Potentiating the Anticancer Properties of Bisphosphonates by Nanocomplexation with the Cationic Amphipathic Peptide, RALA

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00670

Bisphosphonates (BPs) are a class of bone resorptive drug with a high affinity for the hydroxyapatite structure of bone matrices that are used for the treatment of osteoporosis. However, clinical application is limited by a common toxicity, BP-related osteonecrosis of the jaw. There is emerging evidence that BPs possess anticancer potential, but exploitation of these antiproliferative properties is limited by their toxicities. We previously reported the utility of a cationic amphipathic fusogenic peptide, RALA, to traffic anionic nucleic acids into various cell types in the form of cationic nanoparticles. We hypothesized that complexation with RALA could similarly be used to conceal a BP's hydroxyapatite affinity, and to enhance bioavailability, thereby improving anticancer efficacy. Incubation of RALA with alendronate, etidronate, risedronate, or zoledronate provoked spontaneous electrostatic formation of cationic nanoparticles that did not exceed 100 nm in diameter and that were stable over a range of temperatures and for up to 6 h. The nanoparticles demonstrated a pH responsiveness, possibly indicative of a conformational change, that could facilitate release of the BP cargo in the endosomal environment. RALA/BP nanoparticles were more potent anticancer agents than their free BP counterparts in assays investigating the viability of PC3 prostate cancer and MDA-MB-231 breast cancer cells. Moreover, RALA complexation potentiated the tumor growth delay activity of alendronate in a PC3 xenograft model of prostate cancer. Taken together, these findings further validate the use of BPs as repurposed anticancer agents.

Full text of DOI:10.1021/acs.molpharmaceut.5b00670

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Article
Potentiating the anti-cancer properties of bisphosphonates by
nanocomplexation with the cationic amphipathic peptide, RALA.
Ashley S. Massey, Sreekanth Pentlavalli, Richard Cunningham, Cian M McCrudden,
Emma M McErlean, Philip Redpath, Ahlam A Ali, Stephanie Annett, John W McBride,
Joanne McCaffrey, Tracy Robson, Marie Eugenie Migaud, and Helen O. McCarthy
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00670 • Publication Date (Web): 08 Mar 2016
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Potentiating the anti-cancer properties of bisphosphonates by nanocomplexation
with the cationic amphipathic peptide, RALA.
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Ashley S. Massey, PhD, Sreekanth Pentlavalli, PhD, Richard Cunningham, PhD, Cian
M. McCrudden, PhD, Emma M. McErlean, Philip Redpath, PhD, Ahlam A. Ali, PhD,
Stephanie Annett, John McBride, PhD, Joanne McCaffrey, PhD, Tracy Robson, PhD,
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Marie E. Migaud, PhD, Helen O. McCarthy, PhD*
School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast, Northern
Ireland, BT9 7BL
Corresponding author: Helen O. McCarthy; h.mccarthy@qub.ac.uk; 02890972149
Abstract
Bisphosphonates (BPs) are a class of bone resorptive drug with a high affinity for the
hydroxyapatite structure of bone matrices that are used for the treatment of
osteoporosis. However, clinical application is limited by a common toxicity, BP-
related osteonecrosis of the jaw. There is emerging evidence that BPs possess anti-
cancer potential, but exploitation of these anti-proliferative properties is limited by
their toxicities. We previously reported the utility of a cationic amphipathic
fusogenic peptide, RALA, to traffic anionic nucleic acids into various cell types in the
form of cationic nanoparticles. We hypothesized that complexation with RALA could
similarly be used to conceal a BP’s hydroxyapatite affinity, and to enhance
bioavailability, and thereby enhancing anti-cancer efficacy.
Incubation of RALA with alendronate, etidronate, risedronate or zoledronate
provoked spontaneous electrostatic formation of cationic nanoparticles that did not
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exceed 100 nm in diameter, and that were stable over a range of temperatures and
for up to 6 h. The nanoparticles demonstrated a pH responsiveness, possibly
indicative of a conformational change, that could facilitate release of the BP cargo in
the endosomal environment. RALA/BP nanoparticles were more potent anti-cancer
agents than their free BP counterparts in assays investigating the viability of PC3
prostate cancer and MDA-MB-231 breast cancer cells. Moreover, RALA
complexation potentiated the tumour growth delay activity of alendronate in a PC3
xenograft model of prostate cancer. Taken together, these findings further validate
the use of BPs as repurposed anti-cancer agents.
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Key words: Alendronate; Prostate cancer; Bisphosphonates; Cell penetrating
peptides; Self-assembling nanoparticles
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Introduction
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Over the past two decades, bisphosphonates (BPs) have become mainstream
therapeutics in the treatment of diseases with excessive bone turnover1. The
pharmacological properties of BPs are dictated by their distinct mechanism of
action; binding to the calcium-containing bone matrix, release from the matrix by
osteoclasts and intracellular localisation during the bone remodelling process.
