Direct Polymerization of the Arsenic Drug PENAO to Obtain Nanoparticles with High Thiol-Reactivity and Anti-Cancer Efficiency

Article abstract of DOI:10.1021/acs.bioconjchem.8b00032

PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid), which is a mitochondria inhibitor that reacts with adenine nucleotide translocator (ANT), is currently being trialed in patients with solid tumors. To increase the stability of the drug, the formation of nanoparticles has been proposed. Herein, the direct synthesis of polymeric micelles based on the anticancer drug PENAO is presented. PENAO is readily available for amidation reaction to form PENAO MA (4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide) which undergoes RAFT (reversible addition-fragmentation chain transfer) polymerization with poly(ethylene glycol methyl ether methacrylate) as comonomer and poly(methyl methacrylate) (pMMA) as chain transfer agent, resulting in p(MMA)-b-p(PEG-co-PENAO) block copolymers with 3-15 wt % of PENAO MA. The different block copolymers self-assembled into micelle structures, varying in size and stability (D_h = 84-234 nm, cmc = 0.5-82 μg mol^-1) depending on the hydrophilic to hydrophobic ratio of the polymer blocks and the amount of drug in the corona of the particle. The more stable micelle structures were investigated toward 143B human osteosarcoma cells, showing an enhanced cytotoxicity and cellular uptake compared to the free drug PENAO (IC₅₀ (PENAO) = 2.7 ± 0.3 μM; IC₅₀ (micelle M4) = 0.8 ± 0.02 μM). Furthermore, PENAOs arsonous acid residue remains active when incorporated into a polymer matrix and conjugates to small mono and closely spaced dithiols and is able to actively target the mitochondria, which is PENAO's main target to introduce growth inhibition in cancer cells. As a result, no cleavable linker between drug and polymer was necessary for the delivery of PENAO to osteosarcoma cells. These findings provide a rationale for in vivo studies of micelle M4 versus PENAO in an osteosarcoma animal model.

Full text of DOI:10.1021/acs.bioconjchem.8b00032

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Article
Direct Polymerization of the Arsenic Drug PENAO to obtain
Nanoparticles with high Thiol-Reactivity and Anti-Cancer Efficiency
Janina-Miriam Noy, Hongxu Lu, Philip Hogg, Jia-Lin Yang, and Martina H. Stenzel
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00032 • Publication Date (Web): 18 Jan 2018
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Bioconjugate Chemistry
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Direct Polymerization of the Arsenic Drug PENAO to obtain Nanoparticles with
high Thiol-Reactivity and Anti-Cancer Efficiency
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Janina-Miriam Noya, Hongxu Lua, Philip J. Hoggb, Jia-Lin Yangc, and Martina Stenzel*a
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a School of Chemistry, Centre for Advanced Macromolecular Design (CAMD), University of New
South Wales, Kensington, Sydney, NSW 2052, Australia
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b The Centenary Institute and National Health and Medical Research Council Clinical Trials Centre,
University of Sydney, Sydney, Australia
c Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales,
Kensington, Sydney, NSW 2052, Australia
*E-mail: m.stenzel@unsw.edu.au
Abstract
PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid), which is a mitochondria inhibitor
that reacts with adenine nucleotide translocator (ANT), is currently being trialed in patients with
solid tumors. To increase the stability of the drug, the formation of nanoparticles has been
proposed. Herein, the direct synthesis of polymeric micelles based on the anti-cancer drug PENAO is
presented. PENAO is readily available for amidation reaction to form PENAO MA (4-(N-(S-
penicillaminylacetyl) amino) phenylarsonous acid methacrylamide) which undergoes RAFT
(reversible addition fragmentation chain transfer) polymerization with poly(polyethylene glycol
methyl ether methacrylate) (PEGMA) as comonomer and poly(methylmethacrylate) (pMMA) as
chain transfer agent, resulting in p(MMA)-b-p(PEG-co-PENAO) block copolymers with 3-15 wt.% of
PENAO MA. The different block copolymers self-assembled into micelle structures, varying in size
and stability (Dh = 84 -234 nm, cmc = 0.5 – 82 µg mol-1) depending on the hydrophilic to hydrophobic
ratio of the polymer blocks and the amount of drug in the corona of the particle. The more stable
micelle structures were investigated towards 143B human sarcoma cells, showing an enhanced
cytotoxicity and cellular uptake compared to the free drug PENAO (IC50 (PENAO) = 2.7 ± 0.3 µM; IC50
(micelle M4) = 0.8 ± 0.02 µM). Furthermore, PENAOs arsenous acid residue remains active when
incorporated into a polymer matrix and conjugates to small mono and closely spaced dithiols and is
able to actively target the mitochondria, which is PENAO’s main target to introduce growth
inhibition in cancer cells. As a result, no cleavable linker between drug and polymer was necessary
for the delivery of PENAO to osteosarcoma cells. These findings provide a rationale for in vivo studies
of micelle M4 versus PENAO in an osteosarcoma animal model.
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Introduction
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Metal-based drugs have been used as therapeutic agents since the early days of civilisation1. Paul
Ehrlich kick-started their success in 1910 with the development of the first chemotherapeutic
Salvarsan, an arsenical that was used for the treatment of syphilis and African trypanosomiasis
(sleeping sickness).2 But it was not only his work that influenced the modern metal-based medicine
in the 20th century; many other scientists including Robert Koch3, Francis Dwyer4-5, and particularly
Barnet Rosenberg6 are noteworthy for their discovery of the bacteriostatic and anticancer properties
of gold salts, ruthenium polypyridyl complexes, and platinum amine complexes.
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In addition to the above mentioned metal compounds, arsenic-based composites have proven to be
remarkable candidates for biomedical applications due to their binding ability with thiolates of
cysteine residues. Particularly the stable cyclic dithioarsinite complexes formed between arsenic (III)
and closely spaced protein thiols, which results in deactivation of the protein’s function.7-9 Arsenic-
thiol conjugation reaction has been well studied over the last years7, 10-14, and has been used to its
advantages: Peng et al. recently demonstrated that arsenic trioxide can be loaded onto folate
labeled human-serum-albumin by arsenic-sulfur bond formation, which facilitates the therapeutic
effect of arsenic trioxide on chronic myeloid leukemia and xenograft tumor models.15 Inorganic
arsenic trioxide is still the most effective drug against acute promyelocytic leukemia (APL) up to
date.7
A
promising cancer therapeutic is the hydrophilic organoarsenical PENAO (4-(N-(S-
penicillaminylacetyl)amino) phenylarsonous acid) which is emphasized in this work, and was
developed by Hogg and co-workers.16 It is currently being trialed in a phase I/IIa dose escalation
study in patients with solid tumors. PENAO is a second generation trivalent arsenical mitochondria
inhibitor from GSAO (4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid) that deactivates the
mitochondrial adenine nucleotide translocator (ANT) by forming a stable cyclic dithioarsinite
complex with ANT cysteines 57 and 257.17 PENAO has demonstrated to have an anti-tumor growth
effect on many tumors with negligible side-effects at efficacious doses.16, 18-19
However, the efficacy of arsenical-based drugs and most other metal-based drugs is limited by
several factors such as low drug solubility, high systemic toxicity, development of drug resistance,
and rapid deactivation by complexation with proteins or oxidation reactions. For instance, despite
PENAO’s clinical success, its accumulation rate depends on rate of export by the MDR (multidrug
resistance) protein MRP1/2 and efficacy on the cellular levels of glutathione. By inhibiting MRP1/2
and blocking de novo synthesis of glutathione, PENAO’s cell growth inhibition decreased drastically
(from IC50 = 1100 nM to IC50 = 20 nM) in bovine aortic endothelial (BAE) cells.16
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Bioconjugate Chemistry
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To overcome many of these limitations, nanocarriers have been developed. The delivery of
therapeutic compounds using nanotechnology has been widely proposed to improve the solubility
and efficacy, while alleviating the side effects of chemotherapeutic agents by altering their
pharmacokinetics and biodistribution. The delivery of chemotherapeutics via nanocarriers has
shown to prolong circulation time in the blood stream, to increase the specific retention in solid
tumour tissue (enhanced permeability and retention (ERP) effect)20-21, and to avoid the recognition
by the mononuclear phagocyte system.22-23
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Much attention has been drawn to nanoparticle systems for the delivery of inorganic arsenite or
arsenous acid, which are highly toxic to normal tissue. Many of these nanoparticle formulations
improved drug selectivity to diseased tissue, tumor suppression efficacy by alleviating systemic
toxicity,24-25 drug loading efficiency26, and drug accumulation rate in cancer tissues27-30
.
