Cyclic Peptide-Polymer Nanotubes as Efficient and Highly Potent Drug Delivery Systems for Organometallic Anticancer Complexes

Article abstract of DOI:10.1021/acs.biomac.7b01491

Functional drug carrier systems have potential for increasing solubility and potency of drugs while reducing side effects. Complex polymeric materials, particularly anisotropic structures, are especially attractive due to their long circulation times. Here, we have conjugated cyclic peptides to the biocompatible polymer poly(2-hydroxypropyl methacrylamide) (pHPMA). The resulting conjugates were functionalized with organoiridium anticancer complexes. Small angle neutron scattering and static light scattering confirmed their self-assembly and elongated cylindrical shape. Drug-loaded nanotubes exhibited more potent antiproliferative activity toward human cancer cells than either free drug or the drug-loaded polymers, while the nanotubes themselves were nontoxic. Cellular accumulation studies revealed that the increased potency of the conjugate appears to be related to a more efficient mode of action rather than a higher cellular accumulation of iridium.

Full text of DOI:10.1021/acs.biomac.7b01491

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Article
Cyclic peptide-polymer nanotubes as efficient and highly potent
drug delivery systems for organometallic anticancer complexes
Sophie C Larnaudie, Johannes C. Brendel, Isolda Romero-Canelón, Carlos Sanchez-
Cano, Sylvain Catrouillet, Joaquin Sanchis, James P. C. Coverdale, Ji-Inn Song,
Abraha Habtemariam, Peter J. Sadler, Katrina A. Jolliffe, and Sebastien Perrier
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01491 • Publication Date (Web): 20 Nov 2017
Downloaded from http://pubs.acs.org on November 21, 2017
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Cyclic peptide-polymer nanotubes as efficient and highly potent
drug delivery systems for organometallic anticancer complexes
Sophie C. Larnaudie,a Johannes C. Brendel,ǂa,b Isolda RomeroꢀCanelón,a Carlos SanchezꢀCano,a
Sylvain Catrouillet,c Joaquin Sanchis,b James P. C. Coverdale,a JiꢀInn Song,a Abraha Habtemariam,a
Peter J. Sadler,a Katrina A. Jolliffe,*d and Sébastien Perrier*a,b,e
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a Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.
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Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC
3052, Australia.
c Institut Charles Gerhardt Montpellier, Place E Bataillon CC1702, 34095 Montpellier cedex 05, France
d The University of Sydney, School of Chemistry, Building F11, Sydney NSW 2006, Australia.
e Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, U.K.
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Current address: Jena Center for Soft Matter (JCSM), FriedrichꢀSchillerꢀUniversity, Philosophenweg 7, 7743
Jena, Germany.
*s.perrier@warwick.ac.uk; kate.jolliffe@sydney.edu.au
Abstract
Functional drug carrier systems have potential for increasing solubility and potency of drugs while
reducing side effects. Complex polymeric materials, particularly anisotropic structures, are especially
attractive due to their long circulation times. Here, we have conjugated cyclic peptides to the
biocompatible polymer poly(2ꢀhydroxypropyl methacrylamide) (pHPMA). The resulting conjugates
were functionalised with organoiridium anticancer complexes. Small angle neutron scattering and
static light scattering confirmed their selfꢀassembly and elongated cylindrical shape. Drugꢀloaded
nanotubes exhibited more potent antiproliferative activity towards human cancer cells than either free
drug or the drugꢀloaded polymers, whilst the nanotubes themselves were nonꢀtoxic. Cellular
accumulation studies revealed that the increased potency of the conjugate appears to be related to a
more efficient mode of action rather than a higher cellular accumulation of iridium.
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Keywords
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Cyclic peptide, polymer nanotubes, selfꢀassembly, organoiridium complexes, drug delivery.
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Introduction
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Advances in the development of small molecules as highly potent anticancer drugs often face
limitations related to their poor solubility, rapid elimination and limited stability in the body.1ꢀ2 The
use of drug carriers may address these challenges by providing a protective shell that enhances
solubility and retards clearance from the blood stream. Moreover, drug delivery vectors possess
additional means of introducing functionality and increasing the selective accumulation at the specific
target.3 Tuneable in size, the devices can be optimized for passive targeting to tumours via the
enhanced permeability and retention (EPR) effect.4 In addition, most carriers can be functionalized by
a variety of ligands (carbohydrates, peptides, proteins, antibodies, aptamers) to allow for active
targeting of specific cells.5 Overall, drug carrier systems offer potential for improving the therapeutic
efficiency of drugs and reducing their side effects.
It is therefore not surprising that a plethora of potential drug delivery vectors has been reported
including, for example, spherical polymer micelles or vesicles.6 Soꢀfarꢀneglected, but particularly
interesting, are organic nanotubes (NTs) formed by cyclic peptideꢀpolymer conjugates. The nanotubes
are built around a core made of βꢀsheetꢀforming cyclic peptides (CPs).7 CPs presenting an even
number of alternating Dꢀ and Lꢀ amino acids adopt a flat conformation in which the alignment of
amide functionalities allows for the formation of hydrogen bonds between cyclic peptides, promoting
their stacking into rodꢀlike, cylindrical assemblies.8 Cylindrical objects have been shown to exhibit a
longer time of residence in the body than spheres of comparable size,9 and rodꢀlike structures can
show higher activity than spherical particles10 when loaded for example with active peptides,11
antibodies12 or proteins.13 The high aspect ratio and the functionality of these organic nanotubes are
potentially valuable design features for drug delivery.
