Dual Stimuli-Responsive Supramolecular Self-Assemblies Based on the Host-Guest Interaction between β-Cyclodextrin and Azobenzene for Cellular Drug Release

Article abstract of DOI:10.1021/acs.molpharmaceut.9b01142

Health has always been a hot topic of concern, whereas cancer is one of the largest security risks to human health. Although the existing drug delivery systems (DDSs) have been extensively reported and commercially applied, there are still some issues tha

Full text of DOI:10.1021/acs.molpharmaceut.9b01142

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Article
Dual-stimuli responsive supramolecular self-assemblies
based on the host-guest interaction between #-
cyclodextrin and azobenzene for cellular drug release
JianGuo Zhang, Zi-Hao Zhou, Lin Li, Yan-Ling Luo, Feng Xu, and Yashao Chen
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b01142 • Publication Date (Web): 03 Mar 2020
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Revised Graphical Abstract
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78x71mm (300 x 300 DPI)
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Dual-stimuli responsive supramolecular self-assemblies based
on the host-guest interaction between β-cyclodextrin and
azobenzene for cellular drug release
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§
§
§
JianGuo Zhang , Zi-Hao Zhou , Lin Li , Yan-Ling Luo*, Feng Xu*, Yashao Chen
Key Laboratory of Macromolecular Science of Shaanxi Province, School of Chemistry
and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, P.R. China
§
JianGuo Zhang, Zi-Hao Zhou and Lin Li contributed equally to this work.
Corresponding authors’ details:
Yan-Ling Luo and Feng Xu
Fax: +86 29 81530727.
E-mail addresses: luoyanl@snnu.edu.cn (Y. L. Luo) and fengxu@snnu.edu.cn (F. Xu).
Affiliation: Key Laboratory of Macromolecular Science of Shaanxi Province, School of
Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062,
People's Republic of China.
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Abstract
Health has always been a hot topic of concern, whereas cancer is one of the largest
security risks to human health. Although the existing drug delivery systems (DDSs)
have been extensively reported and commercially applied, there are still some issues
that have yet to be well-resolved, including the toxicity, side-effects and targeted
therapy efficiency of drugs. Consequently, it is still necessary to develop a novel, highly
efficient, controlled and targeted DDS for cancer therapy. For this, a supramolecular
polymer, β-CD-g-PDMAEMA@Azo-PCL, was designed and developed through the
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host-guest inclusion complexation interactions between
a
host polymer,
β-cyclodextrin-graft-poly(2-(dimethylamino)ethyl methacrylate) (β-CD-g-PDMAEMA),
and a guest polymer, azobenzene modified poly(ε-caprolactone) (Azo-PCL), and was
characterized by various analysis techniques. The supramolecular assembly was
examined in various pH environments and/or under UV-vis irradiation, showing the
formation of supramolecular assemblies from regular spherical shapes to irregular
aggregates with various hydrodynamic diameters. The 2D NOESY NMR studies
showed the formation of inclusion complexation between Azo-PCL and
β-CD-g-PDMAEMA and between β-CD and the side groups of PDMAEMA. The
supramolecular assemblies could encapsulate doxorubicin to form spherical core-shell
drug-carrying micelles with an entrapment efficiency of 66.1%. The effects of external
environment stimuli on the in vitro drug release were investigated, showing light- and
pH-modulated drug release properties. The cytotoxicity assessment indicated that the
blank supramolecular micelles were nontoxic, whereas the drug-loaded micelles
exhibited comparable or even superior anticancer activity to the anticancer activity of
free DOX and inhibition of cancer cell proliferation. Therefore, the developed
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supramolecular assemblies can potentially be used as drug controlled release carriers.
