Synthesis and Characterization of Nitric Oxide-Releasing Platinum(IV) Prodrug and Polymeric Micelle Triggered by Light

Article abstract of DOI:10.1021/acs.bioconjchem.7b00749

Herein, we report the proof of concept of photoresponsive chemotherapeutics comprising nitric oxide-releasing platinum prodrugs and polymeric micelles. Photoactivatable nitric oxide-releasing donors were integrated into the axial positions of a platinum(IV) prodrug, and the photolabile hydrophobic groups were grafted in the block copolymers. The hydrophobic interaction between nitric oxide donors and the photolabile groups allowed for the loading of platinum drugs and nitric oxide-releasing donors in the photolabile polymeric micelles. After cellular uptake of micelles, light irradiation induced the release of nitric oxide, which sensitized the cancer cells. Simultaneously, photolabile hydrophobic groups were cleaved from micelles, and the nitric oxide-releasing donor was altered to be more hydrophilic, resulting in the rapid release of platinum(IV) prodrugs. The strategy of using platinum(IV) prodrugs and nitric oxide led to enhanced anticancer effects.

Full text of DOI:10.1021/acs.bioconjchem.7b00749

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Article
Synthesis and Characterization of Nitric Oxide-Releasing
Platinum (IV) Prodrug and Polymeric Micelle Triggered by Light
Swapan Pramanick, Jihoon Kim, Jinhwan Kim, Gurusamy Saravanakumar, Dongsik Park, and Won Jong Kim
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00749 • Publication Date (Web): 27 Dec 2017
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Synthesis and Characterization of Nitric OxideꢀReleasing
Platinum (IV) Prodrug and Polymeric Micelle Triggered
by Light
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Swapan Pramanick, †# Jihoon Kim, †# Jinhwan Kim, †‡ Gurusamy Saravanakumar,† Dongsik
Park‡ and Won Jong Kim *†‡
†Center for Selfꢀassembly and Complexity, Institute for Basic Science, 77 Cheongamꢀro,
Namꢀgu, Pohang 37673, Republic of Korea.
‡ Department of Chemistry, Pohang University of Science and Technology (POSTECH), 77
Cheongamꢀro, Namꢀgu, Pohang 37673, Republic of Korea.
# These authors contributed equally to this work.
*Corresponding author. Tel.: +82ꢀ54ꢀ279ꢀ2104; fax: +82ꢀ54ꢀ279ꢀ3399
Eꢀmail address: wjkim@postech.ac.kr
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Abstract
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Herein, we report the proof of concept of photoꢀresponsive chemotherapeutics comprising nitric oxideꢀ
releasing platinum prodrugs and polymeric micelles. Photoꢀactivatable nitric oxide–releasing donors were
integrated into the axial positions of a platinum (IV) prodrug, and the photoꢀlabile hydrophobic groups
were grafted in the block copolymers. The hydrophobic interaction between nitric oxide donors and the
photoꢀlabile groups allowed for the loading of platinum drugs and nitric oxide–releasing donors in the
photoꢀlabile polymeric micelles. After cellular uptake of micelles, light irradiation induced the release of
nitric oxide, which sensitized the cancer cells. Simultaneously, photoꢀlabile hydrophobic groups were
cleaved from micelles, and the nitric oxide–releasing donor was altered to be more hydrophilic, resulting in
the rapid release of platinum (IV) prodrugs. The strategy of using platinum (IV) prodrugs and nitric oxide
led to enhanced anticancer effects.
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Introduction
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Platinum (Pt)ꢀbased drugs represented by cisplatin have been widely used as chemotherapeutics for
various cancers over the last three decades. However, their use is limited by several major problems,
including drug resistance, biomoleculeꢀassociated deactivation, and side effects due to nonꢀspecific
delivery.1ꢀ3 As Ptꢀbased drugs are exploited for the treatment of more than 50% of all cancers,4 research
aimed at addressing these issues is urgently required.
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Compared to active Pt(II) drugs, Pt(IV) prodrugs have two additional coordination sites in their axial
positions, allowing for additional functionalities and enhanced stability against nucleophiles.1ꢀ9 Pt(IV)
prodrugs undergo intracellular bioreduction and afford Pt(II) drugs the opportunity to exert their anticancer
effects. Therefore, the use of Pt(IV) prodrugs have been suggested as a practical way to overcome
biomoleculesꢀassociated deactivation of platinumꢀbased drugs.5ꢀ9
One of the most critical impediments for the application of Ptꢀbased drugs is ineffectiveness in drugꢀ
resistant cells. In an effort to surmount this problem, several molecules, enzymes and nucleic acids have
been simultaneously utilized with Ptꢀbased drugs, which can interfere with DNAꢀrepair systems, prevent
the approach of glutathione, or downꢀregulate the protein expression associated with drug resistance.3,10ꢀ12
However, these strategies have limitations, such as low efficiency and high cost. Accordingly, in this study,
we have focused on a physiological sensitizer, nitric oxide (NO), to enhance the Ptꢀbased drug effect. NO
is a gaseous mediator in vivo that participates in the progression or treatment of various diseases, including
autoimmune diseases, bacterial infections, cardiovascular diseases, cancer, and wound healing.13ꢀ18
Focused on anticancer chemotherapy, NO enhances the efficiency of chemotherapy by reducing the
number of active Pꢀglycoproteins and MDRꢀassociated proteins (MRPs),19,20 depleting glutathione that
inactivates the anticancer drugs,19,21 inactivating hypoxiaꢀinduced factors (HIF) that induces multidrug
resistance (MDR) under anaerobic conditions,19,22 and inhibiting nuclear factor kappa B (NFꢀκB) that
regulates the drug resistance.19,23 These outstanding characteristics have recently attracted the attention of
researchers to exploit it as a sensitizer for drugꢀresistant tumors.19,24ꢀ28
There are several points to consider seriously when selecting NOꢀdonors for the design of Pt(IV)
prodrugs facilitating NO release. First, NO donors should be stable during the loading and cellular uptake
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process because unstable NOꢀdonors have limited potential in the current combined drug and NO delivery
system in anticancer therapy.25ꢀ28 Second, stimuliꢀresponsive NOꢀreleasing donors are strongly preferred,
as the physiological functions of NO are highly dependent on its reacting site and concentration.13ꢀ18 Third,
a NOꢀdonor has to improve the loading efficiency of Ptꢀbased drugs. Among a variety of NOꢀdonors with
distinguished characteristics based on stability, stimuli, and water dispersibility,29ꢀ32 the nitrobenzene was
selected because it exhibits high stability in physiological conditions and rapid NO release under light
irradiation.31ꢀ35 In addition, the lipophilic nitrobenzene was believed to increase the hydrophobicity of the
Pt(IV) prodrug, which could contribute to prodrug loading into the various nanoparticles, including
polymeric micelles and liposomes.3ꢀ9
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To deliver the NOꢀreleasing Pt(IV) prodrugs with minimum side effects and high delivery efficiency, we
exploited photoꢀlabile polymeric micelles since simple light irradiation facilitates the spatioꢀtemporal
trigger of drug release and subsequent targeted therapy.36ꢀ40 In addition, we expected that photoꢀinitiated
micellar degradation with the same light source would be beneficial for oneꢀpot drug and NO release.
Accordingly, 4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl (DMNB) was chosen as a functional moiety for photoꢀlabile
micelles because its hydrophobic group could be cleaved from the polymeric backbone upon exposure of
light with the same wavelength as nitrobenzene,41ꢀ45 causing the disassembly of micelles. Furthermore,
because 2ꢀ(trifluoro)phenol, the byproduct of lightꢀexposed nitrobenzene, is more water soluble than
untreated nitrobenzene due to the hydrogen bonds with water molecules, this photoꢀresponsive phase
transition of nitrobenzene was also expected to contribute to the enhanced photoꢀresponsive drug release.
To summarize, we report a novel strategy for the synthesis of photoꢀtriggered NOꢀreleasing Pt(IV)
prodrugs with photoꢀresponsive nitrobenzene and polymeric micelles with photoꢀlabile DMNB (Figure
1A). After intracellular uptake of the micelles, a light irradiation facilitates the NO release from
nitrobenzene, which alleviates drugꢀresistance of the cancer cells. Simultaneously, the DMNB moiety is
released from block copolymers, and hydrophobic nitrobenzene is altered to be more hydrophilic. These
processes accelerate the disassembly of micelles and reduce the hydrophobic interactions between of Pt(IV)
prodrugs and micelles, resulting in the rapid release of Pt(IV) prodrugs (Figure 1B).