Etidronate is one of the first generation BPs, characterised by a short side-chain
(Fig. 1A), which destabilises osteoclast homeostasis via conversion into an ATP like
molecule, which in turn acts as an inhibitor to a number of ATP-dependent enzymes
and processes.
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The second and third generation of anti-osteoclastic BPs are nitrogen-containing
BPs (N-BPs; e.g. alendronate, risedronate and zoledronate; Fig. 1A), potent
inhibitors of farnesyl pyrophosphate synthase, and significantly more effective than
the first generation BPs in reducing bone remodelling. The anti-osteoclastic activity,
mode of action and selective biodistribution has enabled N-BPs to be utilised as
palliative agents for metastatic bone disease, particularly for breast and prostate
cancer2. In such cancers, where bone metastases are common, the N-BPs reduce
bone resorption and in turn alleviate skeletal related events such as fractures and
bone pain. More recently, however the adjuvant use of BPs with cytotoxic agents
has highlighted their synergistic effects, beyond basic anti-resorptive activities.
Indeed N-BPs exhibit a range of anti-tumour effects in vitro, including inhibition of
tumour growth3, migration, invasion, adhesion and angiogenesis4-6, with synergistic
effects dependent on side chain structure7. Furthermore, a number of clinical trials
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involving more than 20,000 patients observed the positive effects of co-
administration of BPs with hormonal therapies and chemotherapeutics on the
occurrence of bone metastases and recurrence of disease8.
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However, despite more than a decade of research into understanding the molecular
mechanisms of the anti-tumour effects of the BPs, to date the only successful in
vivo applications have been in the prevention of bone metastases9. It is postulated
that the change in the bone microenvironment stemming from the BPs leads to the
positive synergistic pharmacological outcomes, thus supporting the ‘seed and soil’
theory10. There are two factors that limit the translation of in vitro effects to anti-
tumour effects in vivo. Firstly, the presence of a hydroxyl and two phosphonate
groups flanking a carbon atom confers high affinity for the bone matrix and so the
initially circulating BP is not excreted by the kidneys. Secondly, the concentration of
circulating BP required to exert the desired anti-tumour effects is so high that
adverse side-effects will ensue11.
BPs have potential as anti-tumour agents for soft tissue tumours outside the bone
environment. Liposomes have been employed as delivery systems for BPs to
increase circulation times and exploit the anti-tumour effect; liposomal
encapsulation of neridronate increased its cytotoxicity in MDA-MB-231 breast
cancer cells (EC50 of 95 μM, compared to 1.7 μM)12. Furthermore, it inhibited
migration and invasion significantly more than the free drug and at a lower
concentration, and caused a significant reduction in MMP-2 and MMP-9
expression12. Marra et al advanced the liposome model by assessing PEGylated
liposomes for the delivery of zoledronate in both an in vitro and in vivo setting13.
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Upon encapsulation, potentiation factors of greater than one were achieved in all
cell lines assessed, indicating a strong enhancement of tumour cell growth
inhibition13. Intravenous delivery of zoledronate in PEGylated liposomes to a PC3
prostate cancer xenograft model in vivo significantly improved overall survival when
compared to free zoledronate-treated mice13. Anti-tumour effects were further
enhanced when calcium phosphate was used to aid delivery by complexing
zoledronate into nanoparticles prior to encapsulation with PEGylated liposomes14.
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In this study we utilised a peptide delivery system (RALA) to investigate for the first
time the impact of a fusogenic amphipathic peptide on the bioavailability and
potency of BPs, and in particular, alendronate. RALA is an arginine-rich amphipathic
peptide which has shown potential to efficiently deliver nucleic acids in vitro and in
vivo15. RALA consists of a hydrophobic and hydrophilic region, which facilitates
interaction with the lipid bilayers, thus enabling transport of nucleic acids across
cellular membranes. In addition the lower pH in the endosome alters the alpha-
helical conformation of RALA, which enables endosomal disruption and release of
the cargo into the cytosol. We hypothesised that RALA would condense the BP class
of drugs into nano-sized particles, which would significantly improve the potency,
anti-tumour effects and confer significant repurposing potential.
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Experimental
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Analytical TLC was performed with Merck Silica gel 60 F254 plates. Visualisation was
accomplished by UV-light (λ = 254 nm) and/or staining with an anisaldehyde or
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potassium permanganate solution, followed by heating. H, 13C and 2D (H-COSY,
HMQC) NMR spectra were all recorded on Brüker avance DPX 400. The chemical
shifts (δ) are reported in ppm (parts per million). High-resolution mass spectrometry
(HRMS) was recorded on a VG Quattro Triple Quadropole Mass Spectrometer (ES).
pH measurements were performed using a HANNA HI 2211 pH meter.
Synthesis of the BP
All BPs were used in their acidic forms. Risedronic acid16 and zoledronic acid17-19
were synthesised in house using literature procedure while alendronate and
etidronate were obtained from Aroz Technologies (Cincinnati, Ohio, USA). All other
reagents were purchased from Sigma Aldrich and used as supplied without any
further purification.
