PH-Responsive robust polymer micelles with metal-ligand coordinated core cross-links

Article abstract of DOI:10.1039/c4cc01584c

We report on pH-responsive core-shell polymer micelles with catechol-Fe³⁺ coordinated core cross-links, which provide robustness to drug-loaded polymer micelles and allow the facilitated intracellular release of loaded anticancer drugs in respo

Full text of DOI:10.1039/c4cc01584c

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
pH-Responsive robust polymer micelles with
metal–ligand coordinated core cross-links†
Cite this: Chem. Commun., 2014,
50, 4351
Gyu Ha Hwang,a Kyung Hyun Min,a Hong Jae Lee,b Hye Young Nam,b
Gi Hyun Choi,b Byung Joo Kim,a Seo Young Jeonga and Sang Cheon Lee*b
Received 3rd March 2014,
Accepted 6th March 2014
DOI: 10.1039/c4cc01584c
www.rsc.org/chemcomm
We report on pH-responsive core–shell polymer micelles with micelles assembled from linear–linear, linear–dendritic, and
catechol-Fe3+ coordinated core cross-links, which provide robustness linear–hyperbranched block copolymers.11,13 Therefore, the
to drug-loaded polymer micelles and allow the facilitated intracellular polymer micelles bearing pH-labile shell or core-cross-links
release of loaded anticancer drugs in response to an endosomal have been an interesting research target for intracellular delivery
acidic pH.
systems with improved delivery efficacy.8,11,14
Recently, coordination between ferric ions (Fe3+) and catechol
Stimuli-responsive polymer micelles have served a central role ligands has attracted growing interest, because it was found to play
as smart nanocarriers that can trigger the release of anticancer an important role in the hardness and extensibility of the cuticle of
drugs within target cells by sensing intracellular environmental marine mussel byssal threads.15,16 More interestingly, it is noted
signals.1–3 Although they have displayed many advantages, that the catechol ligand and Fe3+ underwent pH-dependent
such as high drug loading capacity and effective accumulation complexation: the mono-catechol–Fe3+ complex dominates at
in tumor tissues by the enhanced permeation and retention pH o 5.6, bis-catechol–Fe3+ complex at 5.6 o pH o 9.1, and
(EPR) effects, the successful outcome in tumor treatments can tris-catechol–Fe3+ complex at pH 4 9.1.17 As expected, at extra-
only be expected in cases where they meet the following cellular pH (7.4), Fe3+ binds with two catechol ligands to form
requirements.4–6 First, they should have high structural stability the bis-complex, whereas, at endosomal pH (B5.0), it forms
during blood circulation. Second, they need to effectively hold mono-complex with one catechol ligand. Therefore, it is a
loaded drugs within their cores before reaching target tumor challenging research target to use this pH-dependent catechol–
tissues. Third, they should be designed to trigger the facilitated Fe3+ complexation in devising a novel polymer micelle nanocarrier
release of loaded drugs in the interior of the target cells.
Currently, diverse cross-linking approaches of micellar
with pH-responsive shell or core-cross-links.
Herein, we describe a strategy to introduce bis-catechol–Fe3+
shells or cores are successfully employed to make the polymer cross-links into a drug-loaded core of polymer micelles and
micelle structure robust by strongly holding the assembled demonstrate that such core-cross-linked polymer micelles
nanostructures.7–9 In addition, various cross-links that are indeed maintain their structural stability and preferentially
stable in blood but become labile in response to intracellular release an anticancer drug in response to endosomal pH. The
environments are utilized as key elements for enhancing delivery bis-catechol–Fe3+ complexes are known to have some of the
efficacy of loaded anticancer drugs.10,11 Of many intracellular highest level of log stability constants (log Ks D 38 to 41) of
signals, the acidic pH in endosomes and lysosomes has been a various metal–ligand chelates.18 In a previous report, it was
target for many intracellular nanocarrier systems.11,12 To date, demonstrated that the binding energy of the coordination bond
the polymer micelles bearing acid degradable linkages, such as between metals and DOPA is only a little lower than that of a
hydrazone, acetal, and ketal, have displayed high delivery covalent bond.19 Therefore, the bis-catechol–Fe3+ core cross-links
efficacy, and the representative examples are the polymer would provide the robustness to the drug-loaded polymer micelles
at blood pH by strongly holding the assembled structures. In
a Department of Life and Nanopharmaceutical Science, College of Pharmacy,
addition, this bis-complex cross-link maintains a stable chelate
structure during blood circulation and act as a diffusion barrier
for release of loaded drug molecules. In contrast, at endosomal
pH, the core cross-links would be disrupted due to the transition
of catechol–Fe3+ coordination from the bis-complex to the mono-
complex, thereby triggering the facilitated drug release.
