Reduction- and pH-Sensitive Hyaluronan Nanoparticles for Delivery of Iridium(III) Anticancer Drugs

Article abstract of DOI:10.1021/acs.biomac.7b00445

The organoiridium(III) complex (Ir(III)) [(η⁵-Cp^xbiph)Ir(phpy) (py)]PF₆ containing π-bonded biphenyltetramethylcyclopentadienyl(Cp^xbiph), C∧N-chelated phenylpyridine(phpy), and pyridine (py) ligands has more pot

Full text of DOI:10.1021/acs.biomac.7b00445

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Article
Reduction- and pH-sensitive Hyaluronan Nanoparticles
for Delivery of Iridium(III) Anticancer Drugs
Zhixiang Cai, Hongbin Zhang, Yue Wei, Yuanyuan Wei, Yanping Xie, and Fengsong Cong
Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017
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Reductionꢀ and pHꢀsensitive Hyaluronan
Nanoparticles for Delivery of Iridium(III)
Anticancer Drugs
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†
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Zhixiang Cai , Hongbin Zhang *, Yue Wei , Yuanyuan Wei , Yanping Xie , Fengsong Cong
†
Department of Polymer Science and Engineering, School of Chemistry and Chemical
Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China
†
†
Department of Biochemistry and Molecular Biology, School of life Sciences and
Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
Eꢀmail: hbzhang@sjtu.edu.cn
KEYWORDS: Hyaluronic acid; glycosaminoglycan; organoiridium complex; nanoparticles;
drug delivery; targeted anticancer efficacy
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xbiph
ABSTRACT: The organoiridium(III) complex (Ir(III)) [(η ꢀCp
)Ir(phpy)(py)]PF containing
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πꢀbonded biphenyltetramethylcyclopentadienyl(Cpxbiph), C^Nꢀchelated phenylpyridine(phpy),
and pyridine (py) ligands has more potent antitumor activity as a new generation of drug than
cisplatin toward various cancer cells. However, poor siteꢀspecific delivery, low solubility, and
poor tumor penetration are common limitations of chemotherapy drugs. To develop CD44ꢀ
targetable, pHꢀ, and reductionꢀresponsive drug delivery systems for Ir(III) drugs, the amphiphilic
hyaluronan (HA)ꢀbased conjugates of HAꢀcystaminꢀpyrenyl (HAꢀssꢀPy) containing disulfide
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bonds and HAꢀpyrenyl (HAꢀPy) were designed. The Ir(III) drug was readily loaded into these
two amphiphilic conjugates and nanoparticles were formed. Dynamic light scattering (DLS)
studies showed that the micelles formed from HAꢀssꢀPy were sufficiently stable under
physiological conditions, but were prone to rapid dissociation in reducing environments (20 mM
glutathione (GSH)). In subsequent confocal microscopy analyses, A549 cancer cells efficiently
internalized HAꢀbased micelles. Moreover, in vitro cytotoxicity assays in A549 cells demonstrat
that Irꢀloaded HAꢀbased nanoparticles have higher cytotoxicity than the free Ir(III) anticancer
drug. Finally, systemic administration of Ir(III)ꢀloaded HAꢀssꢀPy nanoparticles enhanced tumor
inhibition in vivo, and the corresponding biodistribution experiments showed that HAꢀssꢀPy
micelles accumulate in tumors. Overall, our results suggest that HAꢀssꢀPy micelles have a great
potential to be used as an effective Ir(III) drug carrier for targeted cancer therapy.
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INTRODUCTION
Platinumꢀbased anticancer drugs such as cisplatin, carboplatin, and oxaliplatin have received
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, 2
global approval. However, most of these drugs suffer from insolubility in water and platinum
resistance, and they cause several undesirable side effects, including nephrotoxicity and
neurotoxicity. These properties limit the future use of these preparations, despite their current
3, 4
wide application to the treatment of most cancers. With these challenges in mind, considerable
efforts have been made to develop alternative organometallic complexes of platinumꢀgroup
metals, such as ruthenium, osmium, rhodium, and iridium, with reduced side effects and an
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efficacy against a wider range of cancers. In particular, organoiridium(III) complexes (Ir(III))
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ꢀ11
show promising anticancer activities,
and halfꢀsandwich cyclopentadienyl iridium(III)
complexes with potent anticancer activity are also well suited for development as anticancer
12ꢀ16
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agents.
A new generation of anticancer drugs comprising the Ir(III) [(η ꢀ
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xbiph
xbiph
Cp
)Ir(phpy)(py)]PF , containing πꢀbonded biphenyltetramethylcyclopentadienyl (Cp
),
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C^Nꢀchelated phenylpyridine (phpy), and pyridine (py) ligands, had potent antitumor activity in
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a previous study, reflecting potent stimulation of reactive oxygen species production.
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Chemotherapy is the most common treatment for several solid tumor malignancies.
However, the ensuing therapeutic effects remain suboptimal, mostly due to poor solubility and
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bioavailability, nonspecific selectivity.
To address these limitations of chemotherapeutic
agents, many drug delivery approaches have been investigated with the intention of transporting
drugs to tumor targets without damaging healthy tissues and organs. Medical applications of
nanotechnology (nanomedicine) have stimulated the development of various types of drugꢀ
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loaded nanocarriers, such as liposomes,
carbon nanomaterials,
In particular, selfꢀassembled nanoparticles have received an
increasing interest as promising nanocarriers that encapsulate and mediate the sustained release
inorganic materials,
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and polymeric particulates.
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of various hydrophobic anticancer drugs.
Drugs can be entrapped in interior structures or
adsorbed to exterior surfaces of nanoparticles, and most nanoparticles have multiple advantages
over their small molecule counterparts, including improved thermodynamic stability, prolonged
circulation times, reduced systemic toxicity, and improved passive accumulation in cancer cells
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due to enhanced permeability and retention (EPR) effects.