Synthesis of Risedronic acid:
To 3-pyridyl acetic acid hydrochloride (2.0 g, 11.5 mmol, 1.0 eq), in a 100 mL two
necked flask equipped with a reflux condenser, was added neat phosphorous acid
(3.6 g, 43.8 mmol, 3.8 eq) and p-nitrophenol (12.0 g, 86.3 mmol, 7.5 eq). The
mixture was heated to 90 °C for 2 h until all of the solid had melted, forming a thick
suspension. Phosphorous trichloride (7.0 g, 50.0 mmol, 4.3 eq) was added to this
suspension, kept at 90 °C for 6 h, after which a yellow, gel-like material was formed.
After cooling to room temperature (19°C), water (20 mL) was added and the
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mixture was left at reflux for 12 h. Methanol (15 mL) was then added, and the
precipitated product was kept stirring at 0 ° C for 2 h. The white, amorphous
product obtained was filtered under vacuum using Whatman filter paper and
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washed with methanol (100 mL) to yield 3.3 g (80 %) of the titled compound. H
NMR (D2O, 400 MHz): δ ppm 3.37 (2H, t, JHP = 12.1 Hz, CH2Pyr), 7.81-7.87 (1H, m,
Pyr), 8.50 (2H, t, J = 6.0 Hz, Pyr), 8.65 (1H, s, Pyr); 13C NMR (D2O, 125 MHz): δ ppm:
37.0 (CH2Pyr), 73.1-75.7 (t, JCP = 128.3 Hz. PCP), 126.8 (Pyr), 138.8 (Pyr), 139.5 (Pyr),
143.3 (Pyr), 149.8 (Pyr); 31P-NMR (D2O, 162 MHz): δ ppm: 16.6; HRMS (ES+ , m/z)
calcd for (M+H)+ C7H12O7P2: 284.0089; found: 284.0082
Synthesis of imidazol-1-yl-acetic acid hydrochloride:
To a solution of imidazole (0.5 g, 7.4 mmol, 1.0 eq) and Cs2CO3 (2.9 g, 8.8 mmol, 1.2
eq) in dry dimethylformamide (30 mL) was added ethyl bromoacetate (0.9 mL, 8.1
mmol, 1.1 eq) at RT and stirred for 24 h. The inorganic salts were filtered off, and
the filtrate was concentrated. The residue was then taken up in dichloromethane
(40 mL) and washed with H2O (2 x 50 mL). The organic phase was dried over
(MgSO4), filtered through Whatman filter paper, and concentrated to afford ethyl-
1H-imidazole-1-acetate. The crude ester intermediate was solubilised in
dioxane/6M HCl (1:1, 20 mL), refluxed for 4 h. After removal of the volatiles under
reduced pressure, the crude imidazol-1-yl acetic acid was obtained as an orange
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solid deemed pure enough by NMR for the subsequent step, 0.8 g (91 %) yield. H
NMR (D2O, 400 MHz): δ 4.97 (2H, s, CH2COOH), 7.33–7.36 (m, 2H, NCHCHN), 8.63 (s,
1H, NCHN); 13C NMR (D2O, 125 MHz): δ ppm: 49.9 (CH2COOH), 119.6, 123.1
(NCHCHN), 136.0 (NCHN), 170.2 (COOH).
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Synthesis of Zoledronic acid:
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To a suspension of imidazol-1-yl-acetic acid hydrochloride (1.0 g, 8.0 mmol, 1.0 eq)
and phosphorous acid (2.0 g, 23.0 mmol, 3.0 eq) in chlorobenzene (10 mL) was
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added phosphorous oxychloride (2.2 mL, 23.0 mmol, 3.0 eq) at 85 C for 2 h then
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heated to 95 °C for 2.5 h. The reaction mixture was cooled to 60 C and water (20
mL) was added. The aqueous layer was separated, collected and refluxed for 18 h. It
was cooled to 19°C and diluted with methanol (20 mL). The mixture was cooled to
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0-5 C and stirred for 3 h. The precipitated solid was filtered, washed with cold
water (100 mL) followed by methanol (100 mL) and dried under vacuum at 60 oC for
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12 h to afford the zoledronic acid (isolated yield 1.3 g (64 %) as a white solid. H
NMR (D2O, 400 MHz): δ ppm: 4.61 (2H, t, JHP = 9.2 Hz, CH2Ar), 7.29 (1H, s, NCH), 7.44
(1H, s, NCH), 8.63 (1H, s, NCHN); 13C NMR (D2O, 125 MHz): δ ppm: 53.4 (CH2Ar),
72.8 (t, JCP = 135.7 Hz, PCP), 119.1 (NCH), 124.7 (NCH), 136.7 (NCHN); 31P-NMR (D2O,
162 MHz): δ ppm 13.9, HRMS (ES+ , m/z) calcd for (M+H)+ C5H9O7P2: 270.9887;
found: 270.9885.
Preparation of RALA peptide
RALA peptide was produced as an acetate salt by solid-state synthesis (FMOC
chemistry) (Biomatik, UK) and was reconstituted in molecular grade water
(Invitrogen, UK) such that a final concentration of 5.8 mg/ml was achieved.