Keywords: β-Cyclodextrin, Azobenzene, Host-guest interactions, Stimulus
responsiveness
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1
. Introduction
In recent years, supramolecular polymers have attracted extensive attention due to their
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potential applications in the fields of biotechnology, nanosensing, drug delivery and
gene diagnosis and treatment [1-5]. Supramolecular micelles formed by the
self-assembly of polymers usually take on regular spherical core-shell structures. Their
hydrophobic cores can interact with therapeutic drugs physically or chemically, thus
serving as nanoscale containers for encapsulating hydrophobic drugs, while the
hydrophilic shells can interact with solvents to improve the micelle stability and prolong
the blood circulation time [2-4]. This class of supramolecular polymers is highly
oriented and bound together by non-covalent weak interactions, such as multiple
hydrogen bonds, host-guest interactions, π-π stacking, metal coordination bonds and
electrostatic interactions [1,2,5]. Among them, the host-guest interactions have selective
recognizability and abundant responsivity to stimuli. Host-guest molecules are
combined by hydrophobic interactions due to their complementary characteristics in
size and structure, which provides a broad space for the development and application of
various intelligent materials [1-5].
β-cyclodextrin (β-CD) is a natural macrocyclic oligosaccharide composed of seven
D-galactose units connected by 1,4-glycoside bonds, with a truncated pyramidal cavity
structure of hydrophilic outer margins and hydrophobic inner cavities [6,7]. Its unique
molecular structure enables it to complex with a variety of organic compounds, which
in turn increases the solubility of hydrophobic drugs, enhances the stability and
bioavailability of drugs, and reduces the systemic toxicity of drugs; moreover its low
cost, good biocompatibility and biodegradability make it the most widely used host
molecule to build supramolecular polymers. Composite micelles with recognizability,
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reversibility and controllability can be prepared by means of the host-guest interaction
between β-CD and hydrophobic molecules, which provides more possibilities for the
synthesis of anticancer drug carriers. Kang et al prepared a nano-self-assembly with
both thermal and redox responsiveness [8]. Gao et al synthesized a supramolecular
system by noncovalent coupling, which could further self-assemble into nanorods with
good thermal response and fluorescence properties [9].
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Compared with traditional polymers, supramolecular polymers can be joined together
through reversible and dynamic noncovalent interactions, which makes supramolecular
polymer materials have a strong stimulus response performance [10,11].
Supramolecular polymers can have an autonomous response to intracellular
environmental stimuli, including the pH, temperature, redox potential, enzymes,
glucose, ionic strength, etc., and external environmental stimuli (including light,
electricity, magnetism, ultrasound, etc.), to further improve the therapeutic effect of
hydrophobic drugs on tumor tissues and minimize the damage to healthy cells [12]. A
light stimulus is widely used in clinical treatment due to its characteristics of cleanness,
flexibility, controllability, non-contact and low cost [13]. Polymers with photoresponse
performance can convert light signals to chemical signals through photochemical
reactions when the photosensitive groups in the molecule are stimulated by light, thus
resulting in changes in the structure, polarity, solubility, and aggregation behavior of
the polymers [14]. Azobenzene (Azo) contains a large conjugated system and is a
typical photoresponsive group. Azo can change from the trans-configuration to the
cis-configuration under UV irradiation, whereas it can reversibly change from the
cis-configuration to the trans-configuration under visible light irradiation, which makes
the supramolecular polymers containing azo units reversibly self-assemble/dissociate
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under alternating UV and visible light irradiation [15-17]. By means of hydrophobic
interactions and intermolecular van der Waals forces, trans-azobenzene can smoothly
enter into the cavity of β-CD and undergo complexation. However, cis-azobenzene
does not match the structure of β-CD and cannot form complexes. Therefore,
cis-azobenzene is an "active" unit, and trans-azobenzene is an "inactive" unit for drug
delivery and release systems [18,19]. Furthermore, the isomerization amount of
azobenzene is proportional to the UV emission energy, and hence, the controlled
release of drugs can be realized by adjusting the lighting time [20]. Wang et al
designed a novel in vivo drug delivery system by combining photodynamic therapy
with hypoxic-responsive chemotherapy [21]. Gao et al synthesized a photoresponsive
supramolecular polymer brush by means of the host-guest interaction between β-CD
and azobenzene, which provides a new platform for the synthesis of self-assemblies
with different sizes [22]. Bian et al offered a reusable photo-responsive surface based
on host-guest interactions between azobenzene and β-CD for specific cell-controlled
release via alternating UV and visible light irradiation [23]. Yang et al reported a
photo- and temperature-responsive amphiphilic block copolymer micelle assembly,
poly(N-isopropylacrylamide)-block-poly-(2-nitrobenzyl methacrylate), for the
controlled release of hydrophobic guest molecules [24]. These novel findings afford a
new choice for the isolation and analysis of cancer cells, particularly for the controlled
release of anticancer drugs.