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Figure 1. Schematic illustration of (A) the reaction mechanism of the photoꢀactivable NOꢀreleasing Pt (IV)
prodrug and the photoꢀlabile polymeric micelle and (B) its therapeutic mechanism inside cell.
Results and discussion
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Synthesis of NOꢀreleasing Pt (IV) prodrug with nitrobenzene and photoꢀlabile block copolymers
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For the synthesis of photoꢀtriggered NOꢀreleasing Pt(IV) prodrug, amine functionalized nitrobenzene
was prepared prior to the conjugation to Pt(IV) prodrugs. The long alkyl chain was introduced between the
primary amine and nitrobenzene for the efficient reaction with diamminebis(3ꢀcarboxypropanoato)diꢀ
chloroplatinum(IV) (Pt(IV)ꢀdiCOOH) and the enhanced hydrophobicity of the Pt(IV) prodrug. The
presence of the long alkyl chain induced the redꢀshift in the absorbance spectrum of nitrobenzene, in
accordance with previous reports (Figure S1).33,34 The Pt(IV) prodrug with nitrobenzene (Pt(IV)ꢀNO,
compound 8) was synthesized by conjugating carboxylic groups of Pt(IV)ꢀdiCOOH (Figure 2A,
compound 3) to amine functionalized nitrobenzene via EDC/HOBt chemistry (Figure 2B). Successful
synthesis of Pt(IV)ꢀNO was corroborated by 1H NMR , 13C NMR and electrospray ionization mass (ESIꢀ
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MS). In H NMR using DMSOꢀd6, the disappearance of two D2Oꢀexchangable primary amine protons (ꢀ
NH2) of nitrobenzene at δ 3.39 ppm and the appearance of a D2Oꢀexchangable proton triplet at δ 7.79 ppm
demonstrated the formation of a new amide bond (ꢀCOꢀNHꢀ) between nitrobenzene and the carboxylic
groups of Pt(IV) prodrugs. ESIꢀMS results were in accordance with the expected structure of Pt(IV)ꢀNO
([M + H]+ = 1109 m/z and [M + Na]+ = 1131 m/z), and the absorption spectrum of Pt(IV)ꢀNO exhibited
similar spectra with long alkyl chainꢀbearing nitrobenzene. These results demonstrated the successful
synthesis of Pt(IV)ꢀNO. The Pt(IV) prodrug with benzene groups (Pt(IV)ꢀC, compound 13) was prepared
as a control drug incapable of releasing NO by conjugating carboxylic groups of Pt(IV)ꢀdiCOOH to amine
functionalized benzene prepared by Gabriel type synthesis (Figure 2C).
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Figure 2. Synthetic scheme and chemical structures of (A) Pt(IV)ꢀdiCOOH (compound 3), (B) NOꢀ
releasing prodrug (Pt(IV)ꢀNO, compound 8), (C) control prodrug (Pt(IV)ꢀC, compound 13), and (D) photoꢀ
labile polymer (Eꢀpolymer, compound 19) and control polymer (Cꢀpolymer, compound 16).
The block copolymer with photoꢀlabile DMNB (Eꢀpolymer) was synthesized via three steps. First,
poly(ethylene oxide)ꢀbꢀpoly(benzylꢀLꢀglutamate) (PEO114ꢀbꢀPBLG20) was prepared by using macroinitiator
PEOꢀNH2 and monomer γꢀBenzyl ꢀglutamate Nꢀcarboxyanhydride (γꢀBLG NCA) since the ringꢀopening
L
polymerization (ROP) of Nꢀcarboxyanhydride (NCA) has been widely utilized for the synthesis of
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biocompatible and biodegradable polypeptides.46,47 The successful polymerization was confirmed with H
NMR by observing the disappearance of a proton of NCA at δ 4.37 ppm and all peaks corresponding to
PEO and PBLG. The composition of PEO and PBLG was estimated by comparing the integral ratios of the
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peaks at δ 3.64 ppm from ethylene protons (ꢀCH2CH2Oꢀ) of PEO and the peaks at δ 7.26 ppm from
aromatic protons of PBLG. Gel permeable chromatography (GPC) analysis confirmed a single peak
(Mw/Mn = 1.14) which was shifted toward short elution time, indicating the increase in molecular weight
compared to the PEO and successful polymerization (Table 1 and Figure S2). Due to its structural
similarities with Eꢀpolymer, PEO114ꢀbꢀPBLG20 was chosen as a control polymer (Cꢀpolymer) without
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photoꢀresponsiveness (Figure 2D, compound 16). Second, the benzyl groups were removed from PEO114
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bꢀPBLG20 via palladium catalyzed hydrogenation to afford the poly(ethylene oxide)ꢀbꢀpoly(Lꢀglutamic
acid) (PEO114ꢀbꢀPLGA20) with free carboxylic groups. The disappearance of aromatic protons peaks at δ
7.26 ppm and methyl protons adjacent to aromatic rings (ArꢀCH2) at δ 5.10 ppm confirmed the complete
debenzylation of PEO114ꢀbꢀPBLG20. Third, poly(ethylene oxide)ꢀbꢀpoly(4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl Lꢀ
glutamate) (PEO114ꢀbꢀPDMNBLG20) (Eꢀpolymer) was synthesized as a photoꢀresponsive polymer (Figure
2D, compound 19) by conjugating DMNB to the carboxylic groups of PEO114ꢀbꢀPLGA20 via Steglich
esterification. The chemical structure of the block copolymer was confirmed by 1H NMR.
Table 1. Characterization of Eꢀpolymer and Cꢀpolymer
a
c
Sample
[M]0/[I]0
Mnb (NMR)
5000
Mnc (GPC)
6134
Mw/Mn
1.05
PEGꢀNH2
Eꢀpolymer
Cꢀpolymer
ꢀ
20
25
10505
10844
1.34
9380
8373
1.14
a molar ratio of monomer over initiator in the polymerization
b determined by 1HꢀNMR using CDCl3 as solvent.
c determined by GPC using polystyrene as standard and THF as eluent at a flow rate of 1 mg/mL.
Formation and photoꢀdegradability of polymeric micelles
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To investigate the photoꢀdegradability of Eꢀpolymer, the structural change with light irradiation was
observed by 1H NMR. The benzylic methylene protons of DMNB group of PEO114ꢀbꢀPLGA20 have been
disappeared with light irradiation (Figure S3). These results indicate that the DMNB can be released from
Eꢀpolymer by light irradiation, converting the block of the polymer from hydrophobic to hydrophilic.
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The amphiphilicity of Eꢀpolymer and Cꢀpolymer was expected to form selfꢀassembled micelles under
aqueous conditions. The critical micellar concentration (CMC) of Eꢀpolymer and Cꢀpolymer was
determined by the pyrene fluorescence method, which demonstrated their ability to load hydrophobic drug
and possible form of selfꢀassembled structures in aqueous conditions (Figure S4). Accordingly, Pt(IV)ꢀ
NO was loaded into the Eꢀpolymer via the dialysis method to afford the Pt(IV)ꢀNO loaded photoꢀlabile
micelles (Pt(IV)ꢀNO/Eꢀmicelles). Transmission electron microscopy (TEM) depicted spherical
morphologies of Pt(IV)ꢀNO/Eꢀmicelles 25 nm in diameter before ultraviolet (UV) irradiation (365nm, 300
ꢁW/cm2) (Figure 3A). Pt(IV)ꢀNO loaded control micelles (Pt(IV)ꢀNO/Cꢀmicelles), control drugꢀloaded
photoꢀlabile micelles (Pt(IV)ꢀC/Eꢀmicelles) and control drugꢀloaded control micelles (Pt(IV)ꢀC/Cꢀmicelles)
were prepared as control groups (Table 2). The drug loading contents were determined by inductively
coupled plasmaꢀmass spectrometry (ICPꢀMS) and the results are summarized in Table 2. Drug loading
content of Pt(IV)ꢀNO/Eꢀmicelles was higher (27.6%) than those of other formulations, which might be due
to the strong hydrophobic interactions between nitrobenzene and DMNB.