Reconstituted peptide was stored in aliquots at -20°C.
Preparation of BPs
Each BP was reconstituted in molecular grade water to give a concentration of 10
mM. A serial dilution was carried out such that a 1 mM working concentration was
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achieved. The reconstituted BPs were stored at 19°C (room temperature) for
further use.
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Formulation of RALA/BP nanoparticles
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Each BP was complexed with the fusogenic peptide RALA according to a mass ratio
of peptide to BP. For example to achieve a ratio of 10:1, 10 μg of RALA was added to
1 μg of BP in aqueous solution. Samples were allowed to incubate at 19°C for
approximately 30 min before physicochemical characterisation of the nanoparticles.
Nanoparticles were complexed in UltraPure™ DNase/RNase-Free Distilled Water
(Invitrogen), pH 7.0.
Particle size analysis
The mean hydrodynamic particle size measurements for RALA/BP complexes were
performed using Dynamic Light Scattering (DLS) by a Malvern Zetasizer Nano ZS
instrument with DTS software. After incubation of complexes at 19°C for 30 min,
mean size by number was measured at 20°C.
Zeta potential analysis
Zeta potential measurements of RALA/BP complexes were determined by Laser
Doppler Velocimetry using the Malvern Zetasizer Nano ZS instrument and DTS
software (Malvern Instruments, UK). Nanoparticle size and charge were compared
using one-way ANOVA with Dunnett’s multiple comparison test using particle size at
mass ratio 10 as comparator. (Mass ratio 10 was used as a comparator, as this was
the lowest mass ratio at which there was excellent agreement between BPs in
terms of particle size and charge across the range of BPs; mass ratio 10 was used for
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subsequent in vitro and in vivo experiments for this reason; (for comparative figure,
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see Supplementary Figure 1).
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Temperature study
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RALA/BP nanoparticles were formed at a mass ratio of 10:1 and allowed to incubate
at 19°C for 30 min. Malvern Zetasizer Nano ZS with DTS software was used to
measure the mean hydrodynamic size of the complexes over a temperature range
of 4-37°C at 3°C intervals. Nanoparticle sizes were compared using one-way ANOVA
with Dunnett’s multiple comparison test; sizes were analyzed using 4°C, 19°C and
37°C as comparators (cold storage, room temperature and body temperature).
Incubation study
For assessment of the physical stability of the nanoparticles over a period of time,
RALA/BP nanoparticles were prepared at a mass ratio of 10:1 and the mean
hydrodynamic size was measured using Malvern Zetasizer Nano ZS with DTS
software at 30 min intervals starting immediately after particles are prepared and
continuing for 6 h (samples were held at 20°C between intervals). Nanoparticle sizes
were compared using one-way ANOVA with Dunnett’s multiple comparison test
using particle size at 0 min as comparator.
Transmission electron microscopy
RALA/BP nanoparticles were prepared at a mass ratio of 10:1 and loaded onto
carbon reinforced, formvar coated, 200 mesh copper grids (Agar Scientific, UK).
After drying, the grids were stained with 5% aqueous uranyl acetate for 5 min at
19°C. The nanoparticles were imaged using a JEOL 100CXII transmission electron
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microscope at an accelerating voltage of 80 kV and images were captured onto
Kodak 4489 Electron Microscope Film.
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Cell culture and maintenance
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PC3 prostate cancer cells and MDA-MB-231 breast cancer cells (ATCC, Manassas,
VA) were maintained as monolayers in RPMI 1640 medium (Invitrogen, UK) and
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, UK) respectively
supplemented with 10% foetal calf serum (PAA, UK). Cells were maintained in an
incubator at 37°C with 5% CO2 atmosphere. All in vitro cell experiments were
carried out at 37°C in a 5% CO2 incubator. Cultures were routinely assessed for
mycoplasma contamination using Plasmotest reagents (Invivogen, France).
Nanoparticle internalization
Alendronate and zoledronate were complexed (at mass ratio 10) into nanoparticles
using fluorescein isothiocyanate (FITC)-conjugated RALA in the same manner as
described previously. The impact of FITC on the size and charge of nanoparticles
was assessed using Student’s unpaired t-test. PC3s and MDA-MB-231s plated at
50,000 cells per well of 24 well plates were exposed to FITC-RALA/BP nanoparticles,
or free FITC-RALA alone for up to 2 h. Excess extracellular nanoparticles were
removed by washing with PBS, and cells were trypsinized and resuspended in PBS.
The proportion of cells positive for FITC was assessed using a BD FACScalibur flow
cytometer.
In an attempt to demonstrate nanoparticle internalization, PC3s and MDA-MB-231s
grown on glass coverslips (Thermo Scientific, UK) were treated with FITC-RALA/BP
nanoparticles, or free FITC-RALA for 1 h, following which they were fixed and
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mounted onto microscope slides with ProLong® Diamond Antifade Mountant with
DAPI (Life Technologies, UK). Cells were viewed using a Leica SP5 confocal
fluorescence microscope, and analysis was performed using LAS AF Lite software
(Leica). Orthogonal sectioning was performed to determine the cellular localization
of FITC-conjugated particles.