Despite all the above mentioned advantages of these cyclic peptide nanotubes and the advances in the
synthesis of functional materials, so far only a handful of examples of their use as drug delivery
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vectors has been reported.14ꢀ16 Blunden et al. have described the synthesis of such nanotubes bearing
RAPTAꢀC, a moderately active ruthenium anticancer drug. They demonstrated that the attachment of
the drug helped to increase its activity against cancer cells.14
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Here, we present a specifically designed system comprising a selfꢀassembling CP core, a functional
polymer shell and a highly potent organoiridium candidate drug. Particular attention was paid to the
use of biocompatible components in the synthesis and the effective attachment of the metallodrug
through efficient drug conjugation suitable for cancer therapy. In this context, poly(2ꢀhydroxypropyl
methacrylamide) (pHPMA) was chosen, since it has attracted particular interest for drug delivery
applications over the past few decades.17ꢀ18 Several systems derived from pHPMA have been studied
in detail and are currently undergoing clinical trial.19ꢀ20
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In addition to comprehensive optimisation of the delivery vector, the choice of a compatible and
potent drug is critical for designing effective therapeutics. Recently, organoiridium catalysts have
been shown to exhibit high potency towards a wide range of cancer cells21 and through careful choice
of ligands, the efficiency of these complexes can be improved by three orders of magnitude, reaching
subꢀmicromolar values.22 Depending on the cell line and the complex, activity was shown to be ca. 5
to 10 times higher than that of the clinical drug cisplatin, and more than 200 times higher than
RAPTAꢀC. The attachment of this type of complex to a polymeric carrier can be achieved through
incorporation of a suitable metalꢀbinding ligand on the polymer chains.
Based on these considerations, we synthesized a cyclic peptideꢀpHPMA conjugate and loaded it with
an organoiridium anticancer complex. The organoiridium fragment was attached through ligation to a
pyridineꢀcontaining comonomer in the polymer shell. The ability of the drugꢀloaded conjugates to
selfꢀassemble into nanotubes in solution was thoroughly established by scattering techniques, and
their cytotoxicity in vitro was assessed and compared to that of the free drug. For the first time, it was
also tested alongside and a drugꢀloaded polymer control that does not contain the cyclic peptide core,
in order to clearly assess the impact of the selfꢀassembly on the cytotoxicity. Finally, cellular
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accumulation of the three compounds was studied both qualitatively and quantitatively and the
mechanism of action was explored.
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Experimental section
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Materials
4ꢀAminomethyl pyridine (98%), 1ꢀaminoꢀ2ꢀpropanol (95%), methacryloyl chloride (97%), and
deuterated solvents for NMR were purchased from SigmaꢀAldrich. Nꢀmethylmorpholine (NMM, 99
%) was purchased from Alfa Aesar. Potassium carbonate and anhydrous magnesium sulfate (MgSO4)
were purchased from Fisher Scientific. 2,2'ꢀAzobis[2ꢀ(2ꢀimidazolinꢀ2ꢀyl)propane]dihydrochloride
(VAꢀ044) was purchased from Wako Chemicals. Oꢀ(Benzotriazoleꢀ1ꢀyl)ꢀN,N,N’,N’ꢀ
tetramethyluronium hexafluorophosphate (HBTU) was purchased from Iris Biotech. All solvents were
bought from commercial sources and used as received. The cyclic peptide and the chain transfer
agents CPAETC and E(CPAETC)2 were synthesized according to previously reported protocols.23ꢀ24
[(Cp*)ꢀIrꢀ(phpy)(Cl)] and [(Cpxph)ꢀIr(phpy)(Cl)] were synthesized and characterized as previously
described.25ꢀ26
Characterization methods
NMR spectra were recorded on a Bruker DPXꢀ300 instrument. Mass spectra were obtained on an
Agilent 6130B Single Quad. Molecular weights and dispersities of the polymers were assessed by size
exclusion chromatography (SEC) on a Polymer Laboratories PLꢀGPC 50 Plus system in DMF with
0.1% LiBr, using a poly(methyl methacrylate) calibration. Infrared absorption experiments were
performed on a Bruker VECTORꢀ22 FTIR spectrometer. The incremental refractive index, dn/dC,
was determined by measuring the refractive index of the polymer in water at various concentrations
ranging from 0.25 to 2 mg/mL, using a Shodex RI detector operating at a wavelength of 632 nm.
SANS was carried out on the Sans2d small angle diffractometer at the ISIS Pulsed Neutron Source
(STFC Rutherford Appleton Laboratory, Didcot, UK).27ꢀ28 A collimation length of 4 m and incident
wavelength range of 1.75 – 16.5 Å was employed. Data were measured simultaneously on two 1 m2
detectors to give a qꢀrange of 0.0045 – 1.00 Åꢀ1. The smallꢀangle detector was positioned 4 m from
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the sample and offset vertically 60 mm and sideways 100 mm. The wideꢀangle detector was
positioned 2.4 m from the sample, offset sideways by 980 mm and rotated to face the sample. The
wave vector, q, is defined as:
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ꢀ =
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ꢈ
where θ is the scattered angle and λ is the incident neutron wavelength. The beam diameter was 8 mm.