Table 2. Physicochemical characterization of micelles
Sizea (nm)
(dark)
Sizea (nm)
(light)
Drug loading content
(wt.%)
Pt Loading content
(wt. %)
Sample
Pt(IV)ꢀNO/Eꢀ
micelle
27.51±0.128
18.18±0.098
20.08±0.102
4.86±0.023
3.22±0.017
3.52±0.018
75.5±3.1
23.4±1.5
63.1±0.9
36.7±6.0
24.6±0.8
30.9±5.7
Pt(IV)ꢀNO/Cꢀ
micelle
Pt(IV)ꢀC/Eꢀ
micelle
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Pt(IV)ꢀC/Cꢀ
micelle
14.44±0.125
2.53±0.022
24.4±2.1
23.2±2.5
7
8
a determined by particle size analyzer.
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The photoꢀresponsive disassembly of micelles was examined by TEM and dynamic light scattering
(DLS) (Figure 3). There was no change in the morphology or size of lightꢀexposed Cꢀmicelles (Figure
3DꢀF). Although Eꢀmicelles are stable without light irradiation (Figure S5), a majority of Eꢀmicelles
decreased in size or was disrupted after light irradiation for 20 min (Figure 3AꢀC).
Figure 3. Physicochemical characterization of photoꢀresponsive Pt(IV)ꢀNO/Eꢀmicelles and Pt(IV)ꢀNO/Cꢀ
micelles with or without light irradiation. Representative TEM image of Pt(IV)ꢀNO/Eꢀmicelles (A) before
and (B) after light irradiation. (C) Change of hydrodynamic volume of Pt(IV)ꢀNO/Eꢀmicelles before (black)
and after (red) light irradiation. TEM image of Pt(IV)ꢀNO/Cꢀmicelles (D) before and (E) after light
irradiation. (F) Hydrodynamic volume of Pt(IV)ꢀNO/Cꢀmicelles before (black) and after (red) light
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irradiation. Scale bar is 50 nm.
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NOꢀreleasing profile from prodrugꢀloaded micelles
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It is impossible to measure experimentally the NO release from free Pt(IV)ꢀNO in physiological
conditions due to its waterꢀinsoluble characteristics. However, the encapsulation of Pt(IV)ꢀNO with the
micelle was expected to increase the solubility of Pt(IV)ꢀNO, allowing for the measurement of NO release.
Accordingly, the photoꢀresponsive NO releasing behaviour of Pt(IV)ꢀNO/Eꢀmicelles was evaluated by
using a Sievers NOA chemiluminescence analyzer which detects chemiluminescence generated by the
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quantitative reaction between NO and ozone in real time. The ESI/MS and H NMR demonstrated that
axial ligands are not cleaved after light irradiation and the released NO does not form any adducts with the
Pt(IV) prodrugs (data not shown). In particular, Xꢀray photoelectron spectroscopy (Figure S6) clearly
showed that the binding energy Pt4f of Pt(IV)ꢀNO was retained before (75.9 and 79.1 eV of 4f(7/2) and
4f(5/2) levels) and after light irradiation (75.3 and 78.2 eV of 4f(7/2) and 4f(5/2) levels) (Figure S6B,C).
These are similar to those of K2PtCl6 (75.7 and 79.0 eV of the 4f(7/2) and 4f(5/2) levels)48, but different
with those of cisplatin (73.2 and 76.6 eV of the 4f(7/2) and 4f(5/2) levels) (Figure S6A). These results
demonstrate that the species of prodrug is retained after light irradiation, implying the photoꢀstability of the
prodrugs.
As shown in Figure 4A, the pulsatile NO release profile was observed depending on the repeated
exposure of light and dark, clearly demonstrating the lightꢀdependent NO release behaviour of Pt(IV)ꢀNO.
These results indicate that the hydrophobic interactions between block copolymer and nitrobenzene
stabilize the nitrobenzene to release NO efficiently under lightꢀexposed aqueous conditions.
We presumed that the photoꢀresponsive disassembly of Eꢀmicelles would show a more rapid release of
Pt(IV) prodrugs than Cꢀmicelles. In addition, the accelerated drug release was expected during NO release
owing to the photoꢀinduced transition from nitrobenzene to the relatively more water soluble 2ꢀ
(trifluoromethyl)phenol. To ascertain our hypothesis, the photoꢀresponsive release of Pt(IV) prodrugs was
monitored (Figure 4B). The release of drug from Pt(IV)ꢀC/Cꢀmicelles was independent of light irradiation.
However, as postulated, Pt(IV)ꢀC/Eꢀmicelles and Pt(IV)ꢀNO/Cꢀmicelles showed higher drug release in the
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light condition than in the dark condition due to the disassembly of micelles by cleavage of hydrophobic
DMNB or transition of nitrobenzene into hydrophilic 2ꢀ(trifluoromethyl)phenol. Accordingly, Pt(IV)ꢀ
NO/Eꢀmicelles exhibited the most photoꢀresponsive behaviors in drug release. These results suggest that
the utilization of nitrobenzene not only facilitates the loading of Pt(IV) prodrugs in the micelles, but also
improves the photoꢀresponsive drug release behaviors cooperatively with DMNB. After Pt(IV) prodrug is
released from the micelles, it is expected to be transformed into its active Pt(II) form under intracellular
reductive conditions for the efficient anticancer effects. In XPS results (Figure S7), the binding energy Pt4f
of Pt(IV)ꢀNO under intracellular reduction conditions (1 mM ascorbic acid, 24 h)48ꢀ50 was same with those
of cisplatin (73.2 and 76.6 eV of the 4f(7/2) and 4f(5/2) levels) (Figure S6A), which is totally different
from those of Pt(IV)ꢀNO without incubation under reductive conditions (75.9 and 79.1 eV of 4f(7/2) and
4f(5/2) levels). These results imply that the prodrug can exert the Pt(II)ꢀbased anticancer effects after being
released from micelle and NO release.
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Prior to investigating the anticancer effects, the potential of Pt(IV)ꢀNO for intracellular NO delivery was
evaluated by confocal laser scanning microscopy (CLSM) with DAFꢀ2DA, a typical NOꢀdetecting
fluorescence dye (Figure 4C). Cells treated with Pt(IV)ꢀC/Eꢀmicelles showed negligible NO release
similar to nonꢀtreated cells, irrespective of light and dark conditions. However, the cells treated with
Pt(IV)ꢀNO/Eꢀmicelles exhibited increased NO release only when exposed to the light, demonstrating that
Pt(IV)ꢀNO/Eꢀmicelle has the ability to release NO on demand at the intracellular level.
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Figure 4. (A) Realꢀtime NO release profiles of Pt(IV)ꢀNO/Eꢀmicelles under repeated light irradiation and
dark conditions. Red arrow and black arrow represent the light on and off, respectively. (B) Cumulative
release of drug from Pt(IV)ꢀNO/Eꢀmicelles, Pt(IV)ꢀNO/Cꢀmicelles, Pt(IV)ꢀC/Eꢀmicelles, and Pt(IV)ꢀC/Cꢀ
micelles with or without light irradiation. (C) Intracellular release of NO from Pt(IV)ꢀNO/Eꢀmicelle
observed by DAFꢀ2 DAꢀassisted CLSM using MCFꢀ7 cells without and with light irradiation. Green color
depicts fluorescence of DAFꢀ2 DA after reaction with released NO. Scale bar is 50 ꢁm.
