against HeLa cells was examined by
using XTT assays (Figure 2a, Table S1). Treatment with PPNs
prepared using reversible NDBC (B-PPN) or NPDE chemistry
(E-PPN) significantly reduced cell viability. Furthermore, the
cytotoxicity of PPNs was higher than free KLAK peptides that
showed cytotoxic effects consistent with previously reported
results.50 The stronger cytotoxicity of the PPNs than KLAK
peptides can be attributed to the efficient association of the
PPNs with cells when compared with free peptides. In contrast,
the PPNs prepared using nonreversible SPDP chemistry (S-
PPN) resulted in a negligible reduction in cell viability, even at
the highest concentration tested (∼100 μM). Moreover, the S-
PPN particles had a considerably lower cytotoxicity than free
KLAK peptides. The minimal cytotoxicity of S-PPN suggested
that cell death induced by B-PPN and E-PPN was based on the
reversible release of KLAK peptide in the cytoplasm, which
could subsequently induce apoptosis. It is noted that B-PPN
induced a greater reduction in cell viability when compared
with E-PPN when the same equivalent of peptides was used
even though both PPNs featured reversibility in the disulfide
linkages.
RESULTS AND DISCUSSION
■
Preparation and Characterization of PPNs. PPNs were
prepared using MSNs as templates (Scheme 1). Prior to
immobilization on MSNs, KLAK peptides were modified with
2-pyridyldithio moieties by reaction with NDBC, NDEC, or
SPDP. The latter three reagents were synthesized via disulfide
exchange and activation (Supporting Information, Scheme S1).
Subsequent cross-linking by 8-arm-PEG-SH (Mw ∼ 10 kDa)
through disulfide exchange and removal of the templates
resulted in PPNs. Successful preparation of the PPNs was
confirmed by DLS and TEM (Figure 1, Table 1). All three
types of PPNs were well dispersed in aqueous solution and had
a uniform size distribution with an average hydrodynamic
diameter of ∼185 nm (Table 1). Compared with the MSN
templates, which featured an average hydrodynamic diameter of
135 6 nm, the PPNs were slightly larger, probably due to
swelling, owing to the mobility of the PEG chains. The ζ-
Next, to examine the activation of apoptotic processes, the
cells were stained with annexin V-AF-488 and PI after
treatment for 24 h with either the PPNs or free peptides at a
KLAK concentration of 20 μM (Figure 2b, Supporting
Information, Figure S16). Treatment with B-PPN resulted in
∼26% of the cell populations stained with annexin V-AF488 but
not with PI. These results suggest the onset of apoptosis in the
cells. In contrast, the population of such cells (annexin V-
positive, PI-negative) was limited to 5−7% when the cells were
treated with E-PPN, S-PPN, or free KLAK peptides, suggesting
that apoptosis is not induced under the conditions tested
(consistent with the XTT assay). Furthermore, the results
Figure 1. TEM images of (a) MSNs and (c) PPNs prepared using
NDBC chemistry (B-PPN). Size distributions of (b) MSNs and (d) B-
PPNs obtained by DLS.
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Figure 3. (a) Transition of mean fluorescence intensity from HeLa
cells treated with either AF-488-labeled B-PPN or E-PPN at a peptide
concentration of 10 μM, which was measured by flow cytometry. Error
bars represent standard deviations of the means obtained from three
samples. Deconvolution microscopy images of HeLa cells treated with
either (b) B-PPN or (c) E-PPN at a peptide concentration of 10 μM
for 10 h. The green and blue fluorescence represent AF-488-labeled
KLAK peptides and the Hoechst 33342-stained cell nucleus,
respectively.
of 4-DBU33 toward disulfide exchange compared with the
aliphatic disulfide of 2-DEU, thus leading to efficient anchoring
to the cell surface thiols. Additionally, deconvolution
microscopy showed slightly more green fluorescence from the
KLAK peptides upon association with the cells in the samples
treated with B-PPN when compared with that generated by the
samples treated with E-PPN (Figure 3b,c). In combination with
the intracellular distribution study, stronger cellular association
(∼1.5-fold) was observed for B-PPN. However, cellular
association alone cannot completely explain the difference
observed in cytotoxicity (∼4-fold). Therefore, we investigated
the disassembly behavior of the PPNs.