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On the other hand, the pH of tumor microenvironments (6.5~7.0) is slightly lower
than that of normal human tissues (approximately 7.4) due to the rapid growth of
tumor cells and vigorous anaerobic metabolism, which produces a large number of
acid metabolites such as lactic acid [25]. If pH responsive functional groups are
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integrated into the supramolecular polymers, the hydrophilic/hydrophobic balance of
polymer chains can be changed by adjusting the solution pH value and then make the
physicochemical properties of the drug-loaded micelles change; namely, they are stable
in normal physiological conditions and dissociate in acidic environments, thus
achieving the fixed-point release of drugs [26]. Poly(2-(dimethylamino)ethyl
methacrylate (PDMAEMA) is a pH-sensitive polymer that has good antibacterial
properties and molecular weight-dependent cytotoxicity [27,28]. The degree of
protonation of DMAEMA's tertiary amines is different under different pH conditions,
resulting in a change in the hydrophilic/hydrophobic equilibrium of the polymer chain
segments with a change in the pH. It was reported that the dissociation constant (pKa)
of PDMAEMA is approximately 7.1-7.3 [27,29-31]. When the pH value is less than
the pKa, the protonation ability of tertiary amine groups is enhanced, and the solubility
of polymers in aqueous solution is increased, thus showing hydrophilicity. In contrast,
when the pH value is greater than the pKa, the deprotonation of tertiary amines is
enhanced, and the solubility is decreased, thus showing hydrophobicity. Zhou et al
reported a dual pH-sensitive supramolecular star polymer that has a strong inhibitory
effect on the proliferation of tumor cells [32].
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Although the host-guest interaction between β-CD and azobenzene has been
extensively studied, most of the drug carriers are focused on single stimulus-responsive
supramolecular polymers [14,18-21,23]. This class of single stimulus-responsive
supramolecular polymers generally has relatively low release accuracy and exhibits
some side effects; in particular, they cannot sufficiently respond to complex
microenvironment changes in cancer cells in that the change in the behavior of cancer
cells is more often a result of a combinational change in multiple stimuli rather than that
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of a single stimulus [24]. Since dual and/or multiple stimuli responsive supramolecular
polymers can provide a unique opportunity to fine-tune their response to each stimulus
independently, to expand the controlled release modes and comprehensively modulate
drug release profiles via the synergistic response to different stimuli [15], it is highly
necessary to design and develop dual- and/or multi-stimuli responsive supramolecular
drug-loaded micelles. Especially, the synergistic effect between these different stimuli
responsive components is expected to offer more efficient targeted release in cancer
microenvironments in comparison to a single stimulation but maintain better stability in
normal cells and reduce the damage to healthy tissues, thus maximizing the functions of
the supramolecular polymers.
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Against this background, our objective is to construct a supramolecular polymer,
β-CD-graft-PDMAEMA@Azo
modified
poly(ε-caprolactone)
(β-CD-g-PDMAEMA@Azo-PCL), through the host-guest inclusion complexation
interactions between β-CDs and Azo groups; the host polymer β-CD-g-PDMAEMA and
a guest polymer Azo-PCL were synthesized through atom transfer radical polymerization
(ATRP) and ring opening polymerization (ROP), respectively. Herein, hydrophobic PCL
constitutes the core of supramolecular micelles, and hydrophilic PDMAEMA is used as
the shell of supramolecular micelles for sensing pH changes. Azobenzene is a typical
photoresponsive group, which is used to sense the change in light conditions. The drug
loading behavior of supramolecular polymer micelles and the effect of external
environmental stimuli, such as light conditions and pH values, on the drug release rate
are studied using DOX as a hydrophobic drug model. We expect that the developed
supramolecular polymer micelles can accomplish more precise drug release in cancer
microenvironments and maximize the release amount of drugs at lesion or cancer
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locations through the synergistic response to multiple stimuli, thus achieving optimal
bioavailability and targeting efficiency of drugs.