Kyung Hee University, Seoul 130-701, Korea
b Department of Maxillofacial Biomedical Engineering & Institute of Oral Biology,
School of Dentistry, Kyung Hee University, Seoul 130-701, Korea.
E-mail: schlee@khu.ac.kr; Fax: +82 2 960 1457; Tel: +82 2 961 2317
† Electronic supplementary information (ESI) available: Experimental details and
data. See DOI: 10.1039/c4cc01584c
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 4351--4353 | 4351

View Article Online
Communication
ChemComm
In recent years, we have focused on the stimuli-responsive
DTX, an anticancer drug, was loaded into the core of
shell-cross-linked poly(ethylene glycol) (PEG)-b-poly(amino polymer micelles using the dialysis method. The mean hydro-
acid)s polymer micelles and demonstrated the improved anti- dynamic diameter of DTX-loaded non-cross-linked polymer
cancer therapeutic activities.6,7,20,21 In this work, PEG as a micelles (DTX-NPMs), as estimated by dynamic light scattering,
hydrophilic block and poly(L-3,4-dihydroxyphenylalanine) (PDOPA) was 43.5 nm. The loading efficiency and contents of DTX were
as a hydrophobic block were selected as a main component. 48.0% and 4.4 wt%, respectively. Cross-linked PDOPA cores
To prepare a core–shell polymer micelle, a self-assembling were obtained by controlling the pH of an aqueous mixture of
PEG-b-PDOPA copolymer (PEG-PDOPA) was synthesized by FeCl3 and DTX-NPMs to 7.4. For effective coordination, the feed
ring-opening polymerization of di-O,O0-acetyl-L-DOPA-N-carboxy- molar ratio of Fe3+ to catechol ligands was fixed at 1 : 2.
anhydride ((AC2)-DOPA-NCA) in the presence of a CH3O-PEG- Dynamic light scattering and transmission electron microscopy
NH2 macroinitiator (Mn: 5000) and the subsequent deprotection (TEM) measurements showed that the DTX-CLPMs were spherical
process (Fig. S1 in the ESI†). Successful synthesis of PEG-PDOPA and maintained a hydrodynamic diameter of the precursor
1
was confirmed by NMR spectroscopy. In H NMR spectra, the polymer micelles (DTX-NPMs) with a moderate size distribution
resonance peak from acetyl protons (–OCOCH3) of P((AC2)- (Fig. S5 and Table S2 in the ESI†).
ꢀ
DOPA) blocks at 2.26 ppm completely disappeared after a
The pH-dependent complexation behavior between catechol
piperidine-treated deprotection process (Fig. S2 in the ESI†). ligands and Fe3+ was monitored using UV-Visible spectroscopy.
In 13C NMR spectra, the peaks from the carbonyl and methyl Fig. 1a shows that the mono-complex of catechol-Fe3+ observed
carbons of the acetyl groups (–OCOCH3) of P((AC2)-DOPA) blocks at 434 nm dominates at pH 5.0. As pH increased from 5.0 to 7.4
ꢀ
ꢀ
at 168.1 ppm and 20.2 ppm also completely disappeared after and 12.0, the coordination between Fe3+ and catechol ligands
the deprotection process (Fig. S3 in the ESI†). 1H NMR and GPC changed from mono-complex to bis-, and tris-complexes. At pH
analyses showed that the molar composition ratio of EG to DOPA 7.4, the bis-complex assigned to absorption at 520 nm was
in the PEG-PDOPA was 113 : 6, and that PEG-PDOPA had a formed, and, at pH 12.0, the absorption of the tris-complex was
narrow polydispersity (Fig. S4 and Table S1 in the ESI†).
identified at 452 nm.17 Moreover, dramatic color changes of
Scheme 1 illustrates the key idea and working principle of the complex are confirmed by photo images (Fig. 1b) At pH 5.0,
our docetaxel (DTX)-loaded core-cross-linked polymer micelles the color of DTX-CLPMs solution is green. As pH increases, the
(DTX-CLPMs).
color changes to light brown at pH 7.4, and to reddish brown at
PEG-PDOPA would assemble to form the polymer micelles pH 12.0. As pH varies from the physiological pH (7.4) to the
consisting of the drug-loaded hydrophobic PDOPA core and the endosomal pH (5.0), the dominating species would change from
solvated PEG outer shell. The Fe3+ at pH 7.4 induces the formation bis-complex to mono-complex. This indicates that DTX-CLPMs
of the bis-catechol–Fe3+ core cross-links. The cross-links in the can effectively hold DTX within cross-linked cores in blood and
DTX-loaded PDOPA cores may not only enhance micellar stability effectively release DTX in endosomes, where the bis-complex
against micelle-destabilizing conditions but also protect drug cross-links are disrupted to form mono-complexes.