Samples were prepared at a concentration of 5 mg/mL in deuterated phosphate buffer saline (PBS),
and were contained in 2 mm path length quartz cells. Each raw scattering dataset was corrected for the
detectors efficiencies, sample transmission and background scattering and converted to scattering
crossꢀsection data (ꢉΣ/ꢉΩ vs. q) using the instrument software.29 These data were placed on an
absolute scale (cmꢀ1) using the scattering from a standard sample (a solid blend of hydrogenous and
perdeuterated polystyrene) in accordance with established procedures.30
Light scattering measurements were obtained using an ALVꢀCGS3 system operating with a vertically
polarized laser with a wavelength of 632 nm. The measurements were taken at 20°C over a range of
scattering wave vectors.
Synthetic procedures
Synthesis of 2ꢀhydroxypropyl methacrylamide (HPMA)
Potassium carbonate (29 g, 1.1 eq., 0.21 mol) was dispersed in 120 mL of dry DCM. The mixture was
cooled to ꢀ10°C with an iceꢀethanol bath and 1ꢀaminoꢀ2ꢀpropanol (14.5 mL, 1 eq., 0.19 mol) was
added. Methacryloyl chloride (18.5 mL, 1 eq., 0.91 mol) was diluted with 20 mL of dry DCM, and
added dropwise to the previous mixture, while maintaining the temperature at ꢀ10°C. Once the
addition was complete, the reaction was left to warm up to room temperature and stirred overnight.
After filtration and drying over MgSO4, the DCM was evaporated and a white solid was obtained. The
product was dissolved in methanol and washed with hexane, and the methanol phase was evaporated.
The obtained solid was recrystallized from acetone. Yield: 45% (10.2 g). 1HꢀNMR (d6ꢀDMSO, 300
MHz, ppm): δ = 7.82 (broad s, 1H, NH) , 5.65 (s, 1H, CH vinyl), 5.31 (s, 1H, CH vinyl), 4.71 (s, 1H,
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OH), 3.69 (m, 1H, CH), 3.05 (m, 2H, CH2), 1.85 (s, 3H, CH2=C(CH3)), 1.00 (d, J = 6 Hz, 3H, CHꢀ
CH3). 13CꢀDEPTꢀNMR (d6ꢀDMSO, 75 MHz, ppm): δ = 167.7, 139.9, 118.9, 85.1, 46.7, 21.1, 18.6.
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Synthesis of 2ꢀ(3ꢀ(pyridinꢀ4ꢀylmethyl)ureido)ethyl)methacrylate (PUEMA)
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2ꢀIsocyanatoethyl methacrylate (2.2 g, 14.15 mmol) and 4ꢀaminomethyl pyridine (1.53 g, 1 eq., 14.15
mmol) were mixed in dry DCM (10 mL) and left to stir at room temperature for 10 min. The solvent
was evaporated under reduced pressure and PUEMA was collected as a white powder. Yield: 95%
(3.53 g). 1HꢀNMR (CDCl3, 300 MHz, ppm): δ = 8.52 (d, 2H, CHꢀNꢀCH pyridine), 7.19 (d, 2H, CHꢀCꢀ
CH pyridine), 6.09 (s, 1H, CH vinyl), 5.59 (s, 1H, CH vinyl), 5.13 (broad t, 1H, NH urea), 4.97 (broad
t, 1H, NH urea), 4.38 (d, 2H, NHꢀCH2ꢀpyridine), 4.25 (t, 2H, OꢀCH2), 3.53 (q, 2H, OꢀCH2ꢀCH2), 1.93
(s, 3H, CH3). 13CꢀDEPTꢀNMR (CDCl3, 75 MHz, ppm): 166.9, 157.5, 149.2, 148.3, 135.3, 125.5,
121.4, 63.4, 42.4, 39.0, 17.6. FTIR: (ν, cmꢀ1): 3313 (NꢀH stretch, urea), 1720 (C=O stretch,
methacrylate), 1623 (C=C stretch, alkene), 1585 (C=O stretch, urea). MS (ESI): [M+Na]+ calculated:
286.1, found: 285.9.
Copolymerization of HPMA and PUEMA
Chain transfer agent (CTA), monomers, initiator (VA 044) and solvent (30 % water in DMSO) were
introduced into a flask equipped with a magnetic stirrer and sealed with a rubber septum. (See Table
S1 for detailed conditions). The solution was degassed by bubbling nitrogen through it for 15 min,
and then put in an oil bath at 44°C for 24 h in the case of kinetics experiments. Subsequent
polymerizations were stopped after 18 h, when the conversion of PUEMA and HPMA reached > 99%
and 75%, respectively. The polymers were precipitated in iceꢀcold acetone and dried under vacuum.