Hence, due to the potential
applications of these properties to cancer chemotherapies, various synthetic and natural materials
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have recently been developed as nanocarriers for cancer treatment.
In particular,
polysaccharides have promising biocompatibility, biodegradability, hydrophilicity, and ease of
chemical modification, and have been widely used as building blocks for the construction of
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nanoparticle drug delivery systems.
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Despite the advantages of nanoparticles, clinical trials of drugꢀloaded, selfꢀassembled
nanoparticles continue to face technical challenges such as poor stability in the bloodstream
following intravenous injections, and undesirable premature drug release at unwanted sites,
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resulting in a lower therapeutic efficacy. Therefore, drug delivery systems with structures that
respond to external stimuli (including reduction, light, pH, and enzyme activities) are ideal
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candidates for the controlled release of anticancer drugs.
Hence, drug nanocarriers that are
responsive to pH and reducing environments have received tremendous attention as extraꢀ and
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intraꢀcellular environments differ strongly in pH and reduction potential. Specifically, it is well
known that the natural disulfide bondꢀreducing agent glutathione (GSH) is present at higher
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concentrations in the cytoplasm than in the plasma. Hence, nanoparticles with characteristic
disulfide linkages offer an attractive strategy for drug delivery systems, because although
nanoparticles are sufficiently stable under physiological conditions and in blood, rapid reduction
of disulfide bonds leads to the dissociation of nanoparticles inside the cells with high GSH
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concentrations.
In addition, the pH of tumor tissues is lower than that of blood and normal
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tissue, while endosomes and lysosomes have even lower pH values of 5.0ꢀ5.5.
Accordingly,
the pHꢀ and reductionꢀresponsive drug delivery systems have recently received increasing
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attention.
Despite the promise of drug release responses to external stimuli, practical nanocarriers need
to specifically target cancer cells. Hyaluronan or hyaluronic acid (HA) is a naturally occurring
linear polysaccharide comprising alternating repeat units of Dꢀglucuronic acid and Nꢀacetyl
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glycosamine linked by βꢀ(1,4) and βꢀ(1,3) glycosidic bonds. HA has been extensively used in
biomedical and pharmaceutical fields because of its desirable physiochemical properties,
including biodegradability, cytocompatibility, nonimmunogenicity, and high waterꢀbinding
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capacity. In addition, HA has a uniquely high affinity for the CD44 receptor binding.
Previous studies have investigated the use of HA as a delivery vehicle in many drugꢀdelivery
systems, reflecting its interactions with multiple cancer cell types that present abundant HA
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receptors (CD44 and RHAMM).
Several studies report the use of HA and its derivatives
as nanoparticles for cancer imaging and therapy, and in comparison with free anticancer drugs,
these HA nanocarriers have enhanced tumor targeting abilities and therapeutic efficacy, and
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provide strategies for controlled drug release.
In the present study, we developed novel amphiphilic micelles comprising disulfide crossꢀ
linked HAꢀcystamineꢀpyrenyl conjugates (HAꢀssꢀPy) for facile loading (Scheme 1A) and CD44ꢀ
targeted therapy, in conjunction with pHꢀ and reductionꢀtriggered intracellular release of Ir(III)
anticancer drugs (Scheme 1B). The tumor inhibition efficacy of Irꢀloaded HAꢀssꢀPy
nanoparticles was compared with that of Irꢀloaded HAꢀpyrenyl (HAꢀPy) nanoparticles and free Ir
anticancer drugs in tumorꢀbearing mice.
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Scheme 1 Illustration of HAꢀbased micelles as multifunctional platform for the active CD44ꢀ
targeting Ir anticancer drug delivery. (A) Formation of selfꢀassembled nanoparticles comprising
disulfide crossꢀlinked HAꢀcystaminꢀpyrenyl conjugates (HAꢀssꢀPy) for facile loading Ir(III)
anticancer, (B) CD44ꢀmediated uptake of nanoparticles and dualꢀtriggered (pHꢀ and reduction)
intracellular release of Ir(III) anticancer drugs, which triggers apoptosis in cancer cells.
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EXPERMENTAL SECTION
Materials
Sodium hyaluronan (HA, M = 10kDa) was purchased from Shandong Freda Biopharm Co.,
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Ltd, and 4ꢀbromoꢀbiphenyl, 2,3,4,5ꢀtetramethylꢀ2ꢀcyclopentenone (95%), tertꢀbutyllithium
solution (1.3 M in hexane), IrCl ·nH O, 2ꢀphenylpyridine, ammonium hexafluorophosphate,
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silver nitrate, 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide (EDC), Nꢀhydroxysuccinimide
NHS), 1ꢀHydroxybenzotriazole (HOBt), pyrene, cystamine, glutathione (GSH), 1,4ꢀ
(
diaminobutane, 1ꢀpyrenebutyric acid, Dimethyl Sulphoxide (DMSO) and formamide were
purchased from SigmaꢀAldrich. The nearꢀinfrared dye Cy5.5 (99%) was purchased from
Shanghai Seebio Biotech Inc. Nile red and Hoechst 33342 were obtained from Aladdin
(Shanghai, China), and 3ꢀ(4,5ꢀdimethylꢀthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium bromide (MTT),
RPMIꢀ1640 medium, penicillin, streptomycin, fetal bovine serum (FBS) trypsin/EDTA,
phosphateꢀbuffered saline (PBS), and 4% paraformaldehyde were purchased from Shanghai
Ruian Biotechnology Co., Ltd. The A549 cell line was obtained from the Chinese academy of
science. Dialysis membranes (MWCO, 3500 Da) were purchased from Spectrum Laboratories
(Rancho Dominguez, CA, USA). All other reagents and chemicals were of analytical grade and
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were used without further purification. All aqueous solutions were prepared using ultrapure
water (> 18 MU) from a MilliꢀQ Plus system (Millipore).