Most notable for these abovementioned nanocarriers is that they were all prepared by physical
encapsulation of the arsenical compound. Physical entrapment is easy to implement and versatile
for many agents, specifically hydrophobic drugs. However, the loading efficiency is generally low and
drugs can easily leak out of the nanoparticle into the blood stream. In this work, we therefore
applied chemical conjugation of the arsenic drug PENAO to a polymeric micelle to avoid drug leakage
and premature release.
Polymeric micelle systems can be formed from amphiphilic polymers that self-assemble above the
critical micelle concentration (cmc) to core – shell structures. The core is built up by the hydrophobic
part of the block copolymer, allowing to solvate hydrophobic components, while the shell (or
corona) is equivalent to the hydrophilic segment of the block copolymer, and therefore responsible
for its high water-solubility.31
Most anti-cancer drugs are of hydrophobic nature and therefore favor the hydrophobic environment
of the core. When chemical drug-polymer conjugation is utilized, a cleavable linker is often
necessary to enable drug release inside the tumors.32 By using hydrophilic drugs which assemble on
the corona of a micelle, a cleavable linker may not be mandatory. Moreover, specific drug molecules
can act as ligands to increase the probability to interact with its target. Nevertheless, the
conjugation of drugs or ligands on nanoparticle surfaces can implicate difficulties: Stability, size and
immunogenicity are often affected by the attachment of molecules. In addition, conjugated
hydrophilic drugs may exhibit unfavourable pharmacokinetics in terms of absorption, distribution,
and metabolism.33 However, due to the insufficient availability of hydrophilic drugs, little is known
about their effect on micelle surfaces.
Herein we describe the direct synthesis of polymers, based on the organoarsenical drug PENAO,
which were then self-assembled into polymeric micelle structures, as shown in Scheme 1. As PENAO
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acts by reacting with thiols, the activity of the prepared polymers and micelles were tested using
different small synthetic mono and closely spaced dithiols. The micellar delivery systems were tested
in vitro on 2D monolayer cell culture to determine whether PENAO based polymeric nanoparticle’s
cytotoxicity and cellular uptake will have an appreciable outcome towards human 143B
osteosarcoma cells. As the mitochondria is the intended target for the PENAO based polymeric
nanoparticles, their cellular localization was analyzed using confocal microscopy. Although the focus
of this work is to deliver the drug PENAO, it also provides a better understanding of the effect of
hydrophilic drugs on micelle surfaces. Furthermore, the micelle systems can be used in future as
reactive scaffold for cyclic peptides.
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A
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Scheme 1. (A) Synthesis of PENAO MA 4: (i-iii) Synthesis of PENAO: (i) p-arsanilic acid 1 was reacted with bromoacetyl
bromide to obtain the precursor BRAA. (ii) BRAA was reduced with HBr, NaI and H2SO4 to BRAO. (iii) PENAO 2 was
synthesized from BRAO and D-penicillamine. PENAO was then reacted with methacrylic anhydride 3 to PENAO MA 4.
(B) Synthesis of p(MMA)-b-p(PEG-co-PENAO) block copolymers P1 – 4 via the RAFT process using p(MMA) macro RAFT
agents 6. (B) Formation of PENAO micelles (M1 – 4) via self-assembly.
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Bioconjugate Chemistry
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Results and Discussion
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PENAO monomer synthesis
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The drug PENAO (4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid) 2 was obtained
following the three-step procedure of Dilda et al.16 with minor modifications, resulting in 74 % yield
of drug. The PENAO monomer (4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid
methacrylamide, PENAO MA, 4) was synthesized via a nucleophilic substitution reaction of the
secondary amine from PENAOs amino acid residue and methacrylic anhydride 3 (see Scheme 1) and
to the best of our knowledge has not been previously described. Methacrylic anhydride was chosen
as a reagent to obtain a polymerizable methacrylamide handle. The reaction was carried out in
water using less than 2 equiv. excess of sodium hydrogen carbonate to drug. The product PENAO MA
4 was separated as a solid and purified by basic aluminium oxide column filtration to remove
remaining base and during the reaction produced methacrylic acid. PENAO MA was isolated as a
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white solid with a yield of 50 %. H and 13C NMR spectra (see Figure 1 and Figure S2) confirm the
successful synthesis of PENAO MA. The peak f’ in Figure 1A associated with the proton adjacent to
the amine group, shifted significantly from 3.73 ppm to 4.34 ppm, suggesting the successful
amidation of the methacrylamide handle. Additionally, the appearance of the peaks at 5.73 ppm and
5.45 ppm confirm the existence of vinyl protons, which are characteristic of methacrylamide
monomers. Conjugating the amino acid group with a more hydrophobic compound may alter the
hydrophilicity of the PENAO monomer, but the monomer remained soluble in water and methanol.
In fact, PENAO MA could additionally be dissolved in ethanol, while PENAO shows no solubility in this
solvent.
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Figure 1. 1H NMR spectra of PENAO MA 4 (A) and PENAO 2 (B).
PEG-PENAO copolymer synthesis
To exploit PENAO’s growth inhibition towards cancer cells when conjugated to a polymer matrix, a
copolymer was synthesised. The p(PEG38-co-PENAO2) polymer was obtained by the RAFT
technique34, using CPADB as chain transfer agent, AIBN as initiator and poly(ethylene glycol) methyl
ether methacrylate (PEGMA, 7) and PENAO MA 4 as monomers at a feed concentration ratio
[PEGMA:PENAO] : [CPADB] : [AIBN] of 90:10 : 1 : 0.1. The polymerization was carried out in
methanol at 70 °C. After 9 hours, the conversion of the copolymerization was determined by 1H NMR
spectroscopy on the crude polymerization mixtures, revealing a copolymer with 38 repeating units
of PEGMA and 2 repeating units of PENAO MA. Due to the hydrophilic nature of the copolymer, no
particle formation was observed. Instead, p(PEG38-co-PENAO2) could be dissolved in milli-Q water
directly. The copolymer was tested in a SRB assay on 143B sarcoma cells. As previous reported, drug-
conjugated polymers have generally a lower cytotoxicity compared to free drugs.35 This is due to the
slow diffusion of the polymer across the cell membrane compared to the free drug while the
polymers are often too small to display efficient endocytosis. The copolymer p(PEG38-co-PENAO2)
shows negligible cytotoxicity (IC50 = 548.2 µM) towards 143B cells presumably caused by low cellular
uptake (see Figure S4). The incorporation of a drug-conjugated polymer into nano-sized structures
often enhanced the cytotoxicity of the drug, as it improves cellular uptake by undergoing fast and
efficient endocytosis.36 Therefore, an amphiphilic polymer is subsequently prepared that is able to
form polymeric micelles with the drug located within the shell to enable direct contact between the
drug in the shell and the ANT protein located in the mitochondria membrane, which has shown to be
the main target for PENAO.