Conjugation of polymers to CP
The cyclic peptide, pHPMA51ꢀcoꢀPUEMA3.5 (2.5 eq.) and HBTU (3.75 eq.) were solubilized in
DMSO (1.5 mL). NMM (6 eq.) was added to the reaction mixture and it was left to stir at room
temperature for 2 h. After the reaction, DMSO was removed using a stream of N2 and the conjugates
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were dissolved in water and purified from the excess polymer using a centrifugal ultrafiltration unit
with a molecular weight cut off of 30 kDa (Amicon® Ultra centrifugal filter). The isolated conjugates
were freezeꢀdried.
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Complexation of the iridium complexes to the conjugates
[(Cp*)ꢀIrꢀ(phpy)(Cl)] (15.6 mg, 3.02.10ꢀ5 mol) was dissolved in water/methanol 1/1 (12 mL) in a vial
wrapped in aluminium foil, and silver nitrate (5.12 mg, 1 eq., 3.02.10ꢀ5 mol) was added. The mixture
was stirred at room temperature overnight, and centrifuged to remove the silver chloride salts. The
supernatant was decanted in a round bottomed flask and CPꢀ(p(HPMA51ꢀcoꢀPUEMA3.5))2 (25.7 mg, 3
mol eq. of Ir per pyridine unit, 1.44.10ꢀ6 mol) was added. The reaction was left to stir for 2 days, after
which most of the solvent was evaporated, keeping ~ 1 mL. The drugꢀloaded conjugates were purified
from the excess complex on a disposable size exclusion column (PD10, Sephadex G25, GE
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Healthcare) and freeze dried. The drug loading was determined by HꢀNMR in MeOD. The same
protocol was used for the complexation of [(Cp*)ꢀIrꢀ(phpy)(Cl)] to pHPMA93ꢀcoꢀPUEMA7 and of
[(Cpxph)ꢀIrꢀ(phpy)(Cl)] to CPꢀ(p(HPMA51ꢀcoꢀPUEMA3.5))2 and pHPMA93ꢀcoꢀPUEMA7.
Inductively coupled plasma (ICP)
Measurements of trace Ir in biological samples were determined using ICP techniques. Ir standard
solution (iridium chloride, 995 ± 4 µg/mL in 10% v/v hydrochloric acid) was purchased from
Inorganic Ventures. Ultraꢀpure nitric acid (72%) was freshly distilled and diluted using milliQ water
to achieve 3.6% v/v working concentration.
For iridiumꢀcontaining solutions in culture medium, iridium concentration was determined using a
Perkin Elmer Optima 5300 DV Optical Emission Spectrophotometer (ICPꢀOES) with standard
addition of sodium chloride (TraceSELECT) to freshly prepared calibration standards (50ꢀ700 ppb) to
match the sample matrix. Data were processed using WinLab32 V3.4.1 for Windows (Perkin Elmer).
For cell digest samples, iridium concentration (ng × 106 cells) was determined using a Agilent
Technologies 7500 series ICPꢀMS in noꢀgas and Heꢀgas mode. Calibration standards for 193Ir were
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freshly prepared (0.1ꢀ1000 ppb) and an internal standard (167Er, 50 ppb) was used. Data were
processed using ChemStation version B.03.05 (Agilent Technologies, Inc.).
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In vitro testing
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Cell Culture
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Human A2780 ovarian carcinoma cells were obtained from the European Collection of Cell Cultures
(ECACC) used between passages 5 and 18 and were grown in Roswell Park Memorial Institute
medium (RPMIꢀ1640) or Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%
v/v of foetal calf serum, 1% v/v of 2 mM glutamine and 1% v/v penicillin/streptomycin. HOF human
ovarian fibroblasts were obtained from ScienCell Research Laboratories, and maintained in fibroblast
medium supplemented with 2% v/v of foetal calf serum, 1% v/v of penicillin/streptomycin and 1%
v/v of growth factor serum. They were grown as adherent monolayers at 37°C in a 5% CO2
humidified atmosphere and passaged at approximately 70ꢀ80% confluence.
Growth Inhibition Assay
Briefly, 5000 cells were seeded per well in 96ꢀwell plates. The cells were preꢀincubated in the
corresponding drugꢀfree media at 37°C for 48 h before adding different concentrations of the
compounds to be tested. Stock solutions of the Ir(III) complexes themselves were firstly prepared in
5% v/v DMSO and 95% v/v either PBS or cell culture medium, for the ‘conjugated complexes’ or
polymer/conjugate controls the use of DMSO was omitted. The concentration of Ir solutions was
determined by ICPꢀOES before drug administration. In all cases, stock solutions were further diluted
in cell culture medium until working concentrations were achieved, maintaining the total amount of
DMSO below 1%. The drug exposure period was 24 h. After this, supernatants were removed by
suction and each well was washed with PBS. A further 72 h was allowed for the cells to recover in
drugꢀfree medium at 37°C. The SRB assay was used to determine cell viability. Absorbance
measurements of the solubilised dye (on a BioRad iMark microplate reader using a 470 nm filter)
allowed the determination of viable treated cells compared to untreated controls. IC50 values
(concentrations which caused 50% of cell growth inhibition), were determined as duplicates of
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triplicates in two independent sets of experiments and their standard deviations were calculated. Twoꢀ
tailed tꢀtests were performed assuming equal variance.