Therapeutic evaluation of lightꢀresponsive prodrugꢀloaded micelle
The therapeutic potential of Pt(IV)ꢀNO/Eꢀmicelle was evaluated in HCT ꢀ116 and MCFꢀ7 cells (Figure
5, Figure S8, and Table S1). Bare Eꢀmicelles and bare Cꢀmicelles showed negligible cytotoxicity,
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indicating good biocompatibility of the polymers. The increased cytotoxicity of free Pt(IV)ꢀNO was
observed under light irradiation, whereas the therapeutic effects of free Pt(IV)ꢀC were independent of light
irradiation, demonstrating that NO enhanced the sensitivity of Pt(IV) drug. As reported previously,19 the
enhanced anticancer effects might be due to the role of NO in reducing drug resistance via depletion of
glutathione21 and inhibition of Pꢀglycoprotein,19,20 MRPs,19 HIF,22 and NFꢀκB.23 The utilization of micelles
improved the cytotoxicity of Pt(IV) prodrugs compared to the free Pt(IV)ꢀNO and Pt(IV)ꢀC. These results
may be attributed to the amphiphilic characteristics of Pt(IV)ꢀNO and Pt(IV)ꢀC because amphiphilic Ptꢀ
based drugs show poor intracellular uptake due to the entrapment in lipid bilayers of the cellular
membrane.7 That is, these data imply the requirement of drug delivery systems for efficient intracellular
delivery of amphiphilic Pt(IV) prodrugs. Light irradiation has negligible effects on the cytotoxicity of
Pt(IV)ꢀC/Cꢀmicelles. However, Pt(IV)ꢀNO/Cꢀmicelles and Pt(IV)ꢀC/Eꢀmicelles slightly enhanced
anticancer effects under light irradiation due to the drugꢀsensitizing effects or enhanced drug release. As
expected, the Pt(IV)ꢀNO/Eꢀmicelles exerted significantly improved anticancer effects upon light irradiation,
which was likely due to the combined effects of NO and rapid drug release. Among various mechanism of
NO in chemosensitization, we postulated that the lightꢀresponsive NO release in the cells increases the
cellular uptake of platinum drugs by inhibiting drug efflux associated with Pꢀglycoproteins and MRPs,
which enhances the chemotherapeutic effects.19,20 Therefore, we measured Pt contents in the cells after
treatment of each micelle with or without light irradiation (Figure 5C). There is no difference in Pt uptake
profile among nonꢀilluminated micelles, implying that the process of influx and efflux of Pt drug reached
to equilibrium states within 18 h incubation of micelles. Accordingly, it is noteworthy that the improved
uptake of Pt in lightꢀirradiated micelles happened only during 20 min light irradiation and 30 min further
incubation. It is also of importance to note that the possible effect of NO in enhanced drug influx can be
excluded because micelles were washed out from the media before light irradiation. The NOꢀreleasing
drug micelles (Pt(IV)ꢀNO/Eꢀmicelles and Pt(IV)ꢀNO/Cꢀmicelles) showed higher Pt uptake than NOꢀnonꢀ
releasing drug micelles (Pt(IV)ꢀC/Eꢀmicelles and Pt(IV)ꢀC/Cꢀmicelles), respectively, indicating that NO
might make an effect to the efflux system of Pt drug. In addition, the lightꢀresponsive micelles (Pt(IV)ꢀ
NO/Eꢀmicelles and Pt(IV)ꢀC/Eꢀmicelles) showed higher Pt uptake than lightꢀnonꢀresponsive micelles
(Pt(IV)ꢀNO/Cꢀmicelles and Pt(IV)ꢀC/Cꢀmicelles), respectively, implying that light irradiation accelerated
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the drug release into the cells by disruption of drugꢀloaded micelles. These results imply that the improved
anticancer effects of Pt(IV)ꢀNO/Eꢀmicelles are ascribed not only to the lightꢀresponsive burst drug release,
but also to the role of NO in inhibiting drug efflux. The mode of cell death was investigated by Annexin Vꢀ
fluorescein isothiocyanate (FITC)/propidium iodide (PI) assayꢀassisted flow cytometry (Figure S9). We
found that the anticancer effects of Pt(IV)ꢀNO/Eꢀmicelles were through necrosis and late apoptosis (Figure
5D). In contrast, control groups induced an insignificant level of necrosis and apoptosis, which were
correlated with the results of methylthiazole tetrazolium (MTT) assay.
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Figure 5. In vitro anticancer effects of Pt(IV)ꢀNO/Eꢀmicelle with and without light irradiation in (A) HCTꢀ
116 and (B) MCFꢀ7 cell lines. Statistical analysis was performed as compared with the lightꢀexposed
Pt(IV)ꢀNO/Eꢀmicelle (*P<0.05; **P<0.01; ***P<0.001). The platinum concentration is 10 ꢁM. (C) In vitro
cellular uptake of Pt(IV)ꢀNO/Eꢀmicelle, Pt(IV)ꢀNO/Cꢀmicelle, Pt(IV)ꢀC/Eꢀmicelle, and Pt(IV)ꢀC/Cꢀ
micelle with and without light irradiation in MCFꢀ7 cell lines (*P<0.05; **P<0.01; ***P<0.001). The
platinum concentration is 50 ꢁM. (D) Apoptosis analysis of MCFꢀ7 cells by flow cytometry analysis. The
platinum concentration is 10 ꢁM.
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CONCLUSIONS
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In summary, we present herein the novel design of photoꢀresponsive Pt(IV) prodrugs and polymeric
micelles from the synthesis and characterization to biomedical application. The nitrobenzene that is
conjugated to Pt(IV) prodrugs has multiple functions; 1) releases NO in a photoꢀresponsive manner and
subsequently sensitizes cancer cells, 2) improves the loading efficiency of Pt(IV) prodrugs and enhances
the photoꢀresponsive drug releasing behaviors, leading effective anticancer effect. Although the present
study utilizes UV light sources to show its therapeutic potential conceptually, the utilization of
upconversion nanoparticles or twoꢀphoton excitation could be an alternative way and further study to
overcome the limitation of UV light.36,51
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Experimental Section
Materials Cisꢀdiamineplatinum (II) dichloride (99.9%), 4ꢀchloroꢀ1ꢀnitroꢀ2ꢀ(trifluoromethyl)benzene
(99.0%), NꢀBocꢀ1,6ꢀhexanediamine (98.0%), 4,5ꢀdimethoxyꢀ2ꢀnitrobenyl alcohol (98.0%), succinic
anhydride (99.0%), Lꢀglutamic acid γꢀbenzyl ester (99.0%), triphosgene (98.0%), N,N'ꢀ
dicyclohexylcarbodiimide (DCC, 99.0%), 4ꢀdimthylaminopyridine (DMAP, 99.0%), palladium on carbon
(10 wt. % loading, matrix carbon, dry support), Nꢀ(3ꢀdimethylaminopropyl)ꢀN'ꢀethylcabodiimide
hydrochloride (EDC.HCl, ≥98.0%, AT), 1ꢀhydroxybnzoiazole hydrate (HOBt, 97.0%), methanol (99.9 %),
sodium carbonate (anhydrous, powder, 99.99%), N,Nꢀdimethylformamide (anhydrous, 99.8%), N,Nꢀ
diisopropylethylamine (DIPEA, 99.5%), acetonitrile (anhydrous, 99.8%), aniline (ACS reagent, ≥ 99.5%),
hydrogen chloride solution (4.0 M in dioxane), 3ꢀ(4,5ꢀdimemthylꢀthiaolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium
bromide (MTT) and palladium on activated carbon (10% PdꢀC) were purchased from SigmaꢀAldrich Co.
(St. Louis, MO). Methoxy polyhylene glycol amine (mPEGꢀAM, Mn=5,000 g molꢀ1, 90.3%) was
purchased from Sun Biochem, Inc. Nꢀ(6ꢀbromohexyl)phthalimide (97.0%) was purchased from Alfa Aesar.
N,Nꢀdimethylformaimide (DMF, 99.5%) and fluorescence dye pyrene (98.0%) was obtained from Acros
Organics (New Brunswick, NJ, USA). Chloroform (≥99.0%) was purchased from Merck (Darmstadt,
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Germany). Diethyl ether (99.0%), ethanol (94.5%), ethyl acetate (99.5%), hexane (95.0%), tetrahydrofuran
(THF, 99.5%), hydrogen peroxide solution (34.4%), hydrazine monohydrate (80.0%), methylene chloride
(MC, 98.0%), potassium carbonate, sodium bicarbonate (NaHCO3, 99.0%) and sodium sulfate (Na2SO4,
98.5%) were purchased from Samchun pure chemical (Pyeongtaek, Korea). A dialysis membrane with
molecular weight cutꢀoff (MWCO) of 3500 Da was purchased from Spectrum Laboratories (Rancho
Domingues, CA, USA). Dulbecco Modified Eagle’s medium (DMEM) penicillinꢀstreptomycin, fetal
bovine serum (FBS) and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Corning
(Manassas, VA, USA). 4,5ꢀDiaminofluorescein diacetate (DAFꢀ2 DA) was purchased from abcam.