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Cell viability
Cell viability was evaluated by both MTS assay with CellTiter 96 Aqueous One
Solution Cell Proliferation Assay (Promega, UK) and manual counting of the viable
adherent cells using a haemocytometer with Trypan Blue exclusion staining. PC3
prostate cancer cells and MDA-MB-231 breast cancer cells were seeded at a density
of 1 x 104 cells per well and allowed to adhere. Cells were conditioned in OptiMEM
serum-free medium (Invitrogen, UK) for 2 h prior to treatment with solutions of BP
to achieve a final exposure concentration of 5 μM to 2 mM. RALA/BP nanoparticles
were prepared using a mass ratio of 10:1, such that the final concentration of BP
per well was in the range 5 μM to 250 μM. To investigate the impact of RALA
carrying
a
non-toxic cargo, RALA/pEGFP-N1 nanoparticles (which share
physicochemical properties of the RALA/BP particles) were used to treat PC3s, such
that the amount of RALA in each well was equivalent to that found in wells treated
with RALA/alendronate (and approximated the amounts in the other RALA/BP
wells). Cells were incubated for 6 h following treatment before medium was
replaced with complete culture medium and the cells incubated for a further 72 h.
For the MTS assay, treatment was as above. CellTitre reagent was added to each
well to a final concentration of 10%, and plates were returned to the incubator for 2
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h. Absorbance was read at 490 nm using a Bio-Tek Powerwave XS plate reader with
Gen5 software, and any background absorbance was subtracted from readings. Cell
viability was expressed as a percentage of that of the untreated control. Data (for
which dose-response relationships were evident) were normalized using GraphPad
Prism version 6.0g for OS-X (using the equation Y=100/(1+10^((X-LogEC50))) to
recalculate Y values). Non-linear regression analysis was used to fit sigmoidal dose-
response curves to the data, and EC50s for the different conditions were calculated.
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In vivo xenograft model
All protocols conform to the UK Scientific Act of 1986 and were performed under
the project license 2678. The male BALB-C SCID mice were anaesthetised using 4%
isofluorane (Abbott, UK) and injected intradermally on the rear dorsum with 5 x 106
PC3 cells. When the tumour volume reached approximately 100 mm3 the mice were
randomised and divided into 4 groups (n=3/group) and treated with 10 μg
alendronate only, RALA only, RALA/alendronate nanoparticles containing 10 μg
alendronate, or received no treatment. Treatments were thrice weekly for three
weeks, and were by intratumoural injection using a total volume of 100 μl. Digital
calipers were used to measure the length (L), breadth (B) and depth (D) of the
tumours; tumour volume was calculated using the formula -
4
ꢀ = ꢁꢂꢃ
3
where r is half of the geometric mean diameter (GMD), calculated as –
ꢄ
√L*B*D
For analysis of the in vivo results, ANOVA with Bonferroni post hoc test was used.
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Once tumour volume had quadrupled, mice were sacrificed; tumours were removed
and fixed in 10% formalin for 24 h, before embedding in paraffin wax and
sectioning. Standard immunohistochemical procedures were used – xenograft Ki67
content was assessed using anti-Ki67 rabbit polyclonal antibody (ab833 – Abcam,
UK), and horseradish peroxidase-conjugated anti-rabbit secondary antibody (Dako).
Ki67 expression was visualised by addition of diaminobenzidine, and slides were
counterstained in Harris hematoxylin. Whole specimen high-resolution images were
captured, and viewed using PathXL.
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Synthesis and Characterisation of RALA/BP NPs
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Each bisphosphonate (Fig. 1A) was complexed with the amphipathic peptide RALA
(Fig. 1B) according to a mass ratio of peptide to BP.
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RALA /BPs form spherical, nano-sized particles with a positive zeta potential
Characterisation of the RALA/BP nanoparticles confirmed the formation of nano-
sized particles with a positive zeta potential (Fig. 1C-F). This was confirmed by TEM
(Fig. 2). Both the size and charge of the NPs are ideal for cellular entry15. RALA
successfully condensed the three N-BPs alendronate, risedronate and zoledronate
into sub-100 nm sized particles from a mass ratio of 2:1 (RALA/BP) onwards. That
etidronate does not form nanoparticles (<100 nm) below mass ratio 4:1 was not
unexpected; its overall larger negative charge at physiological pH means that
additional RALA is required to achieve a similar degree of neutralization. We did not
assess the impact of pH differential on size/zeta potential across the range of BPs
tested; however, in previous studies, we did not observe any appreciable impact of
pH on the zeta potential of RALA/nucleic acid nanoparticles (unpublished data),
which have similar physicochemical properties to RALA/BP particles. Small
differences in pH may exist in the BP solutions before complexation with RALA, but
we do not anticipate that this impacts on the physicochemical properties of the
resulting nanoparticles.