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Equipotent metal accumulation in cancer cells
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Cell accumulation studies of Iridium complexes were conducted on A2780 ovarian cells. Briefly, 3 x
106 cells were seeded on a Petri dish. After 24 h of preꢀincubation time in drugꢀfree medium at 37°C,
the compounds were added to give final concentrations equal to IC50 (the concentration of Ir solutions
was determined by ICPꢀOES before drug administration) and 24 h of drug exposure were allowed.
After this time, cells were washed, treated with trypsin, counted, and cell pellets were collected. Each
sample was digested in Wheaton vꢀvials using 200 ꢁL of 72% v/v nitric acid at 80°C overnight (72%)
and then diluted with milliQ water to achieve final concentration of 3.6% v/v acid. 193Ir concentration
was determined by ICPꢀMS in both noꢀgas and Heꢀgas mode. These experiments were all carried out
in triplicate and the standard deviations were calculated.
Equimolar metal accumulation in cancer cells
Experiments were carried out as described above with the following modifications: drug
concentrations were equimolar and equal to 0.60 ꢁM. Drug exposure times were kept unchanged
(24 h).
Timeꢀdependent metal accumulation in cancer cells
Experiments were carried out as described above with the following modifications: drug exposure
times were: 1 h, 2 h, 4 h, 6 h, 14 h and 24 h at equipotent concentrations equal to IC50 values.
Metal accumulation in cancer cells at 4°C
Experiments were carried out as described above with the following modifications: plates were placed
at 4°C 20 min prior to dosing at IC50 values, and drug exposure was limited to 4 h.
Cellular metal distribution
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Cell pellets were obtained as described above, and were fractionated using the FractionPREP kit from
BioVision according to the supplier’s instructions. Each sample was digested in Wheaton vꢀvials
using 200 ꢁL of 72% v/v nitric acid at 80°C overnight (72%) and then diluted with milliQ water to
achieve final concentration of 3.6% v/v acid. 193Ir concentration was determined by ICPꢀMS in both
noꢀgas and Heꢀgas mode. These experiments were all carried out in triplicate and the standard
deviations were calculated.
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Results and discussion
Synthesis of the polymer and conjugate carriers
The wellꢀstudied monomer building block 2ꢀhydroxypropyl methacrylamide (HPMA) was chosen as
the main monomer for designing the polymeric drug used in this work. In order to provide a binding
site for the ligation of anticancer iridium complexes, a comonomer was introduced. Pyridine was
chosen as the binding ligand, since it can readily replace the chloride ligand present on the selected
organoiridium precursor. Moreover, organoiridium pyridine complexes themselves exhibit good
anticancer activity (0.1ꢀ10 µM depending on the cell line and the nature of the other ligands).22, 31 2ꢀ
(3ꢀ(Pyridinꢀ4ꢀylmethyl)ureido)ethyl)methacrylate (PUEMA) was therefore synthesized and
characterized (see Supporting Information, Figures S3ꢀ5).
The cyclic peptideꢀpolymer conjugate 2 containing pHPMAꢀcoꢀPUEMA was synthesised using
Reversible Addition Fragmentation Chain Transfer (RAFT) copolymerisation of HPMA with 5 %
PUEMA, leading polymer 1, followed by coupling of the polymer to the chosen cyclic peptide,
cyclo(DꢀLeuꢀLysꢀD
ꢀLeuꢀTrp)2 (Scheme 1 and Table 1).32ꢀ33
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Scheme 1. Synthesis of materials.
Kinetic measurements of the copolymerization using (4ꢀcyano pentanoic acid)yl ethyl trithiocarbonate
(CPAETC) showed that PUEMA was consumed significantly faster than HPMA, indicating a higher
reactivity (Figure S6). The direct consequence of this observation is that the functional monomer
tends to be incorporated first, meaning that most of the pyridine ligands for metallodrug attachment
will ultimately be located towards the αꢀchain end of polymer 1. The bifunctional RAFT agent 1,2ꢀ
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bis((4ꢀcyano pentanoic acid)yl ethyl trithiocarbonate)ꢀethane diamide (E(CPAECTC)2) was used to
provide a non selfꢀassembling polymeric control 3 to the peptideꢀpolymer conjugate 2 (Scheme 1).
The obtained polymers 1 and 3 were well defined, with narrow dispersities of 1.16 and 1.12,
respectively (Table 1 and Figure S7). Using monomer conversions, the final content of PUEMA in
polymer 1 and polymer 3 was estimated to be 6.5 and 7 %, respectively. These values were confirmed
by 1H NMR of the precipitated polymer (Figures S8ꢀ9).
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Copolymer 1 was attached to the cyclic peptide by reacting the amine groups present on the cyclic
peptide with the carboxylic acid endꢀgroup of 1, using
Oꢀ(benzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀ
tetramethyluronium hexafluorophosphate (HBTU) as a coupling reagent. The coupling was complete
after 1 h and the waterꢀsoluble nature of these conjugates enabled straightforward purification by
dialysis. The purified conjugate 2 was wellꢀdefined, with a dispersity of 1.19 (Table 1 and Figure
S10). Interestingly, since the attachment occurs at the αꢀchain end of the polymer, the pyridine units
used for organoiridium attachment are located on average close to the cyclic peptide core, allowing
the HPMAꢀricher shell to provide shielding of the drug from the environment.