Triggered Disassembly of PPNs. The disassembly
properties of the PPNs under reducing conditions were
investigated by monitoring the SLI generated by the PPNs
(Figure 4a). The SLI generated by B-PPNs and E-PPNs was
stable in 10 μM GSH, which mimics the extracellular reducing
potential. Furthermore, the DH and PDI of the two PPNs
remained constant under the same condition, thereby
indicating the stability of the KLAK particles (Supporting
Information, Figure S17). In contrast, the SLI generated by
both B-PPN and E-PPN decreased abruptly under simulated
intracellular reducing conditions of 5 mM GSH, thus suggesting
disassembly of the particles. Notably, B-PPN showed a faster
decrease in SLI when compared with E-PPN. The faster
disassembly of B-PPN could be attributed to faster cleavage of
the aromatic−aliphatic mixed disulfide of 4-DBU when
compared with the aliphatic disulfide.34 Following disassembly,
the release of KLAK peptides is an important step toward
inducing cytotoxic effects. Thus, the KLAK peptide release
profile under reducing conditions was investigated (Figure 4b).
KLAK peptides were released more quickly from B-PPN than
from E-PPN, further confirming the faster cleavage of 4-DBU
than 2-DEU.
Figure 2. (a) Cell viability of HeLa cells after treatment with B-PPN,
E-PPN, S-PPN, or free KLAK peptides for 48 h, as determined by
XTT assays. Error bars represent standard deviations of the means
obtained from four samples. (b) Population of annexin V-AF488
positive, and annexin V-AF488 and PI positive cells after 24 h
treatment with PPNs or free peptides at a KLAK concentration of 20
μM. Approximately 200 cells were analyzed for each sample. The
results are expressed as means standard deviations obtained from
three samples.
suggest that cell death induced by PPN was based on induction
of apoptosis, probably due to the reversible release of peptides
in the cytoplasm. However, significant differences in the cell-
killing efficiency were observed between the two reversibly
cross-linked nanoparticles, i.e., B-PPN and E-PPN. We
therefore investigated the disassembly profile, cellular associa-
tion, and intracellular behavior of these two peptide nano-
particle systems.
Cellular Association of PPNs with Cancer Cells. The
efficiency of cellular association and internalization is directly
related to the cytotoxicity of the PPNs, as apoptotic responses
are downstream of cellular association and internalization.
Therefore, cellular association was evaluated using flow
cytometry and deconvolution microscopy. The AF-488-labeled
KLAK peptide was used to prepare the peptide particles, and
HeLa cells were incubated with these nanoparticles at a peptide
concentration of 10 μM. Flow cytometry revealed that the cells
treated with B-PPN displayed approximately 1.5-fold stronger
fluorescence when compared with the cells treated with E-PPN
after 9 h (Figure 3a), thus indicating more efficient association
with B-PPN than with E-PPN. This result may be attributed to
the higher propensity of the aromatic−aliphatic mixed disulfide
Traceless Peptide Release from PPNs. Even after
peptide release, the molecular pendant group on KLAK
peptides alters the amphipathic nature of a KLAK peptide,
possibly reducing cytotoxicity.41 Therefore, the mechanism of
fragmentation of the molecular pendant groups and subsequent
regeneration of pristine KLAK peptides are of significant
interest. To determine the structure of the released peptide,
MALDI-ToF MS analysis was conducted after disassembly of
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Furthermore, there was no substantial evidence of the existence
of KLAK peptides containing the 4-mercaptobenzyl urethane
(4-MBU, molecular weight: 167.2 g mol−1) moiety. This result
indicates the reversible release of unmodified KLAK peptides
under reducing conditions from B-PPN. In contrast, the
spectrum of E-PPN displayed weak peak signals from the
unmodified KLAK peptides after 2 h of incubation in 5 mM
GSH. Additional peaks relating to the modified KLAK peptides
that contained 2-mercaptoethyl urethane (2-MEU, molecular
weight: 104.1 g mol−1) moieties, as major species, were
observed (Figure 5c). After prolonged incubation for 24 h, the
peptides, which were released from B-PPNs, showed major
peaks, corresponding to free KLAK peptides (Supporting
Information, Figure S18). In contrast, peaks relating to the 2-
MEU moieties-containing KLAK peptides, which were released
from E-PPNs, were clearly observed (Figure 5d). A significant
amount of free KLAK peptide was also detected. These results
suggest that both 4-DBU and 2-DEU moieties can be cleaved
under physiologically relevant reducing conditions (i.e., 5 mM
GSH). However, the removal of the molecular pendant groups
is significantly quicker for the 4-MBU moiety when compared
with that of the 2-MEU moiety. The faster release of
unmodified KLAK peptides under intracellular reducing
conditions would lead to more efficient induction of apoptosis,
possibly resulting in higher cytotoxicity to cells.