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Bioconjugate Chemistry
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PMMA-PEG-PENAO copolymer synthesis
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PEG based polymers maintain widespread use due to its biocompatibility and clinical
establishment.37 The use of p(MMA-b-PEG) block copolymers as drug delivery composites are widely
known and have shown to be reliable candidates due to their ease to self-assemble into micelle
structures as well as being nontoxic at high polymer concentrations.38-40 This system entails
therefore a great platform for the introduction of a new drug PENAO. In addition, it has been
reported that micelles with glassy cores are significantly more stable than micelles with soft cores.41,
wherefore methyl methacrylate (MMA, 5) was chosen as the hydrophobic segment and was
subjected to RAFT polymerization mediated by the chain transfer agent CPADB, at different feed
concentration ratios [MMA] : [CPADB] : [AIBN] of 100/150/200 : 1 : 0.1. Details on reaction solvent
and times, monomer conversions, measured and calculated molecular weight characteristics are
compiled in Table S1. Three different lengths of poly methyl methacrylate p(MMA) were chosen
with the aim of preparing micelles of different sizes. By increasing the hydrophobic content in a
block copolymer, more stable micelles can be obtained.42 In all cases, polymerizations preceded with
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similar conversions (determined from H NMR spectroscopy on crude polymerization mixtures) and
resulted in monomodal, nearly symmetrical molecular weight distributions with low dispersities ƉM ≤
1.09 (see Figure 2B, dashed lines).
The p(MMA)-b-p(PEG-co-PENAO) block copolymers P1 – 4 (see Scheme 1) were obtained via RAFT
polymerization, using the previous described PMMA macro RAFT agents as chain transfer agents,
AIBN as initiator, and PEGMA 7 and PENAO MA 4 as comonomers at a feed concentration ratio
[PEGMA:PENAO MA] : [PMMA] : [AIBN] of 80:20/ 180:20 : 1 : 0.1. Two different ratios of PEGMA to
PENAO MA (80:20 and 180:20) were compiled to synthesize two block copolymers with different
block sizes, but similar amounts of drug (Table 1, entry 1 and 2). The polymerizations were carried
out at 70 °C, using a solvent mixture of methanol and DMSO due to the different solubility of the
hydrophobic macro CTA and hydrophilic PENAO MA. The conversion of the block copolymerizations
1
after 6 hours was determined by H NMR spectroscopy on the crude polymerization mixtures,
resulting in block copolymers with 33 to 76 repeating units of PEGMA and ≤ 7 units of PENAO MA.
The consumption of PENAO MA is slightly lower than that of PEGMA suggesting a gradient structure
of the polymer. It is also interesting to note that the smaller p(MMA) macro RAFT agents with 78
repeating units led to higher conversions of drug monomer, using similar concentration and
polymerization time. The influence of the first block on the reactivity ratio of the subsequent
copolymerization is known as bootstrap effect.43 The hydrophobic p(MMA) block favours the contact
with PEGMA over interaction with the polar PENAO MA. The increased retardation with increasing
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PENAO MA feed ratio prevented the incorporation of more than 30-40 wt.% PENAO MA (polymer
not shown here) into the polymer without significantly affecting the rate of polymerization at high
PENAO MA feed ratios. The block copolymers were purified by dialyses in DMSO and methanol. The
exact monomer molar ratios, solvent ratios, monomer conversions and molecular weights
1
characteristics are listed in Table 1. H NMR und SEC analysis was used to confirm the successful
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chain extension of PEGMA and PENAO MA with PMMA. In all cases, polymers with low dispersities
ƉM ≤ 1.22 were obtained for the block copolymers. The SEC traces shifted from lower to higher
1
molecular weights, signifying a clear change in molecular weight (Figure 2B). H NMR spectra of
purified polymers (example of p(MMA)78-b-p(PEG33-co-PENAO7, P1) shown in Figure 2A conform to
the expected structures. The peaks at 7.65 ppm and 7.83 ppm can clearly be assigned to the phenyl
protons of PENAO MA. Moreover, the peak breadth compared to the monomer peak indicates that
PENAO MA is bounded to the polymer backbone (Figure S3). In addition, the block copolymers were
analysed by ICP-MS, showing a similar weight % of arsenic, compared to the calculated wt. %
determined from 1H NMR (Table 1). Due to PENAO’s high affinity towards thiols it is to mention that
no colour change was observed during the reaction, indicating that the pink coloured RAFT
thiocarbonylthio end group is still intact.
Table 1 Summary of PENAO MA containing block copolymers prepared by the RAFT process at 70 °C for 6 hours
theo. a
SEC b
Monomer feed
(mol ratio in %)
Conversion
(%)
Mn
Mn
PENAO MA wt.%
SEC b
ÐM
Entry Polymer
(kg mol-1) (kg mol-1)
NMR
ICP-MS
p(MMA) -b-p(PEG
-
-
PEGMA (80):
PENAO (20)
PEGMA (41)/
PENAO (34)
78
33
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2
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4
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33.6
40.0
39.1
24.2
25.6
39.5
33.5
1.18
1.19
1.22
1.16
15.34
15.36
co-PENAO ) (P1)
7
p(MMA) -b-p(PEG
PEGMA (89):
PENAO (11)
PEGMA (42)/
PENAO (27)
78
76
8.24
3.65
3.54
7.62
4.14
3.24
co-PENAO ) (P2)
6
p(MMA) -b-p(PEG
-
-
PEGMA (90):
PENAO (10)
PEGMA (42)/
PENAO (15)
135
76
66
co-PENAO ) (P3)
3
p(MMA) -b-p(PEG
PEGMA (90):
PENAO (10)
PEGMA (35)/
PENAO (14)
176
co-PENAO ) (P4)
3
aCalculated from conversion and composition. bDetermined by size exclusion chromatography in DMAc.
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Figure 2. (A) Representative 1H NMR spectrum of p(MMA) -b-p(PEG -co-PENAO ) P1. (B) SEC traces of p(MMA) macro
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RAFT agents (dashed curves) and PENAO containing block co polymers p(MMA)-b-p(PEG-co-PENAO) (solid curves): (P1)
p(MMA) -b-p(PEG -co-PENAO ), (P2) p(MMA) -b-p(PEG -co-PENAO ), (P3) p(MMA) -b-p(PEG -co-PENAO ), (P4)
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p(MMA) -b-p(PEG -co-PENAO ).
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Micelle preparation and their characterization
All block copolymers were only directly soluble in DMSO and DMF, only p(MMA)78-b-p(PEG76-co-
PENAO6) P2 could also be dissolved in methanol, due to its 1 : 1 ratio of hydrophobic to hydrophilic
block. The micellization of the block copolymers p(MMA)78-b-p(PEG33-co-PENAO7) P1 and P2 was
prepared by adding water to a DMSO or methanol solution respectively, while a DMF solution was
used for the block copolymers p(MMA)135-b-p(PEG76-co-PENAO3) P3 and p(MMA)176-b-p(PEG66-co-
PENAO3) P4. Slow addition of water led then to self-assembly into micelles. The micelle solutions
were purified by dialysis in water and the remaining solutions were adjusted to 1 mg mL-1 working
solutions. The size of the micelles was determined by DLS. The block copolymer with the shortest
PEG chain (P1) led to nanoparticles with the smallest size (M1), while P2 – P4 formed nanoparticles
(M2 – M4) with similar sizes within errors despite having increasing hydrophobic block length (
Figure 3 and Table S3). All micelle systems are monodispersed, showing only one population of
particles (Figure 3A). This observation was confirmed when visualizing the size and shape of micelles
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in dry state via transmission electron microscopy (TEM) (Figure 3B). Average diameters measured by
TEM did not deviate significantly to the measured average diameters determined by DLS, indicating
that no aggregate formation is present. Additionally, we observed a rough particle margin. The
higher the ratio of arsenic drug to PEGMA, the rougher the margin appears. Zhang et al.26 observed a
similar phenomenon, when they loading phenylarsine oxide (PAO) into poly(ethylene oxide)-block-
poly[α-(6-mecaptohexyl amino) carboxylate-ε-caprolactone] PEO-b-p(CCLC6-SH) micelles. A control
micelle, formed from p(MMA)176-b-p(PEG)34 P5 was prepared and analysed via TEM, showing
spherical particles with a more even particle margin than p(MMA)-b-p(PEG-co-PENAO) micelles (see
Figure S5). Furthermore, the average particle diameter of p(MMA)176-b-p(PEG)34 determined from
TEM deviates by more than 50 % from the average diameter measured by DLS. Therefore, we
hypothesized that the presence of PENAO MA in the hydrophilic shell influences the surface
morphology of p(MMA)-b-(PEG) particles, which can play an important role during cellular uptake.