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2
. Experimental section
2.1 Materials and reagents
Beta-cyclodextrin (β-CD, 99%) and 6-chlorohexanol (CH, 98%) were purchased
from the Saan Chemical Technology (Shanghai) Co., Ltd., China, and β-CD was dried
in vacuum at 100 °C for 24 h before use. 2-Bromoisobutyryl bromide (BIBB, 98%),
tris(2-(dimethylamino) ethyl) amine (Me TREN, >98%), cuprous bromide (CuBr, 98%)
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and N,N-(dimethylamino)ethyl methacrylate (DMAEMA, 99%) were obtained from the
Aladdin Industrial Corp., Shanghai, China. CuBr was treated before use by the
following method: CuBr was first stirred in glacial acetic acid until it became white and
then filtered. The solid was washed with ethyl alcohol three times, then washed with
diethyl ether two times and finally dried in vacuum at 80 °C for 2 h.
4-(Phenyldiazenyl)phenol or 4-(phenylazo)phenol (Azo, 98%) and ε-caprolactone (ε-CL,
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9%) were supplied by the Macklin Biochemical Co., Ltd., Shanghai, China, and used
as received. Stannous octoate (Sn(Oct) ), potassium iodide (KI), potassium carbonate
(K CO ), chloroform (CHCl ), dichloromethane (CH Cl ), acetone and anhydrous ether
were provided by the Sinopharm Chemical Reagent Co., Ltd., Shanghai, China, and
used directly. Magnesium sulfate (MgSO ) was supplied by the Tianjin Kemio Chemical
Reagent Co., Ltd., Tianjin, China. N,N-Dimethylformamide (DMF), methanol (CH OH),
ethyl alcohol (C OH), ethyl acetate (EA), petroleum ether (PE), and triethylamine
TEA, 99%) were obtained from the Tianjin Fuyu Fine Chemical Co. Ltd., Tianjin,
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China. DMF was distilled before use to remove moisture. Doxorubicin hydrochloride
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(
DOX·HCl, 98%) were afforded by the Macklin Biochemical Co., Ltd., Shanghai,
China, and used as received.
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.2 Experimental procedure
Preparation of the supramolecular polymer β-CD-g-PDMAEMA@Azo-PCL was
achieved through a three-step process, which includes synthesis of a host polymer
β-CD-g-PDMAEMA, a guest polymer Azo-PCL and a supramolecular polymer
Scheme 1 Preparation strategy sketch map of β-CD-g-PDMAEMA@Azo-PCL
supramolecular polymers and their self-assembled micelles.
β-CD-g-PDMAEMA@Azo-PCL, as shown in Scheme 1.
2
.2.1 Synthesis of bromide modified β-CD initiator (β-CD-Br)
The synthesis of bromide modified β-CD initiator, β-cyclodextrin acyl bromide
β-CD-Br), was attained through an acyl bromide reaction of β-CD and BIBB in a molar
(
1
13
ratio of β-CD to BIBB of 1:4. In detail, 5 g (4.4 mmol) of β-CD ( H NMR and C
NMR spectra are shown in Figure S1 of the Supporting information (SI)) was dissolved
in 34 mL of anhydrous DMF in a 100 mL Schlenk flask with stirring, and then, it was
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cooled to 0 °C in an ice bath. An aliquot of 2.175 mL (17.6 mmol) of BIBB was
dissolved in 6 mL of anhydrous DMF, and then, the solution was added dropwise to the
0
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4
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β-CD solution with a syringe under a N
2
atmosphere. The reaction mixture was stirred at
0
°C for 2 h and then at 25 °C for 24 h. The final reaction mixture was precipitated with
an excess of anhydrous diethyl ether and centrifuged. The crude product was washed
with acetone three times and then dried in a vacuum desiccator overnight at 35 °C,
1
giving a purified white solid product β-CD-Br (Mean yield: 60%). H NMR (400 MHz,
DMSO-d
6
, δ in ppm): 4.79 (s, 7H), 4.16-4.40 (s, 7H), 3.50-3.82 (m, 28H), 3.27–3.32 (m,
13
1
7
4H), 2.04 (s, 12H); C NMR (400 MHz, DMSO-d
6
, δ in ppm): 162.8, 102.37, 81.96,
1
13
3.50, 72.84, 72.47, 60.35, 56.49, and 34.73. The H NMR and C NMR spectra are
shown in Figure S2 (SI).