Synthesis of Ir(III) anticancer drugs
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Synthesis of compound 1 tetramethylꢀ(biphenyl)cyclopentadienyl (Cp
H)
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Cp
H was synthesized according to a previously reported protocol with some
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adaptations. Briefly, 4ꢀbromoꢀbiphenyl (4.66 g, 20 mmol) was dissolved in anhydrous
tetrahydrofuran (THF, 80 mL) with stirring, until a clear solution was obtained. Tertꢀbutyllithium
solution (1.3 M in hexane, 12.5 mL, 20 mmol) was then added slowly to an ovenꢀdried threeꢀ
necked flask containing the aboveꢀmentioned solution under N atmosphere at 195 K, and the
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mixture was stirred at this temperature for 3 h. Subsequently, 2,3,4,5ꢀtetramethylꢀ2ꢀ
cyclopentenone (3.32 g, 24 mmol) was added dropwise into the reaction mixture and was then
warmed slowly to room temperature with stirring overnight. After completion (monitored by
TLC), HCl (36%, 10 mL) was added to the orange solution. The mixture was then extracted with
diethyl ether (30 mL × 2) and the combined organic layers were dried using MgSO , filtered, and
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were finally concentrated in vacuo. The residue was recrystallized from chloroform and hexane
(3:1) to produce the desired product as a yellow powder. All yields were obtained after
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recrystallization. Yield: 1.86 g (34%), HꢀNMR (CDCl ): δ = 7.68 (m, 4H), 7.46 (m, 2H), 7.35
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m, 3H), 3.27 (m, 1H), 2.12 (s, 3H), 1.98 (s, 3H), 1.92 (s, 3H), 1.02 (d, 3H, J = 8.0Hz).
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Synthesis of compound 2 [(η ꢀCp )IrCl2]2
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The compound [(η ꢀCp
)IrCl ] was prepared as reported previously with the protocol
2 2
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modifications described below. Briefly, Cp
H (1.37 g, 5 mmol) and IrCl ·nH O (1.19 g,
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mmol) were added to 60 mL of anhydrous methanol. The suspension was then refluxed under
vigorous stirring for 48 h in a N atmosphere. After completion of the reaction, the reaction
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mixture was cooled to room temperature and the resulting orange precipitate was filtered and
thoroughly washed with anhydrous methanol (20 mL × 2) and diethyl ether (20 mL × 2), and
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was finally dried under vacuum at 323 K to produce the desired product. Yield: 1.15 g (43.4%),
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HꢀNMR (DMSOꢀd ): δ = 7.70 (m, 6H), 7.42 (m, 2H), 7.39 (m, 1H), 1.73 (d, 12H, J = 8.0 Hz).
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Synthesis of compound 3 [(η ꢀCp )Ir(phpy)Cl]
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The compound [(η ꢀCp
)Ir(phpy)Cl] was prepared as described in a previous report.
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Briefly, [(η ꢀCp
acetate (0.32 g, 4 mmol) were added to anhydrous CH Cl (20 mL) and the mixture was refluxed
)IrCl ] (1.06 g, 1 mmol), 2ꢀphenylpyridine (0.36 g, 2.4 mmol), and sodium
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in a N atmosphere for 24 h and was then filtered. The filtrate was then evaporated and the
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powder was recrystallized from CHCl /hexane (3:1) to produce the desired product. Yield: 0.66
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g (50%), HꢀNMR (CDCl ): δ = 8.53 (d, 1H, J = 4.0Hz), 7.85 (d, 1H, J = 8.0 Hz), 7.74 (m, 2H),
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.64 (m, 5H), 7.51 (m, 4H), 7.39 (d, 1H, J = 8.0 Hz), 7.19 (t, 1H, J = 8.0 Hz), 7.08 (t, 1H, J = 8.0
Hz), 6.96 (t, 1H, J = 8.0 Hz), 1.85 (s, 3H), 1.82 (s, 3H), 1.80 (s, 3H), 1.62 (s, 3H).
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Synthesis of compound 4 [(η ꢀCp )Ir(phpy)(py)]PF6
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The compound [(η ꢀCp
)Ir(phpy)(py)]PF was synthesized using a published procedure
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)Ir(phpy)Cl] (0.33 g, 0.5 mmol) and AgNO3
with slight modifications. Briefly, [(η ꢀ Cp
(0.09 g, 0.5 mmol) were successively added to anhydrous MeOH (10 mL) and stirred under
reflux in a N atmosphere for 10 h. The precipitate was filtered and pyridine (0.16 g, 2 mmol)
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was added with gentle stirring for 12 h. NH PF (0.33g, 2mmol) was then added and the
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resulting yellow precipitate was collected by filtration. Purification of the crude product provided
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the desired product following recrystallization in methanol/diethyl (1:1). Yield: 0.21 g (25%),
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HꢀNMR (CDCl ): δ = 9.00 (d, 1H, J = 8.0 Hz), 8.54 (d, 2H, J = 8.0 Hz), 7.81 (m, 3H), 7.70 (t,
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2H, J = 8.0Hz), 7.57 (d, 2H, J = 8.0 Hz), 7.45(m, 5H), 7.33 (m, 4H), 7.23 (t, 1H, J = 8.0 Hz),
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.93 (d, 2H, J = 8.0 Hz), 1.90 (s, 3H), 1.82 (s, 3H), 1.66 (s, 3H), 1.61 (s, 3H).