The surface charge of the four p(MMA)-b-p(PEG-co-PENAO) and control p(MMA)-b-(PEG) micelles
were obtained from zeta-potential measurements in water, showing more negative surface charges
for PENAO MA containing particles (ζ = -33.8 - -27.1 mV) compared to non PENAO MA containing
particles (ζ = -17.6) (Figure 3). We also observed the micelle surface to become more negative as a
function of an increase in drug amount. Due to the negative surface charge of the p(MMA)-b-p(PEG-
co-PENAO) particles, aggregation is less likely, which is in agreement with DLS and TEM studies.44
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Figure 3. (A) Representative DLS-measured size distributions of PENAO containing nanoparticles (M1 – M4) and control
micelle and (B) Representative TEM images for PENAO containing nanoparticles (stained with uranyl acetate): M1
p(MMA) -b-p(PEG -co-PENAO ), M2 p(MMA) -b-p(PEG -co-PENAO ), M3 p(MMA) -b-p(PEG -co-PENAO ), M4
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The micelle stability was analysed by the determination of the critical micelle concentration (cmc),
measuring the scattering intensity for different micelle concentrations (c = 0 – 100 µg mL-1) (Figure
S6).45 Micelle stability plays an important role for biological studies. More stable micelle structures
directly correlates with a better cellular uptake.38 As expected, an increase of micelle stability was
observed with an increase in hydrophobic block length for the less drug containing micelles (M3 and
M4), which is in agreement with the literature.46 In contrast, M1 and M2 with cmc values of 68 µg
mL-1 and 82 µg mL-1, respectively, are significantly less stable due to the shorter p(MMA) block
length, but also the higher drug concentration in the shell. It is known that hydrophobic drugs can
improve the thermodynamic stability of micelle systems47 but little is known about the effect of
hydrophilic drugs on non-crosslinked nanoparticle surfaces. Here we hypothesize that the
hydrophilic drug PENAO MA on a polymeric micelle has a detrimental effect on the nanoparticle
stability. We assume that the repulsion of the negatively charged PENAO MA chains results in a
looser micelle structure, allowing water molecules to migrate closer to the hydrophobic core. A
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looser particle structure could also explain the not perfectly round particles, which were revealed by
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TEM analysis (Figure 3B).
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Activity towards thiols and localization in mitochondria
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PENAO acts by forming stable cyclic dithioarsenite complexes with two cysteines in the ANT peptide
loop.17 Polymer-drug conjugation is a well-established technique; however the binding of a drug to a
carrier can render the drug inactive.48 Thus, we exposed P2 as a model bock copolymer, dissolved in
DMSO (at a concentration of 1.85 mmol L-1 [PENAO MA groups]) to model monothiols and closely
spaced dithiols (monothiol: glutathione (GSH), 3-mercaptopropionic acid (MPA), 2.05-2.1 equiv.
relative to [PENAO MA groups]; dithiol: 2,3-dimercaptopropanol (DMP), dithiothreitol (DTT), 1.05
equiv. relative to [PENAO MA groups]). Conversions after an incubation time of 24 h were
determined by Ellman’s assay (thiol concentration plot derived from cysteine hydrochloride
standards, see Figure S7 and S8) on withdrawn reaction samples. The reaction of P2 with GSH and
MPA reached a conversion of 65 % and 66 %, while the conjugation with DMP and DTT revealed
conversions of 90 % and 83 %, respectively. Results are also summarized in Table 2. A high selectivity
of GSAO – a PENAO mimic with the same reactive arsonous acid residue – towards closely spaced
dithiols has been previously reported by Hogg and co-workers.49 The trivalent arsenical affinity
towards dithiols decreases as a function of an increase in size of the formed As-SH ring structure; the
five-membered ring product, formed with DMP reveals a more stable product (dissociation constant
DMP = 130 nM) compared to the seven-membered ring product, formed with DTT (dissociation
constant DTT = 420 nM) (Scheme S3).49 A similar trend was observed, when PENAO was subjected to
the same thiols, using comparable reaction conditions. By exposing DMP and DTT to PENAO,
conversions of 97 % and 80 % were obtained, while the reaction of PENAO and MPA proceeded to a
60 % conversion. Almost full conversion (99 %) was reached, when GSH was conjugated to PENAO.
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The crude PENAO-GSH product was additionally analysed by H NMR spectroscopy, showing no
remaining GSH in solution (Figure S9). It is yet unclear why PENAOs affinity toward GSH is higher
compared to the other examples. It has been previously reported that reduced GSH reacts with
As(V) centres by firstly reducing As(V) to As(III) and then binding to the metalloid centre, while for
instance 1,2-ethanethiol only selectively conjugates to As(III) compounds.50 Dissolved oxygen
deactivates the organoarsonous acids by oxidizing the trivalent to the pentavalent state over time.
Therefore, we believe a small percentage of PENAO’s trivalent As(III) oxidized to its pentavalent
As(V), which may result in lower conjugation efficiency for non-reducing thiols. However, we can
claim that the thiol-arsenic conjugation proceed on PENAO MA containing polymers as well as on
PENAO itself, showing no significant difference in conversion (except PENAO-GSH). With that in
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mind, we analysed the conjugation behaviour on a model micelle system using M3 (concentration =
1 mg mL-1) towards the monothiols GSH and MPA. Similar conversions were achieved (Table 2, Entry
3 and 7), indicating that the conjugation efficiency between PENAO MA and thiol is the same when
on nanoparticle or in free polymer. When increasing the amount of thiols to 10 equiv. to PENAO MA
groups, only a minimal increase in conversion (75 -78 %) was observed (Table 2, Entry 4 and 8).
Additionally, the conjugation product p(MMA)78-b-p(PEG76-co-PENAO-DMP5/6) (Table 2, Entry 10)
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was purified and analysed by SEC analysis, causing the SEC-measured size distribution to shift
minimal to higher molecular weights (Figure S8). We interpreted the negligible shoulder in the
higher molecular weight region, as the result of thiol-mediated cleavage of RAFT thiocarbonylthio
end group producing terminal thiols51 which can undergo crosslinking by reaction with other
polymer chains, or introduce further As–SH conjugation reactions. Nevertheless, no significant
change in width or shape was observed, indicating that side reactions were kept to a minimum. It
seems therefore that the polymers are still highly reactive towards thiols, which is prerequisite for
their activity towards cysteine residues in ANT, which is located on the mitochondria transition pore.
To confirm the localization of the PENAO based micelles into the mitochondria, fluorescein O-
methacrylate was incorporate into the polymer backbone, while the mitochondria of 143B sarcoma
cells was stained with the dye Mito Tracker® Deep Red FM and imaged using confocal microscopy
(Figure 4). The overlapping fluorescence (Figure 4D) from Mito Tracker® Deep Red FM (Figure 4B)
and the fluorescein labelled nanoparticles (Figure 4A) confirmed mitochondrial localization of the
PENAO carrying micelles. Moreover, this outcome demonstrate that the existence of the negative
charges on PENAO nanoparticles (ζ = -33.8 - -27.1 mV) does not hinder the interaction with the
mitochondria and PENAO’s organoarsonous acid residues still act as an active mitochondrial target
when incorporated into a polymeric micelle.
Table 2 Summary of As-SH conjugation products. Conversion was determined by Ellman’s assay.
Entry
Compound
Thiol (equiv.)