release efficiently in extracellular environments (pH 7.4). Upon
Next, we examined whether the bis-catechol–Fe3+ cross-links
endocytosis, coordination of catechol–Fe3+ changes from the in DTX-CLPMs can enhance micellar stability against serum
bis-complex to the mono-complex in response to endosomal proteins, which are known as major species that alter micellar
pH (pH 5.0) to trigger the facilitated intracellular drug release. assembled structures.6,7 The stability of the DTX-CLPMs and
DTX-NPMs in the fetal bovine serum (FBS)-containing solution
was evaluated using dynamic light scattering. Time-dependent
change of the ratio of scattered light intensities (SLI/SLI0) was
monitored in the presence of FBS (Fig. S6 in the ESI†). The DTX-
CLPMs maintained the initial scattered intensity over 90% for
120 min. In contrast, the DTX-NPMs decreased to 91% after
only 10 min, and 86% after 120 min. This result suggests that
Scheme 1 Illustration of bis-catechol–Fe3+ cross-linking in the core of
DTX-loaded PEG-PDOPA micelles at pH 7.4 and triggered release of DTX
by the conversion to the mono-catechol–Fe3+ complex under an endosomal
acidic pH condition (pH 5.0).
Fig. 1 (a) UV-Visible absorption spectra and (b) photo images of aqueous
mixtures of DTX-loaded PEG-PDOPA micelles and Fe3+ at various pH.
4352 | Chem. Commun., 2014, 50, 4351--4353
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
exhibited comparable cytotoxicity of DTX-CLPMs. This result
suggests that DTX release was rather facilitated from the DTX-
CLPMs after endocytosis by the fast transition from the bis-
complex to the mono-complex of the catechol–Fe3+ coordination.
The DTX from DTX-CrEL formulation would release upon placing in
the cell culture media. This is the main reason why DTX-CLPMs have
a relatively low toxicity compared to DTX-CrEL. CrEL may induce
severe toxicity over the DTX concentration of 1 mg mLꢀ1, formulated
by 210 mg mLꢀ1 CrEL. This result showed that DTX-CLPMs could
Fig. 2 (a) DTX release profiles from DTX-NPMs and DTX-CLPMs at the
PBS solution (pH 7.4) and (b) pH-controlled DTX release profiles from evade the side effects of DTX-CrEL formulations. Collectively, the
DTX-CLPMs. Each point represents the mean value of n experiments ꢁ
robust structure and the potential for reducing drug loss in blood
may enhance overall therapeutic efficacy in vivo for DTX-CLPMs
relative to free DTX and DTX-NPMs.
This work was supported by the National Research Founda-
tion of Korea (NRF) grant funded by the Korean government
(MSIP) (No. 2013R1A2A2A01009239 and 2012R1A5A2051388).
S.D. (n = 3).
catechol–Fe3+ core cross-linking could improve the structural
stability of polymer micelles under serum conditions.
The effect of core cross-linking on the DTX release was
evaluated under extracellular pH conditions (PBS, pH 7.4).
Fig. 2a shows that the DTX release from DTX-CLPMs was
greatly inhibited compared to that from DTX-NPMs. At 24 h,
approximately 44.7% of the DTX release was inhibited. This
indicates that the DTX diffusion from the core to the aqueous
media was efficiently protected by the core cross-links formed
at pH 7.4. As shown in Fig. 2b, DTX-CLPMs show contrasting
release profiles at endosomal and physiological pH. The release
rate of DTX at pH 5.0 is higher than at pH 7.4. This result shows
that after reaching the endosomes, core cross-links that were
changed from the bis-complex to the mono-complex could
facilitate the DTX release.
For visualization of cellular entry of DTX-CLPMs, fluorescein
isothiocyanate (FITC)-labeled DTX-CLPMs was monitored in
MCF-7 cells (Fig. S7 in the ESI†). As the incubation time
increased, the green FITC fluorescence intensity gradually
increased and became constant after 1 h. This obviously
indicates the endocytosis of DTX-CLPMs. Viability of breast
cancer MCF-7 cells at DTX-free NPMs, CLPMs, and commercial
Cremophor EL (CrEL) surfactants was estimated. As shown in
Fig. 3a, DTX-free NPMs and CLPMs exhibited no noticeable
cytotoxicities up to 500 mg mLꢀ1, whereas CrEL exhibited severe
cytotoxicity over the concentration of 250 mg mLꢀ1. Fig. 3b
showed the in vitro cytotoxicity of DTX-CrEL, DTX-NPMs and
DTX-CLPMs for MCF-7 cells after 24 h. DTX-CLPMs effectively
inhibited the proliferation of MCF-7 cells, and DTX-NPMs
Notes and references
1 K. Y. Choi, H. Y. Yoon, J.-H. Kim, S. M. Bae, R.-W. Park, Y. M. Kang,
I.-S. Kim, I. C. Kwon, K. Choi and S. Y. Jeong, ACS Nano, 2011, 5,
8591–8599.