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1
General characterization H and13C nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR)
spectra were recorded on Bruker Advance 300 MHz FTꢀNMR with deuterium chloroform (CDCl3) and
DMSO (DMSOꢀd6) as a solvent. The chemical shifts are reported in parts per million (ppm) and referenced
to the CDCl3 singlet at 7.26 ppm, DMSOꢀd6 at 2.50 ppm. 13C NMR spectra were referenced to the center
line of the CDCl3 triplet at 77.0 ppm, DMSOꢀd6 septet at 39.5 ppm. The splitting patterns are designated as
s (singlet), d (doublet), t (triplet), m (multiplet) and br (broad), and coupling constants (J) are reported in
Hertz (Hz). Gel permeation chromatography (GPC) measurements were performed on a Waters Alliance
e2695 GPC system, equipped with three consecutive Styragel® columns (HR1, HR2 and HR4). The
detection was performed on a 2414 refractometer. THF was used as the eluent at a flow rate of 1 mL/min.
A series of polystyrene standards were used for calibration. The molecular weight and polydispersity index
of the polymers were analysed using Empower 2 software. The dynamic light scattering (DLS)
measurements were conducted using a Zetasizer Nano S90 system (Malvern Instruments, Worcestershire,
UK). All measurements were carried out at 25 °C with a 90° detection angle. UVꢀvisible spectra were
recorded on a UV 2550 spectrophotometer (Shimadzu). Electrospray ionization (ESI) mass analysis was
performed on a Thermo Scientific LTQ Velos dual ion trap mass spectrometer equipped with normal ESI
source in the positive ion mode. All sample solutions were prepared in HPLC grade methanol and distilled
water (1:1 v/v). Parameters of ESI were set as electrospray voltage of 2.5 kV and capillary temperature of
250 °C. The spectrum was averaged from 200 scanned spectra obtained using enhanced scan mode.
Samples were analyzed using 15 ꢁM of each solution.
Synthesis of compound 2, [Oxoplatin] Synthesis of oxoplatin was done by employing a procedure
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reported by Y. Shi et al.52 as follows. Briefly, hydrogen peroxide (34.4 wt.%, 75 mL) was added dropwise
to a suspension of cisplatin, 1 (900 mg, 3.00 mmol) in water (45 mL) at 60 °C. After about 4 h, the bright
yellow solution obtained, was cooled at 25 °C for overnight to afford yellow crystals. The crystals was
filtered, washed with iceꢀcooled water and dried in vacuum oven for overnight at 25 °C to obtain oxoplatin
2 (yield: 72.9%, 730 mg).
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Synthesis
of
compound
3,
[Pt(IV)ꢀdiCOOH],
(Diamminebis(3ꢀcarboxypropanoato)diꢀ
chloroplatinum(IV)) Compound 3 was synthesized by employing a procedure reported by M. R.
Reithofer et al.53 with minor modification. Succinic anhydride (903 mg, 9.03 mmol) was added to a
suspension of compound 2 (730 mg, 2.19 mmol) in DMF (10 mL), and the reaction mixture was stirred at
70 °C. After 2 h, a yellow color solution was obtained. DMF was removed from the reaction mixture under
reduced pressure in rotavapor to obtain a sticky mass. This sticky mass was dissolved in acetone and
filtered to afford a clear yellow solution. This solution was concentrated under reduced pressure. On
addition of diethyl ether to this concentrated solution, a pale yellow precipitate was obtained. The
precipitate was filtered, washed with diethyl ether repeatedly and finally dried under vacuum oven at 25 °C
for overnight to obtain compound 3 (yield: 75.2%, 880 mg). 1H NMR (300 MHz, DMSOꢀd6) δ 12.08 (bs,
2H), 6.49 (bs, 6H), 2.49 (m, 4H), 2.37 (m, 4H).
Synthesis of compound 6, [nitrobenzeneꢀBoc] Compound 6 was prepared following the method
described by E. Vittorino et al.54 with minor modification. A mixture of 4ꢀchloroꢀ1ꢀnitroꢀ2ꢀ
(trifluoromethyl)benzene (compound 4, 3.80 g, 16.848 mmol), NꢀBocꢀ1,6ꢀhexanediamine (compound 5,
4.37 g, 20.22 mmol) and sodium carbonate (2.68 g, 25.27 mmol) in anhydrous acetonitrile (90 mL) was
heated to reflux under continuous stirring for 19 h. After cooling down to room temperature, the resulting
suspension was filtered and organic solution was concentrated to sticky solid under reduced pressure. This
sticky solid mixture was purified over silica gel column chromatography using methylene chlorideꢀ
methanol mixture as eluent to obtain pure compound 6 (yield: 52.0%, 3.55 g). 1H NMR (300 MHz, CDCl3)
δ 8.00 (d, J = 9.0 Hz, 1H), 6.89 (d, J = 2.6 Hz, 1H), 6.64 (dd, J = 9.0, 2.6 Hz, 1H), 4.53 (bs, 1H), 4.08 (bs,
1H), 3.20 (t, J = 6.9 Hz, 2H), 3.12 (t, J = 6.3 Hz, 2H), 1.67 (m, 2H), 1.45 (m, 6H), 1.44 (s, 9H). 13C NMR
(75 MHz, CDCl3) δ 156.4, 152.2, 136.2, 129.5, 124.3, 120.7, 112.4, 111.4, 79.5, 43.2, 40.3, 30.1, 28.7,
28.6 (two carbons), 26.3, 26.1 (two carbons). MS (ESI) m/z (M+Na)+: 428.16.
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Synthesis of compound 7, [nitrobenzeneaniline], (4ꢀnitroꢀ3ꢀ(trifluoromethyl) aniline) To a solution of
compound 6 (1.22 g, 3.01 mmol) in anhydrous methylene chloride (10 mL) was added 4 M dioxaneꢀHCl
(10 mL) under cooling condition and then the reaction mixture was allowed to stir vigorously at 25 °C for
18 h. After completion of the reaction (checked by TLC), the organic solvent was evaporated at reduced
pressure, then saturated solution of NaHCO3 was added to neutralize excess HCl. Neutralized solution was
extracted with a mixture of methanolꢀmethylene chloride (5:95 v/v) for three times, organic portion was
washed with brine solution and dried over anhydrous Na2SO4 to afford clear organic solution. This organic
solution was evaporated under reduced pressure to obtain brown sticky solid compound 7 (yield: 98.0%,
900 mg). 1H NMR (300 MHz, DMSOꢀd6) δ 8.07 (d, J = 9.0 Hz, 1H), 7.57 (bs, 1H), 7.05 (s, 1H), 6.80 (dd, J
= 9.0, 2.4 Hz, 1H), 3.39 (bs, 2H), 3.16 (m, 4H), 1.54 (m, 2H), 1.35 (m, 6H).