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RALA/BP nanoparticles show thermal stability over a 6 h time period
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Nanoparticles were formed at a mass ratio of 10:1 for all BPs (Fig. 1C-F) and the
particle size was analysed over a range of temperatures from 4°C to 37°C at 3°C
intervals (Fig. 3A). The significance of size differences was analysed using 4°C (cold
storage), 19°C (room temperature) and 37°C (human body temperature) as
comparators; with the exception of alendronate, when particle size was significantly
larger at 25°C than at 37°C, temperature did not affect particle size in any of the
four BPs in the conditions tested.
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In addition, the RALA/BP NPs were evaluated over a 6 h period (the duration of
cellular exposure in in vitro experiments). All RALA/BP NPs were less than 80 nm in
diameter across temperature range and incubation time (p>0.05; Fig. 3B), with no
aggregation observed which is encouraging for in vivo administration. For each BP,
the mean particle count ranged between 100-300 kcps, indicative of particle
formation and presence. The PDI was consistently between 0.4-0.6 for all BP
nanoparticles.
RALA/BP nanoparticles show stability over 48 h and are pH responsive
Stability of RALA/BP nanoparticles
RALA/BP (10:1 ratio) were analysed using 31P NMR with trimethyl phosphate as
internal standard. All RALA/BP nanoparticles were stable over a period of one week
at neutral pH (one-way ANOVA with Dunnett’s multiple comparison test revealed
no significant variation in phosphorous content between any time point and T0 in
any of the tested BPs (Fig. 4A)). Moreover, the stability of RALA/BP nanoparticles at
the 10:1 ratio was not dependent on the nature of the BPs’ side chains and the
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associated differences in lipophilic and electrostatic properties that each functional
group confers to the BP. This study establishes that RALA can form nanoparticles
with any BP tested (first or second generation) with a relatively low mole to mole
ratio.
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Effect of pH on RALA/BP nanoparticles
NPs were suspended in three aqueous solutions adjusted at different pH as shown
in Fig. 4B-E (pH was adjusted by dropwise addition of 0.1 M HCl or NaOH, as
appropriate). As expected from experience with RALA, these particles were stable at
pH 7 and 9, but particles disassociated at acidic pH15. At this pH, RALA increases its
alpha-helicity, which is essential for disruption of the endosomal membrane to
facilitate release of the cargo into the cytoplasm. Thus the 31P NMRs of all RALA/BP
particles at this pH indicate a complete loss of the phosphonate signal, indicating
that each BP precipitates out of solution along with the RALA peptide.
FITC-RALA/BP nanoparticles are rapidly internalized by cancer cells in vitro
Incubation of FITC-RALA with alendronate or zoledronate resulted in formation of
cationic nanoparticles, similar to particles formed using unconjugated RALA.
Nanoparticles comprising FITC-RALA and alendronate were not significantly
different from those comprising RALA and alendronate, while FITC-
RALA/zoledronate particles were slightly larger and slightly more positively charged
than their unconjugated RALA counterparts (Fig. 5A).
FITC was detectable in PC3s and MDA-MB-231s following 5 min treatment with
alendronate/zoledronate complexed with FITC/RALA, and in cells treated with free
FITC-RALA (Fig. 5B). FITC-RALA treatment produced a diffuse staining pattern in
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both cell types, whereas cells treated with either FITC-RALA/BP complex displayed
more focused cytoplasmic staining. Orthogonal sectioning indicated that FITC
fluorescent signal was produced intracellularly, rather than extracellularly (Fig. 5C).
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RALA/BP nanoparticles demonstrate anti-tumour activity in vitro
PC3 prostate cancer and MDA-MB-231 breast cancer cells were either treated with
free BP or RALA/BP nanoparticles at a range of concentrations for 6 h and then
incubated for 72 h before evaluating cell viability. Complexation with RALA
potentiated the effects of alendronate, etidronate and risedronate, although was
without effect in the case of zoledronate (Table 3 & Figs. 6 & 7); conversely,
complexes formed of RALA and pEGFP-N1 did not negatively impact the viability of
PC3 (Fig. 6A). Perhaps most striking is the anti-cancer effects observed when
etidronate or risedronate, themselves devoid of activity, were delivered using RALA
(Fig. 6A). Of the N-BPs, alendronate was most impressively potentiated by RALA, so
was selected for in vivo studies. Unlike in PC3s, complexation of zoledronate with
RALA slightly increased its potency in MDA-MB-231s; in MDA-MB-231s,
alendronate’s potency was enhanced to a similar degree by RALA complexation as it
was in PC3s (Fig. 7). There was generally good agreement between EC50 values
generated by the two toxicity/viability assays (Figs. 6 & 7).