A412
n (μmol)
Conversion (%)
1
2
3
4
5
6
7
PENAO
P2
GSH (2.05)
GSH (2.05)
GSH (2.05)
GSH (10.0)
MPA (2.1)
MPA (2.1)
MPA (2.1)
0.136
0.686
0.122
0.435
0.912
0.596
0.146
0.294
1.480
0.261
0.931
2.129
1.309
0.316
99
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75
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58
M3
M3
PENAO
P2
M3
8
M3
MPA (10.0)
DMP (1.05)
DMP (1.05)
DTT (1.05)
DTT (1.05)
0.398
0.079
0.330
0.472
0.159
0.848
0.159
0.752
1.089
0.324
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9
PENAO
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12
PENAO
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Nanoparticles
Mitochondria
Merged
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10 µm
Figure 4. Confocal microscopic images of 143B cells. (A) PENAO nanoparticles (labelled with fluorescein O-methacrylate,
incubation time 2 hours); (B) Mitochondria (stained with Mito Tracker® Deep Red FM, incubation time 30 min); (C) Bright
field image of 143B cells; (D) Overlap of green and red fluorescence, indicating mitochondrial localization of the PENAO
containing micelles (indicated with red errors). Scale bar 10 µm.
Cytotoxicity/ cellular uptake studies
With only a few reports on the incorporation of organic arsenicals into synthetic polymers52 little is
known about their cytotoxicity. Wilson et al. recently presented a system of hydrophilic arsenic-
functional polymers, with negligible cytotoxicity towards a series of mouse cell lines.50 Here, we
subjected PENAO, PENAO MA and PENAO MA containing micelles towards human 143B sarcoma
cells and explored their cytotoxicity in a SRB assay, which determine the IC50 value of the compounds
with in vitro cultured cells. PENAO’s cytotoxicity has been intensively investigated towards an
assortment of cell lines showing high growth inhibition with IC50 values around the low micro molar
range.16, 18 The cytotoxicity of free PENAO, PENAO MA and different micelle systems was evaluated
in vitro. After 72 h of incubation, the IC50 of PENAO was determined to be 2.7 ± 0.3 µM. Interestingly,
the PENAO monomer is significant less cytotoxic (IC50 = 77.9 ± 7.6 µM) than PENAO, presumably to
the attachment of the methacrylamide handle. Previous studies have shown that the D-
penicillamine attachment is necessary to improve cellular uptake and to limit the rate of export by
the multidrug resistant protein (MRP1/2).16 All of the here utilized micelle formulations however,
had significant lower IC50 values than the monomer PENAO MA. The more stable M3 and M4 show a
similar or enhanced cytotoxicity to PENAO (IC50 = 2.5 ± 0.04 µM and IC50 = 0.8 ± 0.02 µM,
respectively), while the less stable M1 reveals a higher IC50 value of 8.3 ± 1.6 µM. M2 was due to its
low stability not further investigated for cytotoxicity and cellular uptake studies. The results of the
cytotoxicity assay are depicted in Figure 5. Cellular uptake depends strongly on micelle stability and
particle size. In general it has been consistently shown that smaller particles have a higher rate of
extravasation into permeable tissues and reduced hepatic filtration.53 Cellular uptake studies of
micelle systems and PENAO were analysed by ICP-MS measurements after an incubation time of 24
h, revealing the highest uptake (2.71 %) for the most stable micelle M4. Thus, the higher cytotoxicity
of M4 can be explained by its higher cellular uptake compared to M3 (1.57 %). In this matter, micelle
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stability seems more important than micelle size; the less stable micelle M1 (with an average
hydrodynamic diameter of 84 nm) has the lowest uptake (0.68 %). In comparison, PENAOs cellular
uptake was revealed to be 0.93 %. Cytotoxicity and cellular uptake results are also given in Table S4.
In summary, the most stable micelle system enhanced cytotoxicity and cellular uptake compared to
free PENAO, and this is to best of our knowledge the first covalently bound arsenic containing
synthetic polymer system that enhanced the performance of the free drug and displays improved
proliferation arrest in vitro.
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It is of interest to further investigate the oxidation state and develop more stable and less oxygen
sensitive nanoparticle formulation that efficiently delivers PENAO to solid sarcoma. M4 already
shows promising anti-proliferation activity towards 143B sarcoma cells and polymeric PENAO
nanoparticles are expected to enhance accumulation and retention in target tumour areas and to
lower the toxicity towards normal organs compared to the free drug. By attaching PENAO to a
polymeric matrix instead of using physical encapsulation, drug leakage can be avoided in the
bloodstream. No cleavable linker between polymer backbone and drug was necessary, as the
arsonous acid side remains active for conjugation reaction. PENAO has been intensively investigated
over the last decade16-18, 54-57; however it has never been used in combination with nanoparticles.
The here employed system does not show any degradability in biological environments, it is
therefore important to further develop biodegradable PENAO polymeric nanoparticles that evade
renal filtration and allow subsequent degradation in the human body.
A
B
Figure 5. 72 h Cytotoxicity assay on 143B cells of A) PENAO (IC50 = 2.7 ± 0.3 µM), PENAO MA (IC50 = 77.9 ± 7.6 µM); B) M1
(IC50 = 8.3 ± 1.6 µM), M3 (IC50 = 2.5 ± 0.04 µM), M4 (IC50 = 0.8 ± 0.02 µM).
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In summary, we have demonstrated for the first time that the hydrophilic trivalent organic arsenical
drug PENAO is readily available for amidation modifications with methacrylic anhydride to form a
polymerizable methacrylamide monomer (PENAO MA), which polymerized via the RAFT process with
different sized PMMA macro CTAs and in the present of the hydrophilic comonomer PEGMA. In all
cases, the PENAO containing polymers self-assembled into micelle structures, leaving PENAO on the
particle surface. PENAO’s arsenous acid residue remains active when incorporated into a polymer
matrix and conjugates to small thiols, showing no significant difference in efficiency between PENAO
containing polymers, PENAO containing particles and PENAO itself. Furthermore, the PENAOs moiety
allowed localization into the mitochondria of the nanoparticles, and the more stable micelle
structures induce growth inhibition in sarcoma cells (143B) and compete with the free drug in terms
of cytotoxicity and cellular uptake. This is the first example of PENAO containing nanoparticles,
which show great potential for further investigations into the biomedical arena (e.g. in an
osteosarcoma animal model) in terms of in vivo bio-distribution, anti-cancer efficacy, reduction of
side-effects and protein scaffold ability.
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Experimental
Materials
All chemicals were purchased as reagent grade from Sigma-Aldrich and used as received unless
stated otherwise. Milli-Q water was produced by a Milli-Q water purification system and had a
resistivity of 18.2 MΩ.cm. Sodium methoxide was dried over molecular sieve (4 Å).
Azobis(isobutyronitrile) (AIBN) was recrystallized from methanol and stored at −24 °C. The
monomers methyl methacrylate (MMA) and poly(ethylene glycol) methyl ether methacrylate
(PEGMA, monomer molecular weight 300 g mol−1) were passed through basic aluminium oxide
(Al2O3) to remove inhibitors before polymerization. Deuterated NMR solvents were purchased from
Cambridge Isotope Laboratories. The chain transfer agents 4-cyano-4-[(phenylcarbonothioyl)thio]-
pentanoic acid (CPADB) was prepared as described elsewhere.58 Osteosarcoma cell line (143B) was
from one of our collaborators Prof. Jia-Lin Yang’s laboratory at Sarcoma and Nano-oncology Group
(Lowy Cancer Research Centre).
Analysis Techniques
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Nuclear Magnetic Resonance (NMR) spectroscopic measurements were performed on either a
Bruker Avance III 300 MHz (1H: 300.17 MHz, 13C: 75.48 MHz) or a Bruker Avance III HD 400 MHz (1H:
400.13 MHz, 13C: 100.62 MHz) instrument. Measurements of polymers were done in CDCl3, DMSO-d6
or MeOH-d4. All other samples were analysed in TFA-d1 or D2O. The internal solvent signals δ(CDCl3)
= 7.26 ppm, δ(DMSO-d6) = 2.50 ppm, δ(MeOH-d4) = 3.31 ppm, δ(TFA-d1) = 11.50 ppm, δ(D2O) = 4.79
ppm were used as reference. NMR spectra were processed using either the Bruker TOPSPIN 3.2
software or MestReNova NMR software.