Table 1. Summary of polymers used in this work.
a
b
Mn, th
Mn, GPC
Entry
Material
Ðb
(g.molꢀ1)
8400
(g.molꢀ1)
14 800
29200
p(HPMA51ꢀcoꢀPUEMA3.5)
CPꢀ(p(HPMA51ꢀcoꢀPUEMA3.5))2
pHPMA93ꢀcoꢀPUEMA7
1.16
1.19
1.12
1
2
3
17800
15700
21400
a Determined by 1H NMR. b Determined by SEC using DMF (0.1% LiBr) as eluent, calibrated with pMMA standards.
Complexation of organoiridium anticancer drugs
The attachment of the selected iridium complexes to the polymer 3 and cyclic peptideꢀpolymer
conjugate 2 was achieved following a ligand exchange procedure previously used to synthesize
pyridine analogues of the chlorideꢀcontaining drugs.31 The chloride ligand of the iridium complexes
4a and 4b was first removed using silver nitrate, followed by complexation to the pyridine units of the
polymer chains (Scheme 2). The complexes used in this work were the [(Cp*)Ir(phpy)Cl]
(abbreviated as IrꢀCp*, 4a), which contains pentamethylcyclopentadienyl (Cp*) and C,Nꢀchelated
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phenylpyridine (phpy) as ligands,26 as well as the more hydrophobic [(Cpxph)Ir(phpy)Cl] (IrꢀCpxph,
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4b), in which the Cp* is replaced by an extended phenyltetramethylcyclopentadienyl (Cpxph) ligand.25
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Scheme 2. Synthesis of drugꢀloaded compounds: complexation of organoiridium complexes 4a and
4b to conjugate 2 and polymer 3.
An excess of the iridium complex 4 (3 mol equiv per pyridine site) was used to maximise the drug
loading onto the polymer and peptideꢀpolymer conjugate. After purification by size exclusion
chromatography, the drugꢀbearing compounds (conjugates 2a and 2b, and polymers 3a and 3b) were
1
characterized by H NMR spectroscopy (Figure 1 and Figures S11ꢀ13). The shifts of characteristic
peaks of the complex and of the pyridine units as well as the broadening of the signals associated with
the complex clearly indicate full complexation of the drug to the polymer.
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1
Figure 1. H NMR characterization of the attachment of complex 4a onto conjugate 2, affording
conjugate 2a.
Characterization of supramolecular nanotubes
In order to confirm the selfꢀassembly of the conjugates into tubular structures, the drugꢀbearing
conjugate 2a was dissolved in PBS and small angle neutron scattering (SANS) and static light
scattering (SLS) measurements were performed (Figure 2).
Figure 2. A) Static light scattering profile of conjugate 2a in solution in PBS at different
concentrations. B) Small angle neutron scattering profile of conjugate 2a (orange dots) and its fit
using a cylindrical micelle model (black line).
The SANS data were fitted with a cylindrical micelle model (Figure 2B), which accounts for both the
overall elongated shape provided by the selfꢀassembled cyclic peptide core (characteristic qꢀ1
dependency at low q values: cylinder form factor) and the polymer arms (Gaussian chain form factor
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at high q values).34 By fitting geometrical parameters, reasonable values were obtained starting from a
radius of 5 Å for the peptide core, in accordance with previously reported results (see Supporting
Information, Table S2).8, 35 However, the maximum length of these tubes cannot be fully determined
by these SANS measurements, as the scattering intensity is still increasing at the lowest measured q
values and does not show the formation of a plateau which is indicative of a limited length. Static
light scattering (SLS) measurements were then carried out since this technique allows access to a
larger window of observation (Figure 2A). SLS experiments showed that the molecular weight of the
assemblies was not affected by the concentration of the solution (within the tested range) and this
molecular weight was determined to be 9.74.105 ± 0.37.105 g.molꢀ1 for the drugꢀbearing conjugate.
Using the molecular weight of the unimer and the previously reported distance between adjacent
peptides,7, 35 the average length of the objects can be determined as 21.8 ± 0.9 nm, corresponding to
46 assembled conjugates (see Supporting Information, Figure S14 and Table S3).
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Anticancer activity and cellular accumulation
The antiproliferative activity of the polymers and conjugates was initially determined for A2780
human ovarian cancer cells, as IC50 values, the concentrations at which 50% of cell growth is
inhibited (Figure 3).
Figure 3. Antiproliferative activity in A2780 cells: IC50 values for free organoiridium complexes,
drugꢀbearing polymers, and drugꢀbearing conjugates using IrꢀCp* (dark) and IrꢀCpxph (light) as the
drug. *p < 0.05, **p < 0.01, ***p < 0.001.
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The drugꢀfree control samples (polymer alone and cyclic peptideꢀpolymer conjugates) were nonꢀtoxic
in the tested range of concentrations (Figure S15). However, the organoiridiumꢀcontaining samples
showed high activity. For both drugs, the IC50 of the loaded polymers was slightly higher (however
still in the same order of magnitude) than that of the free drug: 1.90 ± 0.22 ꢁM for the polymer 3a
compared to 1.15 ± 0.04 ꢁM for IrꢀCp*, and 1.80 ± 0.09 ꢁM for the polymer 3b compared to 0.95 ±
0.03 ꢁM for IrꢀCpxph. For IrꢀCp*, no major difference was observed between the polymer and the
conjugate (1.90 ± 0.22 ꢁM for the polymer 3a compared to 1.70 ± 0.03 ꢁM for the conjugate 2a).