RALA/alendronate nanoparticles exhibit anti-tumour activity in vivo
Treatment with RALA only had no significant effect on tumour growth (Fig. 8A;
p=0.0792). In contrast, both alendronate- and RALA/alendronate-treated tumours
exhibited a significant growth delay compared to untreated controls (p<0.0001 and
p=0.0004 respectively). Furthermore, the time taken for tumour volume to
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quadruple was also significantly longer in the RALA/alendronate treated mice,
suggesting that potentiation of anti-cancer properties seen in in vitro assays
translated into the in vivo setting (p=0.0019; Fig. 8B). The endpoint of the
experiment was tumour quadrupling; median survival for the groups were 16 days
(control), 14 days (RALA), 19 days (free alendronate) and 26 days
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(RALA/alendronate) (Fig. 8B).
A significant increase in survival time of
RALA/alendronate nanoparticle treated mice of 56.3% was seen when compared to
the untreated control group (p<0.001). The survival time of this group was also
significantly higher compared to the alendronate only treated group at 32%
(p<0.01) (Fig. 8C). Ki67 expression staining of xenograft sections was suggestive of
an impact of RALA/alendronate on the proliferative capacity of the PC3 cells in vivo
– the intensity of Ki67 staining was considerably stronger in control, RALA and
alendronate samples than in the RALA/alendronate samples (Fig. 8D); given that
RALA/alendronate possessed anti-proliferative capacity in vitro, this finding is
unsurprising.
Discussion
This study has demonstrated for the first time that a peptide delivery system, RALA,
can condense four different BPs into positively charged nanoparticles that are less
than 100 nm in diameter, induce a significant potentiation factor in vitro and
significantly retard tumour growth in vivo.
Major differences in mole: mole ratio between RALA and BP did not prevent the
production of nanoparticles by RALA. In fact, with the exception of etidronate, mass
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ratios (RALA:BP) as low as 2:1 elicited the desired size; this corresponded to a mole
to mole ratio of 0.40 (Table 2). The higher ratio of 4:1 observed for the
RALA:etidronate nanoparticles can be explained by the lack of a charged polar
functional group. Here, while one could see the amino or aromatic amino groups of
the N-BPs as potential destabilisers through repulsive electrostatic interactions with
the positively charged arginine residues of RALA, it is proposed that the side chain
groups allows for additional intramolecular ionic interactions between the BPs
partially coordinated to the RALA peptide, thus decreasing the number of total
arginine side chains required for charge neutralisation. As such, in the case of
etidronate, twice as much RALA was required for nano-sized condensation
compared to the N-BPs. Nanoparticles formed with each BP and at a mass ratio of
10 conferred a positive zeta potential of greater than 10 mV, supporting the
counter-ion condensation theory20. Nanoparticle size depends on the polycation,
and it is clear that arginine-rich RALA carries out its role as an effective condensing
agent21. Particle size is critical in terms of uptake into a cell. Although the process is
dependent upon the cell type, it has been shown that nanoparticles less than 200
nm undergo spontaneous endocytosis, and as such are optimal for tumour cell
uptake21. Indeed, it was interesting to note that MDA-MB-231s seemed to
internalize FITC-RALA/BP nanoparticles more rapidly than PC3s (Fig. 5).
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The positive zeta potential of the nanoparticles facilitates close contact of the
nanoparticles with the target cell due to the presence of negatively charged
proteoglycans on the cell surface22. Non-specific electrostatic interactions between
the positively charged nanoparticles and the slight negative charge of the cell can
result in uptake into endosomes. Studies in macrophages and osteoclasts have
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demonstrated that bone-bound BPs are taken up into these cells by fluid-phase
endocytosis, resulting in containment in membrane-bound vesicles23. This form of
uptake requires acidification of the vesicle to allow neutralisation of the
phosphonate head groups, and facilitates diffusion or active transport across the
endosomal membrane. Uptake into the cell by this method results in a large
fraction of the BP becoming trapped in these intracellular vesicles, meaning only a
small proportion is available to exert an effect in the cytosol. This significantly
reduces the bioavailability of the BPs. As a consequence, the cytosolic bioavailability
of the BPs in skeletal cell lines has not yet been optimised and remains difficult to
explain for the bisphosphonates that are active against non-skeletal cancer cell
lines, if intracellular enzymes are their sole biological targets. Fortunately, the
effects of the BPs, in particular as inhibitors of FPPS, are still substantial so only a
small amount needs to escape from the endosome11.
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In contrast, RALA is extremely efficient in escaping the endosome15. Indeed the 31P-
NMR revealed that at a pH of 4, indicative of the endosome, the particles dissociate
and the BP is released. Therefore the BPs clearly remain in solution, even at an
acidic pH. Previous studies have shown that at a lower pH the hydrophobic and
hydrophilic residues of RALA align, facilitating interactions with the endosome thus
releasing the cargo15. At cytosolic pH, and in presence of other anionic species
entering in competition with the BP for arginine binding, it is likely that the BPs’
interactions with RALA decrease thus increasing the concentration of free BP in this
organelle, where FFPS, the BPs’ biological target, is thought to be present1. This
clearly supports our theory that RALA acts as a versatile delivery vehicle to carry
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charged anionic species across intracellular membranes and release its cargo as the
pH changes.