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Synthesis of compound 8, [Pt(IV)ꢀNO] To a solution of compound 3 (300 mg, 0.56 mmol) in anhydrous
DMF (3 mL) was added EDC.HCl (269 mg, 1.40 mmol) and HOBt (190 mg, 1.40 mmol) under cooling
condition and then the reaction mixture was allowed to stir vigorously at 25 °C. After 30 min., a solution of
compound 7 in DMF (3 mL) was added slowly and then DIPEA (3.7 mL) was added, and the reaction
mixture was allowed to stir at 25 °C for 18 h. After completion of the reaction (checked by TLC), DMF
was evaporated under reduced pressure to obtain solid residue and water was added to that solid to make a
suspension, followed by extraction with a mixture of methanolꢀmethylene chloride (10:90 v/v) for three
times, washing the organic portion with brine solution and finally drying over anhydrous Na2SO4 to afford
clear organic solution. This organic solution was evaporated under reduced pressure to obtain sticky solid
mixture which on purification over silica gel column chromatography using methylene chlorideꢀmethanol
mixture as eluent furnished pure compound 8 (yield: 15.2%, 94.6 mg). 1H NMR (300 MHz, DMSOꢀd6) δ
8.07 (d, J = 9.3 Hz, 1H), 7.79 (t, J = 5.4 Hz, 1H, D2O exchangeable), 7.55 (t, J = 5.1 Hz, 1H, D2O
exchangeable), 7.05 (brs, 1H), 6.80 (dd, J = 9.3, 2.6 Hz, 1H), 6.49 (bs, 3H, D2O exchangeable), 3.16 (dd, J
= 12.3, 6.6 Hz, 2H), 3.01 (dd, J = 12.3, 6.6 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.25 (t, J = 7.2 Hz, 2H), 1.54
(m, 2H), 1.35 (m, 6H). 1H NMR (300 MHz, CD3OD) δ 7.94 (d, J = 9.3 Hz, 1H), 6.89 (d, J = 2.6 Hz, 1H),
6.67 (dd, J = 9.3, 2.6 Hz, 1H), 3.00 (m, 4H), 2.50 (t, J = 6.3 Hz, 2H), 2.35 (t, J = 6.3 Hz, 2H), 1.56 (m,
2H), 1.40 (m, 6H). 13C NMR (75 MHz, CD3OD) δ 182.6, 175.4, 154.7, 136.1, 130.7, 126.0, 122.4, 112.8,
112.2, 44.1, 40.6, 32.7, 32.9, 30.4, 29.8, 27.9 (two carbons). MS (ESI) m/z (M+H)+ :1109.00 and (M+Na)+:
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Synthesis of compound 11, (2ꢀ(6ꢀ(Phenylamino)hexyl)isoindolineꢀl,3ꢀdione) Compound 11 was
prepared following the method described by Y.ꢀP. Pang et al.55 with minor modification. A mixture of
aniline (compound 9, 364 mg, 4.00 mmol), 2ꢀ(6ꢀbromohexyl)isoindolineꢀl,3ꢀdione (compound 10, 1244
mg, 4.00 mmol) and sodium carbonate (552 mg, 4.00 mmol) in anhydrous acetonitrile (20 mL) was heated
to reflux under continuous stirring for 25 h. After completion of the reaction (checked by TLC),
acetonitrile was evaporated under reduced pressure to obtain sticky oily mixture and then water was added
to that mixture to make a suspension, followed by extraction with a mixture of methanolꢀmethylene
chloride (10:90 v/v) for three times, washing the organic portion with brine solution and finally drying
over anhydrous Na2SO4 to afford clear organic solution. This organic solution was evaporated under
reduced pressure to obtain sticky oily mixture which on purification over silica gel column
chromatography using methylene chlorideꢀmethanol mixture as eluent furnished pure compound 11 as a
colorless oil (yield: 31.7%, 410 mg). 1H NMR (300 MHz, CDCl3) δ 7.82 (m, 2H), 7.68 (m, 2H), 7.15 (dd, J
= 8.4, 7.2 Hz, 2H), 6.66 (t, J = 7.2 Hz, 1H), 6.58 (d, J = 8.4 Hz, 2H), 3.68 (t, J = 7.2 Hz, 2H), 3.61 (brs, 1H),
3.08 (t, J = 7.2 Hz, 1H), .72ꢀ1.66 (m, 2H), 1.64ꢀ1.56 (m, 2H), 1.48ꢀ1.35 (m, 4H). 13C NMR (75 MHz,
CDCl3) δ 168.5 (two carbons), 148.5, 134.0, 133.9, 132.2, 129.3 (two carbons), 123.2 (two carbons),
117.1(two carbons), 112.7 (two carbons), 43.8, 37.9, 29.2, 28.5, 26.8, 26.7. MS (ESI) m/z (M+H)+: 323.28.
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Synthesis of compound 12, (1ꢀNꢀPhenylhexaneꢀ1,6ꢀdiamine) Compound 12 was prepared following the
method described by YꢀP. Pang et al.55 with minor modification. To a stirred solution of compound 11 (400
mg, 1.24 mmol) in ethanol (12 mL) was added hydrazine monohydrate (0.496 mL) and stirred for 15 hours
at room temperature. After completion of the reaction (checked by TLC), the white insoluble materials
were filtered off and the filtrate was concentrated under vacuum, the residue was diluted chloroform and
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filtered again. Concentration of the filtrate gave colorless oily compound 12 (Yield: 94.3%, 225 mg). H
NMR (300 MHz, CDCl3) δ 7.17 (dd, J = 8.7, 7.5 Hz, 2H), 6.68 (t, J = 7.5 Hz, 1H), 6.59 (d, J = 8.7 Hz, 2H),
3.11 (t, J = 6.9 Hz, 2H), 2.69 (t, J = 6.9 Hz, 2H), 1.65ꢀ1.56 (m, 2H), 1.48ꢀ1.34 (m, 6H). 13C NMR (75 MHz,
CDCl3) δ 148.5, 129.2 (two carbons), 117.0, 112.7 (two carbons), 43.9, 41.8, 33.1, 29.5, 27.0, 26.7. MS
(ESI) m/z (M+H)+: 193.24.
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Synthesis of compound 13, [Pt(IV)ꢀC] To a solution of compound 3 (120 mg, 0.23 mmol) in anhydrous
DMF (2 mL) was added EDC.HCl (95 mg, 0.49 mmol) and HOBt (67 mg, 0.49 mmol) under cooling
condition and then the reaction mixture was allowed to stir vigorously at 25 °C. After 30 min., a solution of
compound 12 in DMF (2 mL) was added slowly and then DIPEA (0.20 mL) was added, and the reaction
mixture was allowed to stir at 25 °C for 18 h. After completion of the reaction (checked by TLC), DMF
was evaporated under reduced pressure to obtain solid residue and water was added to that solid to make a
suspension, followed by extraction with a mixture of methanolꢀmethylene chloride (10:90 v/v) for three
times, washing the organic portion with brine solution and finally drying over anhydrous Na2SO4 to afford
clear organic solution. This organic solution was evaporated under reduced pressure to obtain sticky solid
mixture which on purification over silica gel column chromatography using methylene chlorideꢀmethanol
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mixture as eluent furnished pure compound 13 (yield: 35.5%, 70 mg). H NMR (300 MHz, DMSOꢀd6) δ
7.80 (t, J = 5.4 Hz, 1H, D2O exchangeable), 7.04 (t, J = 7.5 Hz, 2H), 6.51 (m, 3H), 6.45 (brs, 3H, D2O
exchangeable), 5.49 (t, J = 5.4 Hz, 1H, D2O exchangeable), 2.99 (m, 4H), 2.43 (t, J = 7.5 Hz, 2H), 2.25 (t, J
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= 7.5 Hz, 2H), 1.54ꢀ1.44 (m, 2H), 1.41ꢀ1.23 (m, 6H). H NMR (300 MHz, CD3OD) δ 6.98 (m, 2H), 6.50
(m, 3H), 3.06 (t, J = 6.9 Hz, 2H), 2.95 (t, J = 7.2 Hz, 2H), 2.49 (t, J = 6.3 Hz, 2H), 2.32 (t, J = 6.3 Hz, 2H),
1.51ꢀ1.47 (m, 2H), 1.45ꢀ1.40 (m, 2H), 1.33ꢀ1.25 (m, 4H). 13C NMR (75 MHz, CD3OD) δ 182.6, 175.4,
150.5, 130.1(two carbons), 118.2, 144.4 (two carbons), 45.2, 40.6, 32.8, 32.6, 30.5, 30.4, 28.1, 28.0. MS
(ESI) m/z (M+H)+: 883.32.
Synthesis of compound 15, [γꢀBLG NCA], (γꢀBenzyl Lꢀglutamate Nꢀcarboxyanhydride) Compound 15,
the monomer of γꢀBenzyl ꢀglutamate Nꢀcarboxyanhydride was prepared following the method described
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by S. R. Pan et al. 56 with minor modification as follows. To a solution of γꢀBenzyl ꢀglutamate (BLG,
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compound 14) (3.0 g, 12.64 mmol) in anhydrous THF (70 mL), a solution of triphosgene (1.88 g, 6.32
mmol) in dry THF (30 mL) was added and the reaction mixture was heated to 65 °C. After 30 min., the
suspension turned transparent, heating was continued for another 1.5 h., the reaction mixture was cooled
down to room temperature and solvent was evaporated to dryness under reduced pressure to obtain sticky
solid mass. This sticky mass was dissolved in ethyl acetate and hexane was added slowly to afford white
wooly precipitate. The precipitate was filtered, washed with hexane and dried under vacuum at 25 °C for
overnight to obtain compound 15 (yield: 87.6%, 2.92 g). 1H NMR (300 MHz, CDCl3) δ 7.37 (m, 5H), 6.27
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(bs, 1H), 5.15 (s, 2H), 4.37 (dt, J = 5.1, 0.6 Hz, 1H), 2.61 (t, J = 6.7 Hz, 2H), 2.29 (m, 1H), 2.13 (m, 1H).