Synthesis and characterization of Hyaluronan-Diaminobutane-Pyrene (HA-Py) and
Hyaluronan-Cystamine-Pyrene (HA-ss-Py) conjugates
Synthesis and characterization of diaminobutaneꢀ and cystamineꢀmodified HA
Diaminobutaneꢀ and cystamineꢀmodified HA were synthesized using wellꢀestablished
carbodiimide chemistry. Specifically, 10 mg/mL HA (Mw, 10 kDa, 1 g) in ultrapure water (100
mL) was activated in the presence of EDC (0.96 g, 5 mmol) and HOBt (0.68 g, 5 mmol) with
stirring for 30 min at room temperature. Subsequently, 1,4ꢀdiaminobutane (0.44 g, 5 mmol) was
added dropwise into the solution using a pipette. The mixture was stirred at room temperature for
24 h and was then dialyzed (MWCO, 3500) against deionized water for 3 days. Finally, the HAꢀ
diaminobutane conjugate was obtained as a white powder by lyophilization. Cystamineꢀmodified
HA was prepared as described below. In a typical experiment, EDC (0.96 g, 5 mmol) and HOBt
(0.68 g, 5 mmol) were slowly added to the HA solution (100 mL, 10 mg/mL) with vigorous
stirring for 20 min. Cystamine dihydrochloride (0.57g, 5mmol) was then added and the reaction
was allowed to proceed for 24 h at room temperature. Subsequently, the reaction mixture was
dialyzed for 3 days against ultrapure water and was freezeꢀdried to obtain white floccules. These
o
products were stored at 4 C until further use. The chemical structures of HAꢀdiaminobutane and
1
HAꢀss were characterized using FTꢀIR and HꢀNMR (400 MHz). Ratios of diaminobutane and
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cystamine to HA were determined using HꢀNMR analysis after dissolution in D O. HꢀNMR
2
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analyses were performed using an AVANCE III 400 spectrometer (Bruker Co., Switzerland).
FTꢀIR spectra were recorded using a PerkinꢀElmer Spectrum 100 FTꢀIR spectrometer (Perkin
ꢀ1
Elmer Co., USA) in the range from 4000 to 400 cm according to the KBr sample holder
method.
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Synthesis and characterization of amphiphilic HAꢀPy and HAꢀssꢀPy conjugates
HAꢀPy and HAꢀssꢀPy were prepared by amidation of the carboxyl groups of 1ꢀpyrenebutyric
acid with the amine groups of HAꢀdiaminobutane and HAꢀss, respectively. Briefly, to activate
carboxyl groups, 1ꢀpyrenebutyric acid (0.06 g, 0.2 mmol) was added to DMSO (15 mL) in the
presence of EDC (0.04 g, 0.2 mmol) and NHS (0.02 g, 0.2 mmol) with stirring for 1 h at room
temperature. HAꢀdiaminobutane (0.28 g) and HAꢀss (0.28 g) conjugates were then dissolved in
20 mL of formamide, and 1ꢀpyrenebutyric acid in DMSO was added dropwise. Reaction
mixtures were incubated for 24 h at room temperature with vigorous stirring and were then
dialyzed against excess ultrapure water for 3 days (MWCO, 3500 Da). Finally, suspensions were
o
lyophilized to obtain HAꢀPy and HAꢀssꢀPy conjugates. The products were stored at 4 C for
further use. The chemical structures of HAꢀPy and HAꢀssꢀPy conjugates were determined using
1
1
HꢀNMR and FTꢀIR analysis. Ratios of 1ꢀpyrenebutyric acid were determined using HꢀNMR in
D O/d ꢀDMSO coꢀsolvent (1:1 volume ratio).
2
6
Preparation and Characterization of HA-Py and HA-ss-Py micelles
HAꢀPy and HAꢀssꢀPy micelles were prepared as described below. Briefly, HAꢀPy
conjugates (15 mg) were dispersed in ultrapure water (5 mL) and the solutions were
ultrasonicated for 30 min at 100 W with an ultrasonication probe. Conjugates were then filtered
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through a 0.45 ꢁm membrane filter and were lyophilized. HAꢀssꢀPy micelles were prepared
under the same conditions.
Critical micelle concentrations (CMC) of HAꢀPy and HAꢀssꢀPy were determined using
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fluorescence spectrophotometry with pyrene as fluorescence probe. Briefly, a 10 M pyrene
solution was prepared in acetone, and 1 mL aliquots were transferred into a series of 10 mL
volumetric flasks and the acetone was evaporated for 5 h at room temperature. HAꢀPy or HAꢀssꢀ
−
5
Py solutions with concentrations ranging from 10 to 0.1 mg/mL were added to volumetric
flasks and were stirred for 2 h at room temperature. Fluorescence intensity was recorded using a
fluorescence spectrophotometer and emissions at 374 and 385 nm were determined following
excitation at 330 nm. CMC were estimated as intensity ratios of I374/I385 at low and high
concentrations.
Particle sizes and zeta potentials of HAꢀPy and HAꢀssꢀPy micelles in PBS at pH=7.4 and
5.0, respectively, were detected using dynamic light scattering (DLS) (NanoꢀZS90, Malvern
instruments, UK). All measurements were performed at a constant temperature of 25°C and at an
angle of 90°. Micelles were filtered through 0.45 ꢁm membrane filters before measurements, and
morphological characteristics of HAꢀPy and HAꢀssꢀPy micelles were observed using
transmission electron microscope (TEM) with a JEMꢀ2100F apparatus operated at 200 kV.
Atomic force microscope (AFM) analyses were performed using a Nano Scope IIIa (Veeco,
USA) instrument and images were recorded.
pH- and reduction-triggered disassembly of micelles
Changes in the sizes and size distributions of HAꢀssꢀPy micelles were monitored using DLS
measurements after incubation in PBS buffer (pH=7.4, 0.1 M) containing GSH at 2 ꢁM, 10 mM,
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and 20 mM. Disassembly of HAꢀssꢀPy micelles at different pH values was also characterized
using DLS after incubation in PBS buffer (pH=7.4, and 5.0, respectively). HAꢀPy micelles were
used as a reductionꢀinsensitive control. In a typical experiment, HAꢀPy and HAꢀssꢀPy micelles
were prepared as above and GSH solutions of specified concentrations were added and stirred at
room temperature in volumetric flasks containing reductionꢀsensitive HAꢀssꢀPy micellar
suspensions (0.2 mg/mL). Size distribution of HAꢀssꢀPy micelles were recorded at various time
intervals using DLS measurements. HAꢀPy micelles were used as a reductionꢀinsensitive control.