However the IC50 of the conjugate 2b (0.61 ± 0.02 ꢁM) in A2780 was 3x lower than that of the drugꢀ
loaded polymer 3b (1.8 ± 0.09 ꢁM). This substantial increase in activity between the polymer and the
nanotubes suggests that the selfꢀassembly has a noticeable impact on the behaviour of the carrier.
Furthermore, the conjugate 2b was twice as potent as the free drug IrꢀCpxph. For all the studied
compounds the IC50 values were lower for IrꢀCpxph, which is a more hydrophobic complex than for Irꢀ
Cp*. This result shows the same trend as previously reported data for the complexes themselves25ꢀ26
(the present values are slightly different due to the updated protocol involving confirmation of Ir
concentrations by ICPꢀOES measurements). These organoiridium complexes have not been
previously conjugated to delivery vectors, but it is clear from previous studies that increasing the
hydrophobicity of the cyclopentadienyl ligand enhances their antiproliferative activity.22, 25, 36 The
present report suggests that this is also the case when the complexes are conjugated to polymers and
cyclic peptideꢀpolymer conjugates. For this reason, further studies focussed on the more potent
compounds bearing IrꢀCpxph.
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The antiproliferative activity of the three compounds (IrꢀCpxph as free drug, loaded onto the polymer
(3b), and the conjugate (2b)) was then determined against human ovarian fibroblasts (HOF), a model
for healthy, nonꢀcancerous cells, and compared to that against A2780 ovarian cancer cells (Figure 4).
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Figure 4. Antiproliferative activity of free drug IrꢀCpxph, drugꢀbearing polymer 3b and drugꢀbearing
conjugate 2b in A2780 (cancer, dark) and HOF (healthy, light) ovarian cells. **p < 0.01, ***p <
0.001.
All compounds were found less toxic in normal HOF cells than in A2780. The observed selectivity is
likely attributable to the nature of the drug. This class of iridium anticancer complexes has previously
been shown to exhibit selectivity towards cancer cells, which is believed to rely on interference with
cellular redox homeostasis (the ability of the cells to regulate their levels of reactive oxygen species)
in cancer cells specifically.21
The increased activity of the conjugate 2b compared to the polymer 3b and the free drug in A2780
cells may be related either to enhanced cellular accumulation or to a more efficient mode of action,
for example through a different partitioning of the drug amongst the cell organelles. The possibility of
enhanced accumulation (the balance of the uptake and efflux equilibrium), was investigated by
exposing A2780 cells to the IrꢀCpxph compounds (free drug, polymer and conjugate) at their respective
IC50 values. At regular intervals over 24 h, cells were collected and digested in nitric acid to determine
the amount of iridium accumulated using inductively coupled plasma mass spectrometry (ICPꢀMS)
(Figure 5).
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Figure 5. Iridium accumulated in A2780 cells after 24 h of exposure to the free drug IrꢀCpxph (orange
squares), the drug bearing polymer 3b (green diamonds) and the drug bearing conjugate 2b (purple
circles) at equipotent IC50 conditions.
Figure 5 shows that the kinetics of uptake are different for the three compounds: the maximum
amount of iridium accumulated is reached after only 2 h in the case of the conjugate 2b, after which it
remains the same, while for the polymer 3b, the amount is still increasing after 24 h. In the case of the
free drug, the amount of iridium peaks at 4 h, before decreasing slightly. Such cellular efflux is
common for organometallic complexes.37ꢀ38 These differences in the rate and profile of uptake suggest
that the conjugate interacts differently with the cells. After 24 h of exposure to the free drug, the drugꢀ
bearing polymer and the drugꢀbearing conjugate under equipotent conditions, each at their IC50
concentrations (0.95 ꢁM, 1.80 ꢁM and 0.61 ꢁM, respectively), 21.6 ± 0.7 ng, 28.7 ± 1.6 ng and 9.3 ±
0.2 ng of iridium per million cells were accumulated, respectively. Taking the differences of the IC50
values into account, similar percentages of the total amount of iridium administered are retained: 7.7 ±
0.2 % of the initial amount was accumulated for the drug, 6.5 ± 0.4 % for the polymer and 6.5 ± 0.1 %
in the case of the conjugate. These values are similar to those observed previously for organometallic
drugs.31, 38
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The results suggest that the increased cytotoxicity of the conjugate 2b compared to the other
compounds is not due to enhanced uptake of iridium by the cells, and are further supported by the
amount of iridium accumulated after exposure under equimolar conditions. The cells were incubated
in the presence of the three compounds at the lowest IC50 (0.61 ꢁM). Under these conditions, similar
amounts of iridium were accumulated (10.1 ± 0.1 ng per million cells for the free drug, 10.5 ± 0.2 ng
for the polymer and 9.3 ± 0.8 ng for the conjugate). We conclude that the attachment of the drug to
the polymer or the conjugate does not affect the extent of the accumulation of iridium in the cells,
which suggests that the nanotubes rather exhibit a more effective mode of action, for example through
a different partitioning of the drug amongst the cell organelles.