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Synthesis of compound 16 [Cꢀpolymer], [PEO114ꢀbꢀPBLG20], (Poly(ethylene oxide)114ꢀblockꢀ
poly(benzyl Lꢀglutamate)20) Compound 16, PEO114ꢀbꢀPBLG20 was prepared following the method
described by S. Kumar et al.57 with minor modification as follows. Aminoꢀterminated polyethylene oxide
(PEOꢀNH2) was dissolved in dry DMF under nitrogen atmosphere in a flameꢀdried roundꢀbottomed flask.
In another flask, the BLGꢀNCA monomer (compound 15) was dissolved in dry DMF under nitrogen
atmosphere. Then monomer solution was added dropwise to the polymer solution using syringe. The
resulting mixture was stirred at 25 °C under nitrogen atmosphere for 5 days. After completion of the
reaction (checked an aliquot by proton NMR), DMF was evaporated under vacuum, residue was dissolved
in chloroform and on addition of cold diethyl ether, white solid precipitate of polymeric compound 16 was
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obtained. H NMR (300 MHz, CDCl3) δ 8.32 (NH), 7.26 (aromatic), 5.10 (ArꢀCH2), 3.89 (αꢀCH), 3.64
(CH2CH2O), 3.38 (OCH3), 1.80ꢀ2.27 (βꢀCH2 and γꢀCH2).
Synthesis of compound 17, [PEO114ꢀbꢀPLGA20], (Poly(ethylene oxide)114ꢀblockꢀpoly(Lꢀglutamic acid)20)
Compound 17, PEO114ꢀbꢀPLGA20 was prepared following the method described by S. Kumar et al.57 with
minor modification as follows. Palladium catalyzed (10% PdꢀC) hydrogenation was employed for
debenzylation. To a solution of compound 16 in dry THFꢀMeOH mixture, 10% PdꢀC was added under
nitrogen atmosphere. Then hydrogen balloon was used to replace the nitrogen inside the flask and the
reaction mixture under hydrogen atmosphere, was stirred at 25 °C for 24 h. After completion of the
reaction (checked an aliquot by proton NMR), PdꢀC was filtered off, the resulting solvent was evaporated
under vacuum, residue was dissolved in deionized water and dialyzed in deionized water for 48 h. The
polymer was precipitated by adding cold diethyl ether, the solid was filtered off and then dried under
vacuum at 25 °C for overnight to obtain compound 17. 1H NMR (300 MHz, DMSOꢀd6) δ 12.12 (COOH),
8.17 (NH), 3.90ꢀ4.30 (αꢀCH), 3.34ꢀ3.64 (CH2CH2O), 3.30 (OCH3), 1.70ꢀ2.35 (βꢀCH2 and γꢀCH2).
Synthesis of compound 19 [Eꢀpolymer], [PEO114ꢀbꢀPDMNBLG20], (poly(ethylene oxide)114ꢀblockꢀ
poly(4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl Lꢀglutamate)20) Compound 19, PEO114ꢀbꢀPDMNBLG20 was prepared
following the method described by S. Kumar et al.57 with minor modification as follows. To a solution of
compound 17 in anhydrous DMF was added DCC and DMAP under cooling condition and then the
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reaction mixture was allowed to stir vigorously at 25 °C. After 30 min., a solution of compound 18 in DMF
was added slowly using syringe and the reaction mixture was allowed to stir at 25 °C for 24 h. After
completion of the reaction (checked an aliquot by proton NMR), DMF was evaporated under reduced
pressure to obtain solid residue. The polymer was precipitated by adding cold diethyl ether, the solid was
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filtered off and then dried under vacuum at 25 °C for overnight to obtain compound 19. H NMR (300
MHz, CDCl3) δ 8.39 (NH), 7.57 (ArꢀH), 6.95 (ArꢀH), 5.35 (ArꢀCH2), 3.90 (αꢀCH and ArꢀOCH3), 3.35ꢀ
3.64 (CH2CH2O), 3.37 (OCH3), 1.00ꢀ2.90 (βꢀCH2 and γꢀCH2).
Lightꢀinduced change of chemical structure of Eꢀpolymer 3 mg of Eꢀpolymer was dissolved in CDCl3
and the solution was exposed to 4 h UV irradiation (365 nm, 300 ꢁW/cm2). Its resonance peaks were
recorded by Bruker Advance 300 MHz FTꢀNMR at preꢀdetermined time.
Determination of critical micellar concentration (CMC) Fluorescence pyrene was used to measure the
CMC. In brief, each 1 mL of polymer with various concentrations ranged from 0.5 ꢁg/mL to 250 ꢁg/mL
was mixed with the 1 mL of 1.2 ꢁM pyrene solution. After 24 h shaking at room temperature, the
fluorescence of the solution was measured at the excitation wavelength of 336 nm. The CMC was
calculated from the plots of the intensity ratios at 373 and 383 nm against the logarithm of the
concentration of each polymer.
Preparation of drugꢀloaded micelles Drug loaded micelles were prepared by simple dialysis methods. In
brief, each polymer was dissolved in highꢀperformance liquid chromatography (HPLC) grade THF. To this
solution, a sample of each drug dissolved in the THF was added. Under vigorous stirring, distilled water
was added dropwise and allowed to stir for 30 min at room temperature. After that, the solution was
transferred to a dialysis membrane bag (MWCO=3500) and dialyzed for 24 h against deionized water to
remove the organic solvent and free drug. The micellar solution was further filtered through 0.45 ꢁm
syringe filter to remove the residual aggregates. The resultant solution were lyophilized to get dried drug
loaded micelles.
Drug loading content The amount of drugs in the micelles was determined using an inductively coupled
plasmaꢀmass spectrometry (ICPꢀMS, NexIon300x; PerkinElmer) facility supported by the Pohang Center
for Evaluation of Biomaterials (POCEB) at Pohang Technopark. The calibration curves were made by
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dissolving different concentrations of cisplatin in DPBS. The drug loading content were calculated by
using platinum calibration curves and the following formulae:
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Loading content (wt. %) = ꢀꢁꢂꢃꢄꢅ ꢆꢇ ꢈꢆꢉꢊꢁꢊ ꢊꢋꢌꢃ × 100%
ꢀꢁꢂꢃꢄꢅ ꢆꢇ ꢍꢆꢈꢎꢏꢁꢋ
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Confirmation of speciation of Pt drugs XPS spectra were obtained using an ESCALAB 200iXL
spectrometer (VG scientific Inc, USA) with a nonꢀmonochromatic Mg Kα (1253.6 eV) Xꢀray source.
In vitro measurement of NO release NO release was characterized by using NO analyzer (Sievers 280i
Nitric Oxide Analyzer, NOA, GE analytical instruments, USA) based on a chemiluminescence method.
The system is described in a previous report.58 Briefly, the sample was put in the multiꢀneck roundꢀbottom
flask and then the UV light was irradiated to the flask. The argon flow carries the NO generated from
sample in the flask into the chemiluminescence NO analyzer. Therefore, practically, UV irradiation and unꢀ
vaporized components could not interfere with the signal. Prior to detect NO, the NO analyzer was
calibrated using NO zero filter (0 ppm NO) and a 45.0 ppm NO standard gas. Micelle containing 1 mg of
Pt(IV)ꢀNO was transferred into DPBS solution in the sampling round bottom flask whom the UV light
(365nm, 300 ꢁW/cm2) irradiated. The NO released from sample move into the NO analyzer by using Ar (g)
(200 mLꢂminꢀ1) mobile phase and the chemiluminescence from the reaction between NO and O3 was
quantitatively measured by the NO analyzer.
In vitro drug release The release profile of platinumꢀbased drugs from the micelles was investigated using
dialysis method. Briefly, 3 mg/mL of micelles in PBS solution (pH 7.4) were placed in a dialysis
membrane bag (MWCO=3500) and the dialysis bag was immersed in PBS release media. Each dialysis
bag was exposed to UV irradiation (365nm, 300 ꢁW/cm2) or incubated at dark conditions. At
predetermined time intervals, the release medium was sampled and refreshed. The platinum contents in
sampled medium was determined using ICP Mass Instruments. The percentage of drug release was
calculated by assuming drug loading contents of each micelle as 100 %.