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Encapsulation of Ir(III) anticancer drugs
Ir(III) loaded HAꢀPy (HAꢀPyꢀIr) and HAꢀssꢀPy (HAꢀssꢀPyꢀIr) were prepared using dialysis
methods. Briefly, 15 mg samples of HAꢀPy or HAꢀssꢀPy were added dropwise to 3 mL of
ultrapure water with stirring for 30 min at room temperature to enable complete stabilization.
Subsequently, 10 mg of Ir(III) in DMSO (3 mL) was added to the suspension with stirring at
room temperature for 1 h and dialysis against excess amounts of ultrapure water was performed
using dialysis membranes (MWCO, 3500 Da) at room temperature for 3 days. Dispersions were
filtered through 0.45 ꢁm pore membranes to remove the unloaded Ir, and were then lyophilized
to obtain a yellow powder.
Morphological characteristics of Ir(III)ꢀloaded nanoparticles were observed using TEM and
AFM, and sizes distribution of Ir(III)ꢀloaded nanoparticles at pH 5.0 or pH 7.4 with and without
GSH were characterized using DLS.
Ir loading contents (LE) and encapsulation efficiencies (EE) of Ir anticancer complexes were
determined using UVꢀVis spectrophotometry following dispersion of Irꢀloaded micelles (HAꢀPyꢀ
Ir and HAꢀssꢀPyꢀIr) in ethanol/water (1:1 v/v). A calibration curve was generated from a series of
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Ir solutions (ethanol: water =1:1) with varying Ir concentrations. LE and EE were calculated as
follows:
ꢁꢂꢃꢄꢅꢆꢀꢇꢈꢀꢉꢇꢊꢋꢂꢋꢀꢋꢌꢍꢄꢀꢃꢎꢀꢈꢇꢌꢏꢍꢉꢊꢆꢃꢇꢎ
Loading content (wt%) = ꢀ ꢁꢂꢃꢄꢅꢆꢀꢇꢈꢀꢐꢇꢉꢑꢏꢂꢌꢀꢃꢎꢀꢈꢇꢌꢏꢍꢉꢊꢆꢃꢇꢎ × 100%
(1)
(2)
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ꢁ
ꢂꢃꢄꢅꢆꢀꢇꢈꢀꢉꢇꢊꢋꢂꢋꢀꢋꢌꢍꢄꢀꢃꢎꢀꢈꢇꢌꢏꢍꢉꢊꢆꢃꢇꢎ
Loading efficiency (%) =
×100%
ꢁ
ꢂꢃꢄꢅꢆꢀꢇꢈꢀꢋꢌꢍꢄꢀꢃꢎꢀꢈꢂꢂꢋ
In vitro Release of Ir-loaded nanoparticles
The drug release profiles of HAꢀPyꢀIr and HAꢀssꢀPyꢀIr nanoparticles were investigated
using dialysis techniques. Lyophilized HAꢀPyꢀIr and HAꢀssꢀPyꢀIr nanoparticles containing 5 mg
of Ir(III) were dispersed in PBS buffer (pH=7.4, or 5.0), and nanoparticle suspensions (1 mL)
were transferred to dialysis tubes (MWCO 3500) and were then immersed in PBS buffer
(pH=7.4, or 5.0, 20 mL) containing 0.1% (w/v) Tween 80 to achieve sink conditions. Reductionꢀ
responsive release media was produced by adding GSH to PBS buffer (pH=7.4) at final
concentrations of 0, 10, and 20 mM. In addition, PBS (pH=5.0) containing 20 mM GSH was also
tested as release medium. Release media were gently stirred in a water bath at 37°C, and 2 L
aliquots were collected and replaced with equivalent volumes of fresh media at predetermined
time intervals. Concentrations of Ir(III) released into the aqueous solution from HAꢀPyꢀIr and
HAꢀssꢀPyꢀIr in reductive and acidic environments were quantitatively analyzed using UVꢀVis
spectroscopy, and percentages of drug release were calculated by interpolation from a standard
calibration curve. Release experiments were conducted in triplicate and the results are presented
as means with standard deviations.
In vitro cytotoxicity studies
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Cell culture
A549 lung cancer cells were grown in DMEM containing 10% inactivated fetal bovine
serum (FBS) and 1% penicillinꢀstreptomycin. The same culture media were used in subsequent
confocal microscopic observations and cell viability assays. All cells were maintained in a
humidified incubator at 37°C with 5% CO2.
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The in vitro cytotoxicity of designed Irꢀloaded micelles (HAꢀssꢀPyꢀIr, HAꢀPyꢀIr) and HAꢀ
based micelles (HAꢀssꢀPy, HAꢀPy) against A549 cells was evaluated using the 3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyꢀltetrazolium bromide (MTT) assay. Micelle samples were
3
added at equivalent concentrations. A549 cells were seeded into 96ꢀwell plates at 5 × 10 per
well in 100 ꢁL of culture medium for 24 h and were then washed twice with PBS (pH=7.4) and
treated with various concentrations of HAꢀssꢀPy, HAꢀPy micelles, and free Ir(III), HAꢀssꢀPyꢀIr,
and HAꢀPyꢀIr nanoparticles for 48 h. Culture media were then removed and the cells were
washed twice with PBS to remove the remaining treatments. Subsequently, MTT assay stock
solution in PBS (25 ꢁL, 5 mg/mL) was added to each well and the cells were incubated for 4 h.
Unreacted MTT in the culture medium was carefully removed and purple formazan crystals in
metabolically active cells were dissolved in DMSO (100 µL). Subsequently, absorbance of
dissolved formazan dye was determined at 490 nm in each well using a microplate reader
(EnSpire 2300, PerkinElmer, USA). Cell viabilities were determined according to the resulting
optical density (ODsample) values relative to those in control (ODcontrol), which were cells that
were not treated, and were expressed as percentages of the control. Experiments were conducted
in triplicate and standard deviations of independent experiments are presented as error bars.