2
.2.2 Preparation of the host polymer β-CD-g-PDMAEMA
β-CD-graft-poly(2-(dimethylamino)ethyl methacrylate) (β-CD-g-PDMAEMA) was
synthesized by the ATRP of DMAEMA using β-CD-Br as an initiator and
CuBr/Me TREN as catalytic system in molar ratio of
β-CD-Br/DMAEMA/Me -TREN/CuBr =1:120:3:2. In a typical experiment, β-CD-Br
0.2149 g, 0.15 mmol), 6 mL of anhydrous DMF and DMAEMA (3.033 mL, 18 mmol)
6
a
a
6
(
were successively added into a 50 mL dried Schlenk flask. The mixed solution was
degassed by a freezing–pumping–thawing process to remove oxygen and moisture,
followed by suffusing with a N
fleetly into the reaction flask under the N
freeze–pump–thaw operation was conducted. Then, CuBr (43 mg, 0.3 mmol) was
swiftly added into the flask under N flow by repeating the above operation. The
reaction flask was sealed, and the polymerization reaction was carried out at 90 °C for
2
atmosphere. Me
6
TREN (120 μl, 0.45 mmol) was added
2
atmosphere, and then, an immediate
2
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2 h under magnetic stirring. After the reaction was completed, the final mixture was
diluted with THF and passed through a short neutral alumina column to remove the
copper catalyst. The solvent was then removed by rotary evaporation, and the residual
solution was transferred to a dialysis bag with a MWCO of 2000 to dialyze against 500
mL deionized water for 48 h to remove residual monomers. The dialysate was collected
and lyophilized to give 0.74 g (Mean yield: 24.3%) of white solid β-CD-g-PDMAEMA,
and the monomer conversion was estimated to be approximately 18.6% based on a
1
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0
1
gravimetric method and 27.5% by H NMR. Thus, the theoretical and experimental
-1
1
molecular weights were approximately 4940 and 6620 g mol , respectively. H NMR
400 MHz, CDCl , δ in ppm): 4.04 (t, J=3.6 Hz, 66H), 2.55 (t, J=6.4 Hz, 66H), 2.27 (s,
98H), 1.80-1.89 (m, 66H), 0.89-1.13 (m, 99H), 5.00 (s, 7H), 3.57 (m, 28H), 3.43 (m,
(
3
1
1
5
1
3
4H), and 2.11 (s, 12H); C NMR (400 MHz, CDCl
3
, δ in ppm): 177.42, 176.73, 63.14,
-1
7.13, 54.15, 45.92, 44.73, 29.80, 18.64, and 16.74 [33]; Mn,LLS-GC=12900 g mol and
1
13
PDI=1.39. The H NMR and C NMR spectra are shown in Figure S3 (SI).
2.2.3 Preparation of 6-(4-(phenyldiazenyl)phenoxy)hexan-1-ol (Azo-OH)
Azo-OH was prepared by the Williamson etherification reaction of
4
-(phenyldiazenyl)phenol and 6-chlorohexanol. 4-(Phenyldiazenyl)phenol (4.9565 g,
25.0 mmol), 6-chlorohexanol (5 mL, 37.5 mmol), K CO (5.1815 g, 37.5 mmol), and KI
0.0415 g, 0.25 mmol) were solubilized in 100 mL of dried DMF in a 250 mL
2
3
(
round-bottom flask to obtain a mixed solution. The mixed solution was refluxed at 120
°C for 12 h to afford a red-brown solution. The resulting solution was washed with a
large amount of water to dissolve K
extracted three times using chloroform to remove K
with deionized water thrice to remove DMF. The product was dried with anhydrous
2
CO
3
, and then, the resultant solution was repeatedly
2
CO . The solution was re-extracted
3
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MgSO
4
and filtered; the filtrate was evaporated by rotatory evaporation to remove most
of the solvent. The concentrate was purified by column chromatography using silica gel
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as a stationary phase and petroleum ether and ethyl acetate (v/v=6:1) as an eluent. An
orange flake crystal product was obtained by rotary evaporation and then dried in
1
vacuum at 35 °C for 24 h (Mean yield: 77%). H NMR (400 MHz, CDCl
3
, δ in ppm):
7
.95–7.81 (m, 4H), 7.55–7.38 (m, 3H), 7.04–6.94 (m, 2H), 4.04 (t, J=6.5 Hz, 2H), 3.66
(t, J=6.5 Hz, 2H), 1.83 (dq, J=8.1, 6.5 Hz, 2H), 1.63–1.63 (m, 2H), 1.55–1.42 (m, 4H),
13
1.39 (s, 1H); C NMR (400 MHz, CDCl
3
, δ in ppm): 161.69, 152.78, 146.86, 130.39,
1
1
29.08, 124.81, 122.58, 114.72, 68.22, 62.76, 32.65, 29.19, 25.89, and 25.58. The H
1
3
NMR spectrum with integrals and C NMR spectrum are incorporated in Figure S4
SI), showing that Azo-OH was sufficiently purified.