Detection of intracellular nitric oxide release human breast carcinoma cells (MCFꢀ7, KCLB30022) were
cultured in DMEM with 10% FBS, 100 U/mL penicillin/streptomycin and incubated at 37 °C in a 5% CO2
incubator. Cells were seeded on 12ꢀwell culture plate at an initial density of 2 x 104 cells/well and
incubated overnight at 37 °C in a 5% CO2 incubator. Each sample was treated at a concentration of 10 ꢁM
prodrug to the cell and incubated for 6 h, followed by treating DAFꢀ2 DA. After 30 min incubation, UV
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light (365nm, 300 ꢁW/cm2) was irradiated for 20 min to the cells. Cells were washed with DPBS and fixed
with 10% neutral buffered formalin solution overnight at 4 °C. The fixed cells were mounted in
Vectashield antifade mounting medium containing DAPI (Vector Laboratories). The images were pictured
by an Olympus FVꢀ1000 instrument and analyzed with Olympus Fluoview version 1.5 viewer software.
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In vitro cell cytotoxicity test Human colon cancer cells (HCT 116, KCLB10247) and human breast
carcinoma cells (MCFꢀ7, KCLB30022) were cultured in DMEM medium containing 10% FBS and 1%
penicillin/streptomycin. Cells were grown and maintained in a humidified atmosphere with 5% CO2 at 37
oC. The cytotoxicity was evaluated by the standard MTT assay. In brief, cells were seeded onto 96ꢀwell
plates at a density of 5×103 cells/well. After 24 h, each sample (cisplatin, nitrobenzene, Pt(IV)ꢀNO, Pt(IV)ꢀ
C, bare Eꢀmicelle, bare Cꢀmicelle, Pt(IV)ꢀNO/Eꢀmicelle, Pt(IV)ꢀNO/Cꢀmicelle, Pt(IV)ꢀC/Eꢀmicelle, and
Pt(IV)ꢀC/Cꢀmicelle) was treated to the cells with doseꢀdependent manner. After 18 h, the cells were
irradiated by UV (365nm, 300 ꢁW/cm2) or incubated at dark for 20 min. After 30 h incubation, each well
was washed with DPBS and 200 ꢁL of fresh medium with 20 ꢁL of 5 mg/mL MTT solution was added to
each well. After 4 h, the medium was removed and resultant formazan crystals were dissolved by DMSO
solution. The absorption was measured at 570 nm using a microplate spectrofluorometer (VICTOR3 V
Multilabel Counter, PerkinꢀElmerꢀWellesley, MA, USA). The nonꢀtreated and nonꢀirradiated cells were
used to represent 100 % cell viability. The results are presented as mean ± standard deviation (n = 3).
In vitro cellular uptake of Platinum MCFꢀ7 cells were seeded onto 6ꢀwell plates at a density of 2×105
cells/well. After 24 h, each sample (Pt(IV)ꢀNO/Eꢀmicelle, Pt(IV)ꢀNO/Cꢀmicelle, Pt(IV)ꢀC/Eꢀmicelle, and
Pt(IV)ꢀC/Cꢀmicelle) was treated to the cells at a concentration of 50 ꢁM Pt. After 18 h, the cells were
irradiated by UV (365nm, 300 ꢁW/cm2) or incubated at dark for 20 min. After 30 min, cells were washed
with 1 mL DPBS and harvested with trypsin solution. After counting cell numbers, harvested cells were
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dispersed in 1 mL DPBS. After 60% nitric acid was added and subsequently evaporated at 150 C for 2
days, 5 mL 1% nitric acid is mixed. The platinum contents in sampled medium was determined using ICP
Mass Instruments
Fluorescenceꢀactivated cell sorting (FACS) analysis MCFꢀ7 cells were seeded in 6ꢀwell culture plates at
an initial density of 2 × 105 cells/well and incubated overnight in the CO2 incubator. Cells were then
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treated for 18 h with micelles at a concentration of 10 ꢁM prodrug in culture medium, and then irradiated
with UV light (365nm, 300 ꢁW/cm2) for 20 min. The cells were further incubated for 6 h, harvested by
trypsinꢀEDTA, and examined by an Annexin VꢀFITC apoptosis detection kit, using provided protocol for
flow cytometry for 10,000 events in each experiment. Annexin V (+)/PI (ꢀ) was counted for apoptosis,
Annexin V (ꢀ)/PI (+) was counted for necrosis, and both (+)/(+) was counted for necrosis/late apoptosis.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS publications website at DOI:
1
UVꢀVis spectra of Pt(IV)ꢀNO, GPC of Eꢀpolymer and Cꢀpolymer, H NMR of Eꢀpolymer under UV
irradiation, CMC graph of Eꢀpolymer and Cꢀpolymer, Eꢀmicelle stability test with DLS, XPS of Pt(IV)ꢀNO
before and after UV irradiation, XPS of Pt(IV)ꢀNO before and after incubation under redox conditions,
cytotoxicity test, and Annexin VꢀFITC/PI stainingꢀassisted flow cytometry analysis of MCFꢀ7 cell treated
with each sample
AUTHOR INFORMATION
*Eꢀmail: wjkim@postech.ac.kr. phone: +82ꢀ54ꢀ279ꢀ2104. fax: +82ꢀ54ꢀ279ꢀ3399.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the Bio & Medical Technology Development Program of the National
Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRFꢀ
2017M3A9F5030930) in Korea.
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ABBREVIATIONS
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Pt, Platinum; MRPs, MDRꢀassociated proteins; HIF, hypoxiaꢀinduced factors; MDR, multidrug resistance;
NFꢀκB, nuclear factor kappa B; DMNB, 4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl; Pt(IV)ꢀdiCOOH, diamminebis(3ꢀ
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carboxypropanoato)diꢀchloroplatinum(IV);
EDC,
Nꢀ(3ꢀdimethylaminopropyl)ꢀN'ꢀethylcabodiimide
hydrochloride; HOBt, 1ꢀhydroxybnzoiazole hydrate; ESIꢀMS, electrospray ionization mass; PEO114ꢀbꢀ
PBLG20, Cꢀpolymer, poly(ethylene oxide)ꢀbꢀpoly(benzylꢀLꢀglutamate); γꢀBLG NCA, γꢀBenzyl Lꢀglutamate
Nꢀcarboxyanhydride; ROP, ringꢀopening polymerization; NCA, Nꢀcarboxyanhydride; PEO114ꢀbꢀPLGA20,
poly(ethylene oxide)ꢀbꢀpoly(Lꢀglutamic acid); PEO114ꢀbꢀPDMNBLG20, Eꢀpolymer, poly(ethylene oxide)ꢀbꢀ
poly(4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl ꢀglutamate); TEM, transmission electron microscopy; UV, ultraviolet;
L
ICPꢀMS, inductively coupled plasmaꢀmass spectrometry; DLS, dynamic light scattering; DAFꢀ2 DA, 4,5ꢀ
Diaminofluorescein diacetate; XPS, Xꢀray photoelectron spectroscopy; CLSM, confocal laser scanning
microscopy; FACS, Fluorescenceꢀactivated cell sorting; cisplatin, cisꢀdiamineplatinum (II) dichloride;
DCC,
N,N'ꢀdicyclohexylcarbodiimide;
DMAP,
4ꢀdimthylaminopyridine;
DIPEA,
N,Nꢀ
diisopropylethylamine; MTT, 3ꢀ(4,5ꢀdimemthylꢀthiaolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium bromide; DMF, N,Nꢀ
dimethylformaimide; THF, tetrahydrofuran; MC, methylene chloride; NaHCO3, sodium bicarbonate;
Na2SO4, sodium sulfate; MWCO, molecular weight cutꢀoff; DMEM, Dulbecco Modified Eagle’s medium;
FBS, fetal bovine serum (FBS); DPBS, Dulbecco’s phosphate buffered saline; 1H NMR and 13C NMR, 1H
and13C nuclear magnetic resonance spectroscopy; CDCl3, deuterium chloroform; ppm, parts per million;
GPC, Gel permeation chromatography
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Delivery of Platinum Anticancer Drugs. Adv. Drug Delivery Rev. 65, 1667ꢀ1685.
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Oberoi, H. S., Nukolova, N. V., Kabanov, A. V. and Bronich, T. K. (2013) Nanocarriers for
Platinum Drugs and Other Macromolecular Metal Complexes for Cancer Treatment. Prog. Polym. Sci. 39,
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