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In vitro cellular uptake and intracellular release of of nile red (NR) labeled HA-Py and HA-
ss-Py micelles
The in vitro cellular uptake and intracellular release studies of micelles was investigated in
A549 cells using confocal laser scanning microcopy (CLSM). The fluorescent probe nile red was
encapsulated in micelles, and nile red loaded HAꢀPy and HAꢀssꢀPy micelles were prepared with
a similar protocol to that for Ir(III)ꢀloaded nanoparticles. A549 cells were plated on microscope
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slides in 6ꢀwell plates at 1 × 10 cells/well with culture medium (2 mL) containing 10% FBS,
penicillin (100 IU/mL), and streptomycin (100 ꢁg/mL), and were incubated at 37°C in a
humidified atmosphere containing 5% CO for 24 h. Subsequently, nile red loaded HAꢀPy or
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HAꢀssꢀPy nanoparticles (100 ꢁL) were added to all the wells and were incubated for another 6 or
24 h. For competitive inhibition studies, A549 cells were preꢀtreated with free HA polymer (10
mg/mL) for 2 h and were then incubated with nile red loaded HAꢀssꢀPy nanoparticles to confirm
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CDꢀ44 mediated cell uptake. Culture media were then removed and the cells were rinsed twice
with PBS and fixed with 4% paraformaldehyde solution for 20 min. Finally, cell nuclei were
stained with Hoechest 33342 for 10 min and the intracellular uptake of nanoparticles was
determined using confocal laser scanning microscopy (Leica, UK) with excitation and emission
wavelengths of 352 and 452 nm for Hoechest 33342, and 488 and 530 nm for nile red,
respectively.
In vivo animal experiments and tumor models
Female Balb/c nude mice of 4ꢀ5 weeks of age were purchased from the Chinese Academy of
Sciences (Shanghai). All animal experiments were performed in compliance with the guidelines
approved by the Animal Ethics Committee of Shanghai Jiao Tong University. Tumorꢀbearing
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mice were prepared by subcutaneously injecting 200 µL cell suspensions containing 1 × 10
A549 cells in PBS into the right armpit regions. Tumor volumes were allowed to reach
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approximately 200 mm before experiments were carried out.
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In vivo antitumor efficiency of HA-Py-Ir and HA-ss-Py-Ir nanoparticles
Antitumor efficiencies of nanoparticles were evaluated in A549 tumorꢀbearing mice. Mice
were randomly divided into treatment groups, and received intravenous injections of HAꢀPy
micelles, HAꢀssꢀPy micelles, or HAꢀPyꢀIr, HAꢀssꢀPyꢀIr, or free Ir(III) via tail veins at doses of 5
mg/kg Ir equivalents (200 µL), respectively, 10 times at 3 day intervals. Hydrophobic drugs are
regularly dissolved in DMSO for biologic experments, both in vitro and in vivo in experimental
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models.
Free Ir was dissolved in 10% DMSOꢀphysiological saline considering the poor
water solubility of Ir in water. In parallel, a control group received injections of a 10% DMSOꢀ
physiological saline. The protocol for antitumor efficiencies of nanoparticles was designed as
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described in a previous report. Tumor sizes were measured using vernier calipers once every 3
days and the body weights of mice were recorded every 3 days throughout the experiment.
3
Tumor volumes (V) were calculated using the following formula: V (mm ) = 1/2 × longest
2
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diameter (mm) × shortest diameter (mm) . At the end of the experiment, the mice were
sacrificed and tumors were excised, weighed, and photographed.
In vivo and ex vivo imaging of Cy5.5-loaded HA-Py and HA-ss-Py nanoparticles
To monitor the in vivo distributions of nanoparticles, the NIR dye Cy5.5 was loaded into
HAꢀPy and HAꢀssꢀPy micelles and Cy5.5 loaded nanoparticles were prepared using a dialysis
method according to the protocol for nile red loaded nanoparticles. A549 tumorꢀbearing mice
were injected intravenously with 200 µL of Cy5.5ꢀloaded HAꢀPy or HAꢀssꢀPy selfꢀassembled
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nanoparticles via the tail veins at doses of 5 mg/kg. Fluorescent images of mice were obtained at
appropriate wavelengths (λ , 670 nm and λ , 700 nm) using an in vivo imaging system (Caliper
ex
em
IVIS Lumia II, Caliper life science, USA) at 1, 2, 4, 6, 8, and 12 h after injections. Calculations
were performed using the region of interest (ROI) function of Analysis Workstation software
(ART; Advanced Research Technologies Inc., Montreal, Canada).
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Six hours after the injections, A549 tumorꢀbearing mice were sacrificed and tumor tissues
and major organs, including the heart, liver, spleen, lung, and kidney, were excised.
Subsequently, ex vivo fluorescent images of the tumor tissues and major organs were obtained
and analyzed using the imaging system as described above. Tissue distributions of nanoparticles
were quantified by measuring the fluorescence intensities at regions of interest (ROI).
Statistical analysis
Data are expressed as means ± standard deviations. Differences between experimental and
control groups were identified using 2ꢀtailed Student’s tꢀtests and were considered significant
when p < 0.05.
RESULTS AND DISCUSSION
5
xbiph
Synthesis and characterization of [(η -Cp
)Ir(phpy)(py)]PF6
xbiph
5
xbiph
5
xbiph
5
Initially, complexes 1 Cp
H, 2 [(η ꢀCp
)IrCl ] , 3 [(η ꢀCp
)Ir(phpy)Cl], and 4 [(η ꢀ
2
2
xbiph
Cp
)Ir(phpy)(py)]PF were synthesized with moderate yields as described previously (Figure
6
5
1
A). Reaction details are described in the “Materials and Methods” section. The compound [(η ꢀ
xbiph
Cp
)Ir(phpy)(py)]PF is one of the most potent anticancer complexes, and structures of
6
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intermediates 1ꢀ3 and Ir(III) anticancer drug 4 were confirmed using HꢀNMR spectroscopy
(Figures 1B).