(
2
.2.4 Preparation of the guest polymer Azo-PCL
Azobenzene initiated polycaprolactone (Azo-PCL) as a guest polymer was prepared by
ROP of ε-CL using Azo-OH as an initiator. Representatively, Azo-OH (0.200 g, 0.67
mmol), ε-CL (2.975 mL, 26.85 mmol), and a catalytic amount of Sn(Oct) (0.033 mL,
.10 mmol) were quickly added into a 50 mL dried Schlenk flask. The system was
2
0
degassed by a freeze–pump–thaw operation repeated three times to eliminate oxygen
and ultimately suffused with nitrogen gas. The mixture was heated to 120 °C to start the
polymerization with persistent stirring. After 24 h, the reactor was exposed to air to
terminate the reaction, offering a brownish red viscous liquid. The resulting solution was
diluted with CH
2
Cl , precipitated into an excess of cold methanol twice, and centrifuged.
2
The resultant product was dried in a vacuum desiccator at 35 °C overnight, giving a
1
yellow solid, which was named Azo-PCL (Mean yield: 85%). H NMR (400 MHz,
CDCl , δ in ppm): 7.86 (m, 4H), 7.45 (m, 3H), 6.97 (m, 2H), 4.06 (t, J=6.7 Hz, 130H),
3
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.63 (t, J=5.6 Hz, 4H), 2.30 (t, J=7.5 Hz, 128H), 1.80 (m, 2H), 1.61 (m, 256H), 1.38 (m,
1
3
32H); C NMR (400 MHz, CDCl
3
, δ in ppm): 173.50, 161.56, 152.71, 146.62, 130.29,
1
1
1
1
1
1
1
1
1
1
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28.98, 124.70, 122.49, 114.63, 68.06, 64.10, 63.62, 34.08, 32.29, 28.54, 28.30, 25.69,
-1
-1
5.49, 25.29, and 24.54; Mn,NMR=7700 g mol , Mn,LLS-GC=8000 g mol and PDI=1.20.
1
13
The H NMR and C NMR spectra are incorporated in Figure S5 (SI).
2
.2.5 Preparation of the supramolecular micelles
β-CD-g-PDMAEMA@Azo-PCL supramolecular assemblies were prepared by a
host-guest inclusion complexation interaction between β-CD and azobenzene at molar
ratios of β-CD-g-PDMAEMA:Azo-PCL of 0.5:1, 1:1 and 1.5:1 through a dialysis
technique, as illustrated in Scheme 1. Briefly, 15 mg of β-CD-g-PDMAEMA and 28.9
-3
mg (3.75×10 mmol) of Azo-PCL were separately dissolved in DMF to form a 5 mg
-1
mL solution and stirred for 30 min. Then, the Azo-PCL solution was added dropwise
to the β-CD-g-PDMAEMA solution and stirred for 30 min. Deionized water (2 mL) was
−1
added dropwise into the mixed solution at a rate of 5 μl s under vigorous stirring until
the solution became turbid to induce the formation of supramolecular micellar
assemblies.