Application of PPNs for Therapeutic Delivery. To
investigate the application of PPNs for therapeutic delivery,
DOX, as a representative small molecule drug, was incorpo-
rated into the PPNs by covalent conjugation to PPNs using the
above established reversible disulfide linkages. DOX was first
modified with either NDBC or NDEC and then immobilized
onto MSNs together with KLAK peptides. After cross-linking
and template removal, DOX-loaded PPNs were obtained, as
confirmed by the red coloration of the pellets and DLS and
TEM analyses (Supporting Information, Figure S19, Figure 6a,
and Table 2). Both the DOX-free and DOX-loaded PPNs had
comparable DH, PDI, and ζ-potential values. Even after DOX
loading, the PPNs maintained colloidal stability under
physiological salt conditions (Supporting Information, Figure
S20). The amount of DOX loading could be readily tuned by
adjusting the feed ratio of DOX and KLAK peptides during
immobilization (Table 2). The particles prepared at KLAK/
DOX molar ratios of 1.0:1.3 and 1.0:0.3 are denoted as
DOX1.3@B-PPNs (or DOX1.3@E-PPNs) and DOX0.3@B-PPN,
respectively. The DOX-loaded peptide nanoparticles disas-
sembled selectively in simulated intracellular conditions (5 mM
GSH) (Supporting Information, Figure S21). DOX was also
readily released in such a simulated intracellular reducing
environment (5 mM GSH). In contrast, release was
significantly limited in 10 μM GSH, which simulates the
extracellular reducing environment (Figure 6b). A faster release
of DOX was observed from DOX1.3@B-PPN when compared
with that from DOX1.3@E-PPN. This result was consistent with
the disassembly study conducted on B-PPN and E-PPN.
Notably, an initial burst release was not observed, as is often
reported for other systems and results in the cytotoxicity to
normal cells.51 Thus, selective drug release in intracellular
reducing conditions can be realized using the present system,
thereby reducing risks of undesirable side effects to normal cells
from an initial surge in drug concentration upon drug
administration. In this regard, the selective cytotoxicity to the
cancer cells may be further improved by incorporation of
amine-containing targeting ligands, which will be investigated in
Figure 4. (a) Variations in the scattering light intensity generated from
B-PPNs and E-PPNs in 5 mM and 10 μM GSH. Error bars represent
standard deviations of means obtained from three samples. (b) Release
of KLAK peptides from B-PPNs and E-PPNs in 5 mM and 10 μM
GSH.
the PPNs in 5 mM GSH. As a control, the free KLAK peptide
was analyzed. The spectrum displayed peaks at m/z of ∼1526
([M + H]+) and ∼1548 ([M + Na]+) (Figure 5a). After 2 h of
incubation, peaks relating to unmodified KLAK peptides were
clearly detected from the peptides from B-PPNs (Figure 5b).
Figure 5. Molecular weight analysis of the peptides released from the
PPNs. MALDI-ToF MS spectra of (a) free KLAK peptides and KLAK
peptides released from (b) B-PPNs after 2 h of incubation in 5 mM
GSH, and E-PPNs after (c) 2 h and (d) 24 h of incubation in 5 mM
GSH.
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Figure 6. Physicochemical characterization and bioactivity evaluation of DOX-loaded PPNs. (a) Size distribution and TEM image (inset) of
DOX1.3@B-PPN. (b) DOX release profiles from DOX1.3@B-PPN and DOX1.3@E-PPN in 5 mM and 10 μM GSH. (c) Cytotoxicity of free DOX and
DOX-loaded PPNs prepared using different disulfide linkages (DOX1.3@B-PPN, DOX0.3@B-PPN, DOX1.3@E-PPN) and different DOX loadings.
Error bars represent standard deviations of the means obtained from six samples. Deconvolution microscopy images of cells treated with (d)
DOX1.3@B-PPN or (e) DOX1.3@E-PPN at 2 μM DOX for 24 h. Red and blue fluorescence represent DOX and Hoechst 33342, respectively.