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Size exclusion chromatography (SEC) was performed on a Shimadzu system equipped with a 50 ×
7.8 mm guard column and four 300 × 7.8 mm linear phenogel columns (105, 104, 103 and 500 Å)
operating at a flow rate of 1 mL min−1 using N,N’-dimethylacetamide (HPLC grade, 0.05% w/v BHT,
0.03% w/v LiBr) as eluent. The system was calibrated with a series of narrow molar mass distribution
poly(methyl methacrylate) with molar masses ranging from 0.58–1820 kg mol−1.
Dynamic light scattering (DLS) was performed on aqueous solutions (approximately 1 mg mL-1
polymer in milli-Q water) which were analysed by a Malvern Zetasizer Nano ZS instrument equipped
with a 4 mV He-Ne laser operating at λ = 632 nm and noninvasive backscatter detection at 173°.
Measurements were carried out in a disposable cuvette at 25°C, provided 15 scans equilibration
period prior to each set of measurements. For a given sample, a total of three measurements were
conducted with the number of runs, attenuator, and path length being automatically adjusted by the
instrument, depending on the sample quality.
Transmission Electron Microscopy (TEM) was carried out on a JEOL 1400 TEM with a beam voltage
of 100 kV and a Gatan CCD for acquisition of digital image. Samples were prepared by depositing 2
µL of the aqueous solution mixture onto a copper grid and incubated for 2 min. Excess of the
solution mixture was removed and the grids were air dried and negatively stained with 5 µL uranyl
acetate (UA) solution for 5 min.
UV-vis measurements were performed on a Varian Cary 300 Scan spectrophotometer equipped with
a Cary temperature controller and a Peltier heating element in quartz cuvettes of 1 cm path length.
The measurements were performed at a scan rate of 3000 nm min-1, a wavelength range of 200-800
nm, and a standard temperature of 25 °C. All samples were diluted with PBS (pH 8) and incubated
with 50 µL Ellman’s reagent solution (0.01 mol L-1 in PBS) for 30 min previously to measurement.
Cell Culture. 143B (osteosarcoma) cells were grown as monolayer cultures in cell culture flasks by
using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum
(FBS), 1 % glutamax and 100 U mL-1 penicillin. The cells in flask were grown in an incubator with a
humidified atmosphere at 5 % CO2 at 37 °C. The medium was routinely changed every 3 days. For
cell passage and subculture the cell culture media were removed, and cells that have reached
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confluence were washed with phosphate buffered saline (PBS) and detached by trypsin/EDTA
treatment. The cells were then collected, centrifuged, resuspended in the new culture medium and
recultured in new flasks.
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Inductively coupled plasma-mass spectrometry (ICP-MS) was performed on a Perkin-Elmer NexION
300D inductively coupled plasma-mass spectrometer (Perkin-Elmer, Norwalk, CT, USA) to determine
quantitative determination of arsenic. All experiments were carried out at an incident radio
frequency power of 1500 W. The plasma argon gas flow of 15 L min−1 with an auxiliary argon flow of
1.2 L min−1 was used in all cases. The nebulizer gas flow was adjusted to maximize the ion intensity
at 1.02 L min−1, as indicated by the mass flow controller. The element/mass detected was 91AsO (DRC
mode) and the internal standard used was 103Rh. The replicate time was set to 1000 ms, and the
dwell time was set to 100 ms. Peak hopping was the scanning mode employed and the number of
sweeps/readings was set to 10. A total of 3 replicates were measured at the normal resolution.
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Experiments
Synthesis of 4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid (PENAO). PENAO was
synthesised in 3 steps according to the procedure of Dilda et al.16 with some modifications: (i) p-
Arsanilic acid (10.06 g, 46.09 mmol) was suspended in 50 mL milli-Q water and 50 mL of a sodium
carbonate solution (7.32 g, 69.09 mmol). The slightly yellow solution was cooled to 0-5°C in an ice
bath and bromoacetyl bromide (18.59 g, 8.0 mL, 92.12 mmol) was added over a period of 10 min.
The reaction was maintained for
1 h below 20 °C. The precipitated product 4-(2-
bromoacetylamino)benzenearsonic acid (BRAA) was collected by filtration and washed with water.
(ii) The wet cake was then added to a mixture of 75 mL MeOH and 75 mL 48 % hydrobromic acid.
Sodium iodide (0.666 g, 4.43 mmol) was added, followed by dropwise addition of a sodium sulfate
solution (12.59 g, 88.76 mmol in 65 mL milli-Q water). The heterogeneous mixture was maintained
at room temperature for 2.5 hours and then filtered. The residue was suspended in a mixture of
methanol (75 mL), 48 % hydrobromic acid (30 mL) and milli-Q water (45 mL), followed by the
addition of 75 mL milli-Q water and 75 mL 5 % sodium carbonate solution. The product 4-(2-
bromoacetylamino)benzenearsonous acid (BRAO) was filtered and dried under reduced pressure
(yield: 62 %). 1H NMR (400 MHz, TFA-d1): δ/ppm = 7.98 (dd, 4 H, phenyl protons), 4.16 (s, 2 H, BrCH2-
). (iii) Under nitrogen atmosphere, 15 mL of anhydrous 25 wt. % sodium methoxide (dried over
molecular sieve, 4 Å) and 15 mL anhydrous methanol were weight into a schlenkflask. D-
penicillamine (0.51 g, 3.43 mmol) was added and the yellow solution was stirred for 5 min at room
temperature. Under nitrogen atmosphere BRAO (1.00 g, 3.11 mmol) was then added to the solution
and the mixture was stirred for 20 min. The pH was adjusted using 5 % sulfuric acid in methanol to
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obtain a final pH of 3.6. The heterogeneous solution was filtered and the solvent was removed under
reduced pressure. The slightly yellow solid was redissolved in methanol and added dropwise to 50
mL of rapidly stirred acetone and left to stir overnight at room temperature. The product was
filtered and dried under reduced pressure to obtain PENAO as colourless solid (yield: 74 %). 1H NMR
(400 MHz, D2O): δ/ppm = 7.75-7.73 (d, 2 H, -NHphenyl proton, meta), 7.60-7.58 (d, 2 H, -NHphenyl
proton, ortho), 3.73 (s, 1 H, H2NCHCOOH), 3.62 (q, 2 H, -S(CH2)CO-), 1.61, 1.40 (s, 6 H, -HCC(CH3)2).
13C NMR (100.62 MHz, D2O): δ/ppm = 171.10 (-COOH), 171.00 (-CONHCH-), 144.45 (-NHCphenyl),
138.91 (-CAs(OH)2), 130.10 (-NHphenyl, meta), 121.71 (-NHphenyl, ortho), 61.38 (-NHCHCOOH),
46.82(-CHC(CH3)2), 33.17 (-SCH2-), 26.87, 23.17 (-CHC(CH3)2).
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Synthesis of 4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide (PENAO
MA). PENAO (450 mg, 1.15 mmol) and sodium hydrogen carbonate (186.9 mg, 2.23 mmol) were
dissolved in 7 mL milli-Q water and the solution (pH 7) was cooled to 0-5 °C in an ice bath.
Methacrylic anhydride (204.5 mg, 202.5 µL, 1.33 mmol) was added dropwise with vigorous stirring.