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In order to evaluate this partitioning, equipotent uptake experiments were repeated and the cell pellets
collected after 24 h. The iridium content of the membrane, cytosol, cytoskeleton and nucleus fractions
was determined by fractionation of the cell compartments, and the results are shown in Figure 6A.
The total amount of iridium in each fraction follows the trend previously observed for whole cells: the
amount of iridium increases in the order conjugate < drug < polymer. The percentage of iridium in the
membrane fraction increases slightly in the order drug (56 %) < polymer (65 %) ≈ conjugate (69 %),
which may indicate that the polymerꢀconjugated drugs favour an endocytosisꢀmediated pathway,
since endosomes and lysosomes are collected in the membrane fraction. To confirm that the polymer
and conjugate follow an energyꢀdependent mechanism, accumulation experiments were undertaken at
4°C, conditions known to block endocytosis processes (Figure 6B).39 At 4°C the free drug
accumulated to an extent of 3.8 ± 0.2 ng Ir per 106 cells, which corresponds to about 15% of the
amount accumulated at 37°C, suggesting that energyꢀindependent pathways (such as passive
diffusion) play at least a partial role in the cellular accumulation of the free drug, in accordance with
previous reports.38 The polymer 3b accumulated to a lesser extent, with 1.9 ± 0.3 ng Ir per 106 cells. In
contrast the conjugate 2b did not accumulate significantly in these conditions, supporting the
hypothesis that cell entry involves energyꢀdependent mechanisms.
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Figure 6. A) Iridium content of the membrane, cytosol, cytoskeleton and nucleus fractions of A2780
cells after 24 h of exposure to the IrꢀCpxph compounds at equipotent IC50 concentrations. B) Cellular
accumulation of Ir after 4 h of exposure to the IrꢀCpxph compounds at equipotent IC50 concentrations at
4°C and 37°C.
Conclusions
We report the synthesis of novel cyclic peptideꢀpolymer conjugates able to carry organoiridium
anticancer complexes. 2ꢀHydroxypropyl methacrylamide (HPMA) was copolymerized with a novel
pyridineꢀcontaining monomer which provides a specific binding site for the complexation of highly
potent candidate anticancer complexes [(Cp*)Ir(phpy)Cl] and [(Cpxph)Ir(phpy)Cl]. The copolymer
was then conjugated to selfꢀassembling cyclic peptides using HBTU coupling. The selfꢀassembly of
these conjugates was studied by static light scattering and small angle neutron scattering, which
revealed that the building blocks form short cylinders about 20 nm in length on average. These drugꢀ
bearing nanotubes exhibited comparable or increased toxicity towards human ovarian cancer cells
compared to the free drug. Remarkably, their toxicity towards a healthy cell model was lower than the
free drug, suggesting a degree of selectivity towards cancer cells. Interestingly, the nanotubes also
showed higher toxicity towards cancer cells when compared to the drugꢀbearing polymers used as a
nonꢀassembling control and more conventional example of a drug delivery vector. The analysis of the
amount of iridium accumulated in the cells after equipotent and equimolar uptakes revealed that a
similar percentage of iridium enters the cells in each case, indicating that the drugꢀbearing conjugates
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do not enhance the iridium uptake, but rather exhibit a more efficient mode of action. Investigations
of the mechanisms of entry and partitioning profile of the drug into different organelles revealed that
energyꢀdependent mechanisms of cell uptake account for a higher fraction of the accumulated iridium
in the case of the polymer and conjugate than for the free drug.
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Supporting information
Characterization of monomers and polymers, polymerization kinetics, SANS and SLS analysis, cell
viability in presence of drugꢀfree compounds.
Acknowledgements
We thank the Royal Society Wolfson Merit Award (WM130055; S.P.), the MonashꢀWarwick
Alliance (S.C.L.; S.P.; J.S.) and the ARC (DP140100241; K.A.J.; S.P.), CRUK/EPSRC
(C53561/A19933; C.S.ꢀC.; P.J.S.; S.P.), ERC (TUSUPO 647106; S.C.; S.P.) for financial support.
J.C.B. thanks the German Science Foundation (DFG) for granting a full postdoctoral fellowship (BR
4905/1ꢀ1). We thank ISIS for neutron scattering beam time and Dr. Sarah Rodgers for instrument
training and support.
Authors contributions
Conceptualization – S.C.L., J.S., P.J.S., K.A.J, S.P.; Methodology – S.C.L, J.C.B., I.R.ꢀC.; Resources
– A.H.; Formal Analysis – S.C.L, J.P.C.C., S.C.; Investigation – S.C.L., I.R.ꢀC., C.S.ꢀC, JꢀI.S.;
Writing – Original Draft, S.C.L; Writing – Review & Editing, all authors.
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Title: Cyclic peptide-polymer nanotubes as efficient and highly potent drug delivery systems for
organometallic anticancer complexes
Authors: Sophie C. Larnaudie, Johannes C. Brendel, Isolda Romero-Canelón, Carlos Sanchez-Cano,
Sylvain Catrouillet, Joaquin Sanchis, James P. C. Coverdale, Ji-Inn Song, Abraha Habtemariam, Peter J.
Sadler, Katrina A. Jolliffe, and Sébastien Perrier
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