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O
dry THF
o
IrCl
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Br +
-
80 C
Ir
1
Cl
Cl
Cl
2
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2
3
) AgNO3
) py
PF
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N
) NH PF
4
6
2
MeOH, H O
CH COONa CH Cl
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2
Ir
Ir
Cl
N
N
N
3
4
(A)
1
xbiph
(
a) HꢀNMR spectra of Cp
H
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(
b) HꢀNMR spectra of [(η ꢀCp
)IrCl2]2
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xbiph
(c) HꢀNMR spectra of [(η ꢀCp
)Ir(phpy)Cl]
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(
d) HꢀNMR spectra of [(η ꢀCp
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(
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1
Figure 1. (A) Synthesis route for complexes 4 [(η ꢀCp
)Ir(phpy)(py)]PF . (B) the HꢀNMR
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spectra of complexes 1, 2, 3 and 4
Synthesis and characterization of HA-Py and HA-ss-Py conjugates
Figure 2A shows the protocol for the synthesis of HAꢀPy and HAꢀssꢀPy conjugates. In this
investigation, we developed a novel and tumorꢀtargeting delivery vehicle that can be loaded with
Ir(III) anticancer complexes and that can rapidly release them inside target cells. The ensuing
reductionꢀsensitive amphiphilic HAꢀssꢀPy conjugates were designed and prepared as follows:
Firstly, HAꢀss was synthesized by chemical conjugation of cystamine as a donor of disulfide
bonds to hydrophilic HA polymers using EDC chemistry. Subsequently, pyrene groups, which
are πꢀrich moieties for the loading and active release of Ir, were introduced to HAꢀss conjugates
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with free amines of cystamine through amide formation in the presence of EDC and NHS,
resulting in the formation of HAꢀssꢀPy conjugates. As a control, reductionꢀinsensitive HAꢀPy
was synthesized by the conjugation of 1ꢀpyrenebutyric acid to the HA polymer using the
symmetrical linker 1,4ꢀdiaminobutane in a carbodiimideꢀmediate coupling reaction.
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The chemical structures of HAꢀdiaminobutane, HAꢀss, HAꢀPy, and HAꢀssꢀPy conjugates
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were characterized using HꢀNMR (Figure 2B). Compared with HꢀNMR spectra of HA, distinct
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peaks were detected at δ = 1.65, 2.80, and 2.95 ppm and at δ = 2.78 and 2.93 ppm from HꢀNMR
spectra of HAꢀdiaminobutane and HAꢀss and were attributed to methylene protons from 1,4ꢀ
diaminobutane and cystamine, respectively. These data confirmed the successful synthesis of
HAꢀdiaminobutane and HAꢀss conjugates. In quantitative analyses, grafting efficiencies of 1,4ꢀ
diaminobutane and cystamine were about 0.22 and 0.20 from the integration ratio of the peak of
the methylene proton to that of the Nꢀacetyl glucosamine proton peak of HA, which appeared at
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.0 ppm. Successful synthesis of HAꢀPy and HAꢀssꢀPy conjugates were confirmed according to
specific peaks from aromatic rings of pyrene (δ = 8.07ꢀ8.47; Figure 2B). According to integral
ratios, degrees of substitution of pyrene groups in HAꢀPy and HAꢀssꢀPy conjugates were 0.16
and 0.15, respectively.
The synthesis of HA derivatives was also characterized using FTꢀIR spectra (Figure 2C). In
comparison with the spectra of HA, FTꢀIR spectra of HAꢀdiaminobutane and HAꢀss showed an
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additional peak at 1730 cm , which was assigned to amide stretching vibration bands.
Compared with the absorption spectra for HAꢀdiaminobutane and HAꢀss, spectra for HAꢀPy and
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HAꢀssꢀPy included additional peaks at 847 cm , and these corresponded with double bonds
from pyrene groups. These data confirmed that pyrene was successfully conjugated to the
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backbone of HAꢀdiaminobutane and HAꢀss and that HA derivatives were successfully
synthesized.
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HA-butanediamine
HA-Py
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Figure 2. (A) The synthetic route for HAꢀPy and HAꢀssꢀPy conjugates. (B) HꢀNMR spectra of
HA, HAꢀss, HAꢀdiaminobutane, HAꢀPy and HAꢀssꢀPy conjugates. (C) FTꢀIR spectra of HA,
HAꢀss, HAꢀdiaminobutane, HAꢀPy, and HAꢀssꢀPy conjugates.
Preparation and characterization of HA-ss-Py and HA-Py micelles and Ir-loaded
nanoparticles
Reductionꢀsensitive HAꢀssꢀPy and reductionꢀinsensitive HAꢀPy conjugates formed micelles
via selfꢀassembly in aqueous solution due to hydrophobic interactions between the pendant
hydrophobic groups. HAꢀssꢀPy and HAꢀPy micelles were prepared using a simple ultrasonic
method. In general, the amphiphilic properties and selfꢀassembly behaviors of micelles are
characterized by measuring CMC using pyrene as a hydrophobic fluorescent probe. In Figure
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A, fluorescence intensity ratios (λ / λ ) at λ = 330nm are plotted against conjugate
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ex
concentrations, and CMC were estimated from threshold concentrations of micelles. These
experiments showed that the HAꢀssꢀPy and HAꢀPy conjugates had low CMC of about 2.4 and
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.8 mg/L, respectively. These low CMC indicate that the present conjugates form particularly
stable micelles under highly diluted conditions.