The mixture was stirred for 2 h while maintaining the temperature of 0-5 °C then stirred overnight at
room temperature. The crude solution was added dropwise to 100 mL of rapidly stirred acetone,
redissolved in methanol and precipitated again into 50 mL of rapidly stirred acetone. The solvent
was removed by decanting and the precipitated colourless solid was redissolved in methanol and
filtered through an alumina oxide packed column, before it was dried under reduced pressure (yield
50 %). 1H NMR (400 MHz, D2O): δ/ppm = 7.80-7.78 (d, 2 H, -NHphenyl proton, meta), 7.69-7.67 (d, 2
H, -NHphenyl proton, ortho), 5.73 (q, 1 H, HHC=C(CH3)-), 5.45 (q, 1 H, HHC=C(CH3)-), 4.34 (s, 1 H, -
HNCHCOOH), 3.56 (q, 2 H, -S(CH2)CO-), 1.92 (s, 3 H, HHC=C(CH3)-), 1.50, 1.43 (s, 6 H, -HCC(CH3)2). 13
C
NMR (100.62 MHz, D2O): δ/ppm = 175.28 (-COOH), 171.80 (-CONHCH-), 170.90 (-H2CCONH-), 140.72
(-NHCphenyl), 138.83 (-CAs(OH)2), 130.92 (-NHphenyl, meta), 130.45 (H2C=C(CH3)), 121.67 (-
NHphenyl, ortho), 121.47 (H2C=C(CH3)-), 61.94 (-NHCHCOOH), 48.69 (-CHC(CH3)2), 33.31 (-SCH2-),
26.78, 24.61 (-CHC(CH3)2), 17.60 (H2C=C(CH3)).
Copolymerization of PEGMA and PENAO MA. PEGMA (147.3 mg, 0.49 mmol, 90 equiv.) and PENAO
MA (25.0 mg, 0.055 mmol, 10 equiv.), RAFT agent CPADB (1.52 mg, 0.0055 mmol, 1 equiv.), AIBN
(0.90 mg, 0.00055 mmol, 0.1 equiv.) and methanol (0.5 mL, c = 1.08 mol L-1) were mixed in a glass
flask, then sealed with a rubber septum, purged with nitrogen for 20 min and placed into a
preheated oil bath at 70 °C for 9 h. The polymerization was stopped by quenching to room
temperature and monomer conversion and theoretical molecular weights, Mntheo., were determine
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by a H NMR spectroscopic measurement of a reaction sample (50-100 µL) diluted with CD3OH (450
µL) by comparison of polymeric signals with the vinyl signals of the residual monomer. The polymer
was isolated by dialysis against methanol (regenerated cellulose membranes, MW cut-off 6000-8000
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g mol-1) and dried under reduced pressure. The copolymer poly[(polyethylene glycol methyl ether
methacrylate)-co-4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide)],
(p(PEG38-co-PENAO2)) was analysed via SEC chromatography. Conversion (PEGMA) = 41 %,
conversion (PENAO) = 15 % Mntheo = 12.6 kg mol-1, MnSEC = 17.6 kg mol-1, ĐMSEC = 1.13). The number of
repeating units (PEGMA = 38 units; PENAO = 2 units) of p(PEG38-co-PENAO2) was calculated from the
monomer conversion obtained from 1H NMR spectroscopy.
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Polymerization of methyl methacrylate (MMA). In a typical experiment, MMA (100, 150, 200
equiv.), RAFT agent CPADB (1 equiv.), AIBN (0.1 equiv.) and solvent (acetonitrile, approx. 1.5 mL
mmol-1 of monomer) were mixed in a glass flask, which was sealed with a rubber septum, purged
with nitrogen for 25 min and placed into a preheated oil bath at 70 °C for 16-16.5 h. After cooling,
polymers were isolated by precipitation into a diethyl ether-hexane mixture (2 : 1), redissolved in
chloroform and precipitated again. Details on polymerization times, and polymer characterization by
SEC and NMR are summarized in Table S1.
Chain extension of PMMA with PEGMA and PENAO MA. PENAO containing block copolymers were
prepared by RAFT polymerisation according to the following general procedure. A mixture of PENAO
monomer and PEGMA (0.67-1.24 mmol in total), p(MMA) macro RAFT agent (CTA, 1 equiv.), and
AIBN (0.1 equiv.) were dissolved in a solvent mixture of DMSO and methanol (approx. total
concentration of 0.98-1.13 mol L-1) and combined in a glass flask equipped with a magnetic stir bar.
The absolute mass of all reactant and concentrations are given in Table S2. The flask was sealed with
a rubber septum, purged with nitrogen for 25 min and placed into a preheated oil bath at 70 °C for 6
h. The polymerization was stopped by quenching to room temperature. Monomer conversion and
theoretical molecular weights, Mntheo., were determine by a 1H NMR spectroscopic measurement of a
reaction sample (50-100 µL) diluted with DMSO-d6 or MeOH-d4 (450 µL) by comparison of polymeric
signals with the vinyl signals of residual monomers. Polymers were purified by dialysis in DMSO and
then methanol (regenerated cellulose membranes, MW cut-off 6000-8000 g mol-1). Conversion, SEC
data, solvent ratio and mol % feed are given in Table 1.
Chain extension of PMMA with PEGMA. PEGMA was chain extended via the RAFT process according
to the following procedure. PEGMA (0.56 mmol, 200 equiv.), p(MMA) macro RAFT agent (p(MMA)176
,
1 equiv.), and AIBN (0.2 equiv.) were dissolved in acetonitrile (total concentration of 1.12 mol L-1).
The solution was purged with nitrogen for 25 min and then polymerized at 70 °C for 7 h. The
polymerization was stopped by quenching to room temperature and monomer conversion and
theoretical molecular weights, Mntheo., were determine by a 1H NMR spectroscopic measurement of a
reaction sample (50-100 µL) diluted with CDCl3 (450 µL) by comparison of polymeric signals with the
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vinyl signals of the residual monomer. The polymer was purified two times by precipitation into a
diethylether – hexane mixture (4 : 1), dried under reduced pressure and analysed via SEC
chromatography. The block copolymer poly[(methyl methacrylate)-b-(polyethylene glycol methyl
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theo
ether methacrylate)] (p(MMA176)-b-p(PEG40) , conversion = 20 %, Mn = 29.9 kg mol-1, MnSEC = 22.7
kg mol-1, ĐM = 1.15) was used as a control polymer. The number of repeating units (40 units) of
SEC
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p(PEG) was calculated from the monomer conversion obtained from 1H NMR spectroscopy.
Formation of Micelles. The poly[(methyl methacrylate)-b-(polyethylene glycol methyl ether
methacrylate)-co-4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide)] or
p(MMA)-b-p(PEG-co-PENAO) block copolymers or p(MMA)-b-p(PEG) block copolymers were
dissolved in a suitable solvent (DMSO, MeOH or DMF) and milli-Q water was added under rapidly
stirring via a syringe pump using a rate of 0.2 mL/hour. The solutions were dialysed against milli-Q
water for 2 days with frequent water change. The hydrodynamic diameter and zeta potential was
measured by dynamic light scattering. The exact polymer feed, solvent ratios, particle sizes,
distributions and zeta potentials are listed in Table S3.
Critical Micelle Concentration (CMC). The CMC was determined by dynamic light scattering
measuring the scattering intensity for different micelle concentrations. Briefly, the aqueous micelle
solutions (1 mg mL-1) were serially diluted with milli-Q water to obtain concentrations of 0 – 100 µg
mL-1. For each sample, a total of two measurements with a 10 scans equilibration period prior to
each set of measurements were conducted. The number of runs was set to 11 (run duration 10 sec.),
attenuator and path length was fixed to 8 and 4.65.
Procedure for quantitating sulfhydryl groups using a cysteine hydrochloride standard – Ellman’s
assay. A thiol calibration plot was prepared using 2.5 mL phosphate buffer (0.1 mmol L-1, pH 8,
containing EDTA (1 mmol L-1, 29.2 mg, 0.10 mmol)), 250 µL cysteine hydrochloride standard solution
(0 - 1.5 mmol L-1 in PBS) and 50 µL 5’5-dithio-bis-(2-nitrobenzoic acid) (DTNB) solution (10 mmol L-1
in PBS). The samples were mixed and incubated for 30 min at room temperature. The UV
absorbance of the thiolate ion was then measured at λ = 412 nm (see Figure S7). The thiol
concentration was determine from the calibration equation λ412 = 1.183[SH] + 0.01203.