2 E. Burakowska, S. C. Zimmerman and R. Haag, Small, 2009, 5,
2199–2204.
3 P. Hu and N. Tirelli, Bioconjugate Chem., 2012, 23, 438–449.
4 N. Fomina, J. Sankaranarayanan and A. Almutairi, Adv. Drug Delivery
Rev., 2012, 64, 1005–1020.
5 K. Y. Choi, K. H. Min, H. Y. Yoon, K. Kim, J. H. Park, I. C. Kwon,
K. Choi and S. Y. Jeong, Biomaterials, 2011, 32, 1880–1889.
6 K. H. Min, H. J. Lee, K. Kim, I. C. Kwon, S. Y. Jeong and S. C. Lee,
Biomaterials, 2012, 33, 5788–5797.
7 A. N. Koo, K. H. Min, H. J. Lee, S. U. Lee, K. Kim, I. C. Kwon, S. H. Cho,
S. Y. Jeong and S. C. Lee, Biomaterials, 2012, 33, 1489–1499.
8 L. Wu, Y. Zou, C. Deng, R. Cheng, F. Meng and Z. Zhong, Biomaterials,
2013, 34, 5262–5272.
9 R. Shrestha, M. Elsabahy, S. Florez-Malaver, S. Samarajeewa and
K. L. Wooley, Biomaterials, 2012, 33, 8557–8568.
10 H. He, S. Chen, J. Zhou, Y. Dou, L. Song, L. Che, X. Zhou, X. Chen,
Y. Jia, J. Zhang, S. Li and X. Li, Biomaterials, 2013, 34, 5344–5358.
11 S. J. Lee, K. H. Min, H. J. Lee, A. N. Koo, H. P. Rim, B. J. Jeon,
S. Y. Jeong, J. S. Heo and S. C. Lee, Biomacromolecules, 2011, 12,
1224–1233.
12 H. P. Rim, K. H. Min, H. J. Lee, S. Y. Jeong and S. C. Lee, Angew.
Chem., Int. Ed., 2011, 50, 8853–8857.
´
13 (a) E. R. Gillies and J. M. J. Frechet, Bioconjugate Chem., 2005, 16,
361–368; (b) M. Prabaharan, J. J. Grailer, S. Pilla, D. A. Steeber and
S. Gong, Biomaterials, 2009, 30, 5757–5766; (c) S. Lee, K. Saito, H.-R.
Lee, M. J. Lee, Y. Shibasaki, Y. Oishi and B.-S. Kim, Biomacromolecules,
2012, 13, 1190–1196.
14 Y. Li, W. Xiao, K. Xiao, L. Berti, J. Luo, H. P. Tseng, G. Fung and
K. S. Lam, Angew. Chem., Int. Ed., 2012, 51, 2864–2869.
15 H. Xu, J. Nishida, W. Ma, H. Wu, M. Kobayashi, H. Otsuka and
A. Takahara, ACS Macro Lett., 2012, 1, 457–460.
16 H. Zeng, D. S. Hwang, J. N. Israelachvili and J. H. Waite, Proc. Natl.
Acad. Sci. U. S. A., 2010, 107, 12850–12853.
17 N. Holten-Andersen, M. J. Harrington, H. Birkedal, B. P. Lee,
P. B. Messersmith, K. Y. C. Lee and J. H. Waite, Proc. Natl. Acad.
Sci. U. S. A., 2011, 108, 2651–2655.
18 S. W. Taylor, G. W. Luther III and J. H. Waite, Inorg. Chem., 1994, 33,
5819–5824.
19 H. Lee, N. F. Scherer and P. B. Messersmith, Proc. Natl. Acad. Sci.
U. S. A., 2006, 103, 12999–13003.
20 H. J. Lee, S. E. Kim, I. K. Kwon, C. Park, C. Kim, J. Yang and S. C. Lee,
Chem. Commun., 2010, 46, 377–379.
21 A. N. Koo, H. J. Lee, S. E. Kim, J. H. Chang, C. Park, C. Kim, J. H. Park
and S. C. Lee, Chem. Commun., 2008, 6570–6572.
Fig. 3 (a) Viability of MCF-7 cells at various concentrations of NPMs,
CLPMs, CrEL after 24 h. (b) In vitro cytotoxicity of DTX-CrEL, DTX-NPMs,
DTX-CLPMs for MCF-7 cells after 24 h (n = 3).
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 4351--4353 | 4353