Particle size distributions and zeta potentials of nanoparticles play important roles in
intravenous activities. Figure 3B and 3C show the DLS results with TEM and AFM images for
HAꢀssꢀPy and HAꢀPy micelles. DLS results showed that reductionꢀsensitive HAꢀssꢀPy micelles
had an average particle size of 260 ± 20 nm and a low polydispersity index (PDI) of 0.241 in
PBS (pH=7.4). Under the same conditions, HAꢀPy micelles had a mean particle size of 250 ± 15
nm and a PDI of 0.238 (Table 1). The ζ potentials of HAꢀssꢀPy and HAꢀPy micelles indicated
negative charges of −24.6 ± 0.4 and −25.9 ± 0.5 mV, respectively, reflecting charged carboxylic
groups of HA polymers on micelle surfaces. These negatively charged surfaces inhibit the
aggregation of micelles by electrostatic repulsion and provide longꢀterm stability in aqueous
solution. Compared to the sizes of HAꢀssꢀPy and HAꢀPy micelles at pH=7.4, no size changes
were also evident for HAꢀssꢀPy (average particle size = 267 ± 22 nm) and HAꢀPy micelles
(average particle size = 248 ± 13 nm) at pH=5.0 (Figure 3(B)). TEM and AFM micrographs
revealed that these micelles were generally spherical, with sizes that were in accordance with
DLS results. The average size of HAꢀssꢀPy and HAꢀPy micelles was approximately 120 nm
(Figure 3C). TEM and AFM images indicated micelle sizes that were smaller than those
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determined by DLS, likely reflecting swelling of hydrophilic backbone in aqueous solution.
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Figure 3. (A) CMC determination of HAꢀPy and HAꢀssꢀPy micelles. (B) DLS results of HAꢀssꢀ
Py and HAꢀPy micelles. (C) AFM and TEM images of HAꢀssꢀPy and HAꢀPy micelles
Stability of HA-based micelles
In order to investigate the stability of HAꢀbased micelles, we determined changes in the
sizes of HAꢀPy and HAꢀssꢀPy micelles using DLS in the presence or absence of GSH at 2 µm,
10 mM, and 20 mM. As shown in Figure 4A, HAꢀssꢀPy micelle particle sizes increased
significantly from 260 nm to 350 nm and 580 nm after 24 h in the presence of 10 and 20 mM
GSH, respectively. These observations suggest that cleavage of disulfide linkages led to the
aggregation of micelles. However, no size changes were evident for HAꢀssꢀPy micelles after 24 h
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in the presence of 2 µM GSH, which is the GSH concentration in extracellular fluids. In
contrast, sizes of HAꢀPy micelles did not vary with GSH concentrations after 24 h, suggesting
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that HAꢀPy micelles have a good aqueous stability during circulation. As shown in Figure 4B,
HAꢀssꢀPy micelle particle sizes increased significantly with time in the presence of 20 mM GSH,
and these observations were accompanied by decreased scattering intensities.
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Preparation and characterization of Ir(III)-loaded HA-ss-Py and HA-Py nanoparticles
Because the Ir(III) anticancer drug has diphenyl structures, and HAꢀbased micelles contain
pyrene structures, the Ir anticancer drug can be loaded into HAꢀssꢀPy and HAꢀPy micelles via π–
π stacking interactions. Thus, encapsulation of Ir(III) anticancer drug was achieved using simple
dialysis. Because nanoparticle sizes and ζ potentials of drugꢀloaded nanoparticles are key factors
in intravenous administration, we performed further DLS studies (Figure 4C) and showed
average hydrodynamic diameters of HAꢀssꢀPyꢀIr and HAꢀPyꢀIr nanoparticles of 210 ± 3 and 230
±
5 nm (pH=7.4) with low polydispersity indexes of 0.084 and 0.100, respectively. After loading
the Ir(III) anticancer drug, nanoparticles sizes were diminished to 30ꢀ40 nm, reflecting the
compact structure of π–π stacking interactions between Ir(III) anticancer and pyrene substituents.
In addition, ζ potentials of HAꢀssꢀPyꢀIr and HAꢀPyꢀIr nanoparticles were negative at pH=7.4
(−25.1 ± 0.2 and −21.0 ± 0.3 mV, respectively), offering significant repulsion between
nanoparticles and increased stability (Table 1). To investigate the stability of HAꢀssꢀPyꢀIr and
HAꢀPyꢀIr nanoparticles, we determined the changes in the sizes of HAꢀPyꢀIr and HAꢀssꢀPyꢀIr
nanoparticles using DLS in the presence of GSH of 0, 2 µM, 10 mM, and 20 mM at pH=7.4. As
shown in Figure 4C, upon addition of 10 mM and 20 mM GSH, HAꢀssꢀPyꢀIr nanoparticles
exhibited significant increase in particle size. In contrast, little size changes were observed for
HAꢀPyꢀIr nanoparticles, and for HAꢀssꢀPyꢀIr nanoparticle in the presence of 0 and 2 µm GSH.
Moreover, the DLS results also showed that the HAꢀssꢀPyꢀIr and HAꢀPyꢀIr nanoparticles were
destabilized at pH=5.0 forming relatively large micelles of ca 300 nm. These results implied that
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the HAꢀssꢀPyꢀIr and HAꢀPyꢀIr nanoparticles might be triggered to release Ir(III) anticancer drug
in acidic environment, which was further confirmed in the following in vitro pHꢀsensitive release
experiments.
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In further experiments, we examined nanoparticle morphologies of HAꢀssꢀPyꢀIr and HAꢀPyꢀ
Ir using TEM and AFM (Figure 4D). TEM and AFM micrographs showed spherical
morphologies with particle sizes of about 100 nm, and the differences in comparison with DLS
data likely reflect swelling of nanoparticles in aqueous solution.
The functionalization process of Ir(III) anticancer drug loaded HAꢀssꢀPy and HAꢀPy were
monitored by UVꢀVis absorption spectroscopy. As shown in Figure 4E, Ir anticancer drug in
ethanol/water (1:1 v/v) showed one absorption at 263 nm. When Ir loaded on either HAꢀssꢀPy or
HAꢀPy formed HAꢀssꢀPyꢀIr or HAꢀPyꢀIr nanoparticles, the absorance peaks at 263nm slightly
shifted to 266 nm, indicative of interactions between Ir and Py groups in HA conjugates. The
optical results indicated that Ir(III) had been loaded on the HAꢀbased micelles as expected.
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