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With PC3 cells, neither risedronate nor etidronate, delivered as free drug, were
capable of inducing growth inhibition; however, both showed good potency when
delivered using RALA. These results confirm that these two bisphosphonates do not
modulate extracellular biological targets relevant to cell survival in PC3, although
further studies are required to confirm this. The present study also demonstrates
that for these two BPs, facilitating intracellularisation re-establishes these BPs as
anti-proliferative agents, and this in par with their ability to inhibit key cytosolic
enzymatic processes24. We have not determined the efficiency of BP encapsulation
by RALA. When used to complex nucleic acids, RALA proved to be highly efficient,
encapsulating more than 92% of the available pDNA15. In the cases of alendronate,
and particularly zoledronate, it is conceivable that free BP could partially contribute
to the cytotoxicity observed. However, given the lack of cytotoxic potency of
etidronate and risedronate, we can confidently conclude that encapsulation with
RALA affords the BPs their cytotoxic potency. Therefore, this study suggests that
delivery of the BP to the cytosolic compartment results in potent anti-cancer
effects, an entirely different mechanism of action and altered pharmacological
profile for these two BPs, which requires further elucidation.
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There is mounting evidence that some of the observed zoledronate pharmacological
effects could be linked to cell surface receptors, including CD7325. It is most likely
that this is facilitated through an alternative mechanism of cellular entry, as we
have previously demonstrated that RALA/DNA nanoparticles predominantly enter
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cells via clathrin-mediated endocytosis15 Future studies could be designed to
elucidate whether intracellular delivery of zoledronate that bypasses the CD73
receptor opens up an avenue of therapeutic efficacy to those cells previously
resistant to zoledronate induced cell death. Finally, it is also important to note that
in order to target tumour cells and spare normal tissue, a targeting peptide could be
added on the N-terminus of RALA, designed in a way to recognise overexpressed
receptors.
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Translation of in vitro findings into the in vivo setting is a frequent stumbling block.
In these initial studies, we injected RALA/alendronate directly into the tumour as
proof of principle that the enhanced potency could be translated. Ideally, the
RALA/alendronate would be delivered systemically, and studies are ongoing to
achieve this.
The observed in vivo growth delay is highly significant, as we propose that
formulating the BP with RALA alters the BP from a bone specific anti-resorptive drug
to an anti-tumour agent. Indeed it is likely that complexation with RALA masks the
bone mineral affinity of alendronate; 31P NMR analysis of RALA/alendronate mixed
with 0.1 mg/ml hydroxyapatite (HA) produced a spectrum similar to that produced
with RALA/alendronate alone, while a second peak was evident on the spectrum
when 31P NMR was used similarly for free alendronate mixed with HA
(Supplementary Figure 2). This indicates an interaction between free alendronate
and HA that was not evident when alendronate was complexed with RALA. It is
possible that once the BP has escaped the tumour environment, it no longer exerts
an anti-tumour effect, and could revert to its bone resorptive activity, therefore an
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optimised dosing regimen is necessary. Furthermore, the dose of BP required to
provoke a therapeutic effect would be much lower in the RALA system to achieve
anti-resorptive effects, thus potentially negating the limiting side effects of jaw
necrosis associated with zoledronate. Fortunately, such dose-limiting side-effects
are not associated with alendronate, so it is ideally placed as a candidate BP for
further evaluation, development and repurposing, utilising RALA.
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Another argument towards the potential of the RALA system in modulating the
pharmacological profile of bisphosphonates lies in the fact that many BP-containing
compounds have been synthesised and shown extremely promising inhibitory
properties against FPPS and GGPS enzymes for instance, but had very poor anti-
proliferative activity when evaluated against cell lines. The RALA system offers
completely new pharmacological opportunities for this now well established large
class of compounds, for which only a few have found a therapeutic niche as anti-
resorptive agents.
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ASM was funded by the Royal Pharmaceutical Society of Great Britain
RC was funded by the EPSRC EP/H031065/1
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Supporting Information:
S1 – The size and charge of all four BPs when complexed with the RALA peptide at a
range of mass ratios from 2 – 20. Data demonstrates that from a mass ratio of 4
upwards all NPs were less than 100 nm and were positively charged.
S2 – This 31P NMR confirms that when RALA is present there is no free alendronate
present to interact with the HA in this instance. This demonstrates that all of the BP
is complexed by RALA.
References
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Kwaasi, A. A.; Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M. W.; Boyde, A.;
Kashemirov, B. A.; McKenna, C. E.; Russell, R. G. The relationship between the
chemistry and biological activity of bisphosphonates. Bone 2011, 49 (1), 20-33.
2. Vassiliou, V.; Kardamakis, D. The management of metastatic bone disease with
the combination of bisphosphonates and radiotherapy: From theory to clinical
practice. Anticancer Agents Med Chem. 2009, 9 (3), 326-335.
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3. Paterson, A.; Powles, T. J.; Kanis, J. A.; McCloskey, E.; Hanson, J.; Ashley, S.
Double-blind controlled trial of oral clodronate in patients with bone metastases
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