Thiol conjugation. Procedure for PENAO or PENAO block copolymers conjugation reaction: PENAO
(1 equiv.) or p(MMA)78-b-p(PEG76-co-PENAO6) block copolymer (1 equiv. of PENAO MA groups) was
dissolved in PBS (pH 8) (for PENAO) or DMSO (for p(MMA)78-b-p(PEG76-co-PENAO6)), (approx. total
concentration of 6.0 mmol L-1), and 50 µL thiol stock solution (2.05 - 2.1 equiv. for monothiols
(glutathione or 3-mercaptopropionic acid) or 1.05 equiv. for dithiols (2,3-dimercaptopropanol or
dithiotreitol), dissolved in PBS or DMSO) was added. The mixture was stirred overnight at room
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temperature. The conversion of the As-SH conjugation was determine via Ellman’s assay: 100 µL of
sample and 50 µL 5’5-dithio-bis-(2-nitrobenzoic acid) (DTNB) solution (10 mmol L-1 in PBS) were
added to 2.50 mL phosphate buffer (0.1 mmol L-1, pH 8, containing EDTA (1 mmol L-1, 29.2 mg, 0.10
mmol)). PENAO-GSH conjugation was additionally analysed via 1H NMR spectroscopy (see Figure S9).
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Procedure for PENAO nanoparticle conjugation reaction: 1 mL of micelle solution (containing approx.
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1 mg of polymer p(MMA)135-b-p(PEG76-co-PENAO3) were reacted with glutathione or 3-
mercaptopropionic acid (2.05 - 2.1 equiv. to PENAO MA groups) overnight at room temperature. For
the Ellman’s assay, 250 µL of sample and 50 µL 5’5-dithio-bis-(2-nitrobenzoic acid) (DTNB) solution
(10 mmol L-1 in PBS) were added to 2.5 mL phosphate buffer (0.1 mmol L-1, pH 8, containing EDTA (1
mmol L-1, 29.2 mg, 0.10 mmol)).
All solutions were mixed and incubated for 30 min at room temperature before the UV absorbance
of the thiolate ion was measured at λ = 412 nm. The thiol concentration was determine from the
calibration equation λ412 = 1.183[SH] + 0.01203 (see Figure S8). Results are listed in Table 2.
IC50 determination via Sulforhodamine B Assay. Cytotoxicity test and SRB assay of polymeric
micelles, PENAO and PENAO MA were determined by a standard sulforhodamine B colorimetric
proliferation assay (SRB assay). 143B (osteosarcoma) cells were seeded at a corresponding density
of 8,000 cells in 100 µL DMEM media per well in 96-well cell culture plates followed by the addition
100 μL of DMEM culture medium per well and incubated at 37 °C in a 5 % CO2 atmosphere for 24 h.
The specimens were sterilized by UV irradiation for 20 min before serially diluting (sequential halved
dilution) with sterile water. For the cytotoxicity assay, the medium in the cell culture plate was
discarded, and 100 μL of fresh 2 × concentrated DMEM medium was added to each well of the 96-
well culture plate. The sample (PENAO, PENAO MA, M1, M3, or M4) was added into the plate
(position 3 A – D to 11 A – D) at 100 μL per well, respectively. Sterile water (100 µL) was added to the
non-treated cells into the plate (position 1 A – H, 2 A – H, 3 E – H to 11 E – H, 12 A – G) as a control.
The cells were incubated with the micelles or free drug for 72 h, and the cell viability was
determined using SRB assay. The incubation with micelles or drug was terminated by the addition of
cold trichloroacetic acid (TCA) (10 % w/v) for 30 min at 4 °C. After a complete washing with
deionised water (5 times), the TCA-fixed cells were stained with 100 μL of 0.4 % w/v sulforhodamine
B (SRB) solution in 1 % acetic acid (w/v) for 15 min. After staining, unbound dye was removed by
washing with 1 % acetic acid for five times, and plates were air-dried. Finally, the SRB was solubilized
with 200 μL of 10 mM Tris buffer to dissolve bounded dye, and the optical density was determined
by using a microplate reader at the wavelength of 490 nm. Dose−response curves were plotted
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accordingly where the values were expressed as percentage of control (non-treated cells were used
as controls). The optical density was used to calculate cell viability.
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Cellular Uptake via ICP-MS. 143B cells were seeded into a 25 cm2 flask at 50 x 104 cells per flask and
incubated for 48 h. The DMEM medium was replaced by 2 x concentrated DMEM medium and the
cells were treated with PENAO polymer micelles (M1, M3, M4) at equal micelle concentrations and
free PENAO (c = 1500 nmol L-1). After 24 h incubation at 37 °C at a 5 % CO2 atmosphere, the medium
was removed and the cells were rinsed with cold PBS (5 mL x 5), trypsinized, collected and rinsed
with 9 mL milli-Q water. After drying, the cells were incubated with 50 µL HNO3 (70 %, v/v) at 25 °C
for 5 h and then diluted with 10 mL milli-Q water. Arsenic content uptake was determined using
inductively coupled plasma mass spectroscopy (ICP-MS). A five-point standard curve was plotted
between intensity versus a serial dilution of a certified reference standard ranging from 0.2, 0.5, 1,
10, 100 ppb.
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Mitochondrial particle localization via confocal microscopy. To be able to track the mitochondrial
localization of the PENAO micelles, 0.2 eq. fluorescein O-methacrylate was copolymerized with the
comonomers PENAO MA and PEGMA using p(MMA)176 as macro RAFT agent and the polymer F-P5
theo
SEC
SEC
(p(MMA)176-b-p(PEG71-co-PENAO2), Mn
= 40.1 kg mol-1, Mn = 34.8 kg mol-1, ĐM = 1.32) was
then self-assembled into micelle structure F-M5, according to the earlier described procedure. The
exact polymer feed, solvent ratios, particle sizes, distributions and zeta potentials are listed in Table
S3. For imaging, 143B cells were seeded into a fluoro dish at 10 x 104 cells in 2 mL DMEM medium
per dish and incubated at 37 °C at a 5 % CO2 atmosphere for 24 h. The medium was discarded and
the cells were washed with PBS (1 mL x 3) and treated with 100 µg mL-1 F-M5 micelle solution in 1
mL DMEM medium for 2 h. The medium was discarded and the cells were washed with Hanks'
Balanced Salt Solution (HBSS, 1 mL x 3) before 1 mL Mito Tracker® Deep Red FM dye (500 nM in
HBSS) was added. The cells were incubated for another 30 min with the dye, washed with HBSS (1
mL x 2) and imaged using a Zeiss LSM780 confocal microscope. The excitation wavelengths for
micelles and Mito Tracker were set as 488 and 633 nm, respectively. The observation was using a
100 × oil objective lens (N.A=1.4) and the images were captured and processed using ZEN software.
Acknowledgements
The authors acknowledge Ms Sylvia Ganda and Mr Alberto Piloni for Transmission Electron
Microscopy assistance. We also like to that the staff of the UNSW Electron Microscopy Unit (EMU)
for help. The Mark Wainwright Analytical Centre in acknowledged for performing inductively
coupled plasma-mass spectrometry experiments.
There are no conflicts of interest to report
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Details on the MMA polymerization and the chain extension, the formation of micelles, the
calculations of the thiol content as well as the As content in the cells can be found in the supporting
info. The supporting info includes furthermore NMR analysis of the monomer, cytotoxicity of the
statistical copolymer, TEM and stability studies by DLS on the micelle.
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Abbreviations
(4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) (PENAO); poly(methylmethacrylate)
(pMMA); poly(polyethylene glycol methyl ether methacrylate) (PEGMA); methanol (MeOH),
dimethylsulfoxide (DMSO); 2,2'-Azobis(2-methylpropionitrile) (AIBN); dynamic light scattering (DLS);
nuclear magnetic resonance (NMR); critical micelle concentration (cmc); number-average molecular
weight (Mn), dispersity (Ð), zetapotential (ζ), polydispersity index from DLS (PdI); inductively
coupled plasma mass spectroscopy (ICP-MS), transmission electron microscopy (TEM),
ORCID
Martina Stenzel: 0000-0002-6433-4419
Philip Hogg: 0000-0001-6486-2863
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in vitro
Mitochondrial localization
Growth inhibition in sarcoma cells
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