Table 2. Physicochemical Properties of PPNs Loaded with DOX
hydrodynamic diameter, DH
ζ-potential
feed KLAK/DOX molar
ratio
loaded KLAK/DOX molar
ratio
a
a
a
chemistry
(nm)
PDI
(mV)
DOX0.3@B-PPN
DOX1.3@B-PPN
DOX1.3@E-PPN
NDBC
NDBC
NDEC
188 22
0.16 0.10
0.14 0.10
0.17 0.10
2
2
2
1
1
1
1.0:0.3
1.0:1.3
1.0:1.3
1.0:0.8
1.0:2.3
1.0:2.4
187
8
188 12
a
Determined in phosphate buffer (pH 7.4, 10 mM). The results are expressed as the mean standard deviation obtained from four samples.
our future work. The cytotoxicity of the DOX-loaded PPNs was
examined by XTT assays (Figure 6c). DOX1.3@B-PPN
prepared from NDBC chemistry exerted higher cytotoxicity
against HeLa cells when compared with DOX1.3@E-PPN
prepared from NDEC chemistry. Deconvolution microscopy
analysis revealed the intracellular distribution of DOX. In the
cells treated with DOX1.3@B-PPN, strong DOX fluorescence
was observed in the nucleus, indicating significant accumulation
of DOX in the nucleus (Figure 6d). In contrast, such
localization was less pronounced in cells treated with
DOX1.3@E-PPN (Figure 6e). The lower nuclear distribution
of DOX released from DOX1.3@E-PPN could be attributed to
slower cleavage of the disulfide bonds and subsequent
fragmentation of the 2-MEU moiety over the 4-MBU moiety,
thus resulting in slower accumulation of DOX in the nucleus.
These results indicate that the selection of reversible disulfide
chemistry significantly alters the intracellular distribution and
activity of therapeutics loaded within PPNs. Such a
phenomenon may prove useful in the design of nanocarrier
systems with tunable intracellular therapeutic activity. 4-DBU is
suitable when abrupt increases in drug concentration (release)
is required, whereas 2-DEU is more appropriate when gradual
sustained drug release is desired.
PPNs allow codelivery of KLAK peptides and DOX, possibly
imparting a tunable cytotoxicity to the DOX-loaded peptide
nanoparticles. To examine the effect of codelivery of KLAK
peptides on cytotoxicity, DOX0.3@B-PPN with a higher KLAK/
DOX ratio was prepared (Table 2). DOX0.3@B-PPN exhibited
stronger cytotoxicity when compared with DOX1.3@B-PPN at a
given DOX concentration. This result was attributed to the
higher content of KLAK peptide (3-fold) in DOX0.3@B-PPN
when compared with that in DOX1.3@B-PPN, thus leading to a
greater delivery of KLAK peptides together with DOX.
Notably, the degree of the enhanced cytotoxicity was much
greater than expected from the cytotoxicity study of DOX-free
B-PPN (Figure 2a). For example, approximately 25% difference
in the cell viability was observed between DOX0.3@B-PPN and
DOX1.3@B-PPN at 2 μM DOX, which corresponds to a KLAK
peptide concentration of 0.9 μM for DOX1.3@B-PPN, and 2.5
μM for DOX0.3@B-PPN. Meanwhile, DOX-free B-PPN
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CONCLUSION
■
Functional peptide particles, or PPNs, from KLAK peptides
were engineered via template-assisted assembly using reversible
cross-linking chemistries based on disulfides. The resulting
peptide nanoparticles exerted tunable cytotoxicity against
cancer cells, depending on the reversible chemistries. A marked
difference was observed in the cytotoxicity of the PPNs
prepared using reversible 4-DBU and 2-DEU chemistries, with
the PPNs prepared using 4-DBU chemistry exhibiting stronger
cytotoxicity than those prepared using 2-DEU chemistry. The
stronger cytotoxicity displayed by the former PPNs could be
attributed to faster disulfide cleavage and subsequent
fragmentation of molecular pendant groups on the KLAK
peptides, which resulted in regeneration of pristine peptides.
The facile conjugation and loading of a small molecular drug
into the peptide particles was also demonstrated using DOX
with a single conjugation site. The release of DOX and hence
the cytotoxicity of the DOX-loaded peptide nanoparticles were
also tunable by the conjugation chemistry used. Furthermore,
the use of KLAK peptides as constituent materials afforded
codelivery of KLAK peptides and DOX, thereby providing the
DOX-loaded peptide nanoparticles with tunable cytotoxicity,
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b11302.
■
S
Scheme S1, Figures S1−S21, Table S1 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*fcaruso@unimelb.edu.au
ORCID
■
2888.
Jiwei Cui: 0000-0003-1018-4336
Yi Ju: 0000-0003-0103-1207
Frank Caruso: 0000-0002-0197-497X
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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■
̈
This research was conducted and funded by the Australian
Research Council (ARC) Centre of Excellence in Convergent
Bio-Nano Science and Technology (Project CE140100036)
and the ARC under the Australian Laureate Fellowship (F.C.,
FL120100030) scheme. This work was performed in part at the
Materials Characterisation and Fabrication Platform (MCFP)
at the University of Melbourne and the Victorian Node of the
Australian National Fabrication Facility (ANFF).
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