After
the
stirring
was
performed
for
4
h,
the
β-CD-g-PDMAEMA@Azo-PCL solution was transferred into a dialysis bag (MWCO:
2000) for dialysis at room temperature for 48 h to completely remove DMF. Fresh
deionized water was replaced hourly for the first 3 h and then once every 6 h. A fraction
of the dialysate was collected and lyophilized for 24 h, affording cream-colored or beige
supramolecular polymer β-CD-g-PDMAEMA@Azo-PCL; another part of the dialysate
1
was collected in a 50 mL volumetric flask for further measurements. H NMR (400
MHz, CDCl
3
, δ in ppm): 6.94-7.82 (m, 9H), 3.99 (t, J=5.6 Hz, 196H), 3.60 (t, J=6.7 Hz,
4
H), 2.51 (t, J=6.7 Hz, 66H), 2.23 (m, 326H), 1.76 (m, 66H), 1.59 (m, 256H), 1.32 (m,
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1
6
1
2
34H), and 0.82-0.98 (m, 99H); C NMR (400 MHz, CDCl , δ ppm): 177.43, 173.73,
3
4.27, 63.14, 57.13, 54.11, 45.91, 44.74, 34.21, 29.81, 28.43, 25.61, 24.67, 18.60, and
6.64. The details are described in Figure 1.
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.3 Measurements and characterization
1H nuclear magnetic resonance (NMR) and two-dimensional nuclear Overhauser effect
1
spectroscopy of NMR (2D NOESY H NMR) analyses were performed on a 400 MHz
JNM-ECZ400S/L1 spectrometer (JEOL Corp., Japan) using CDCl
3
or DMSO-d as the
6
solvent and tetramethylsilane (TMS) as the internal standard substance. Fourier
transform infrared spectra (FT-IR) were recorded on a Tensor II Fourier transformation
infrared spectrophotometer (Bruker Corp., Germany), and samples were pressed into
KBr pellets. A laser light scattering gel chromatography system (LLS-GC, VISCOTEK
TM, Marvin Corp., UK) was used to determine the number-averaged molecular weight
(M
n
) and polydispersity index (PDI=M
w
/M ). The calibration was conducted by using
n
polystyrene (PS) standards, and the refractive index increment (dn/dc) values of the PS,
-1
host and guest samples were determined to be 0.185, 0.181 and 0.087 mL g ,
respectively, at 35 °C using chromatography grade THF containing 2.5% TEA as the
mobile phase. Before measurement, the dried polymers were dissolved in THF/TEA to
-
1
form a concentration of 2 mg mL . The polymer solutions were filtered through a 0.22
μm needle-type Teflon filter (organic, Φ13 mm). The measurement was performed at a
-1
flow rate of 1.0 mL min at a column temperature of 35 °C.
The fluorescence spectra were recorded on a fluorescence spectrophotometer
(
FluoroMax-4, HORIBA Scientific, Japan) using pyrene as a probe to measure the
critical micelle concentration (CMC). In detail, 5.0565 mg of pyrene was accurately
weighed, dissolved in acetone, and brought to volume in a 50 mL brown volumetric
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flask, offering a pyrene solution concentration of 5×10 mol L . The pyrene solution (6
μl) was placed into a 10 mL sample tube, and the acetone was completely evaporated in
1
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an oven. Then, 5 mL of micelle solution with a concentration (C) of 1×10 ~5×10 mol
-1
L was added to the sample tube containing pyrene and magnetically stirred for 12 h for
fluorescence spectrum scanning. The excitation wavelength was set as 330 nm, the
wavelength range was from 350 to 550 nm, and the slit width was 1 nm. The CMC
values were obtained by calculating the fluorescence intensity ratio (I
3
/I
1
) at 384 nm (I )
3
and 373 nm (I ) from the emission spectra, plotting the I /I -logC curves, and
1
3 1
determining the concentration corresponding to the intersection of the two tangents.
The ultraviolet–visible (UV–vis) absorption spectra were recorded on a UV-6100S
UV–vis spectrophotometer (Maipuda Instrument Co., Ltd., Shanghai, China) to measure
the light responsiveness and loading ability for the drug of the prepared supramolecular
polymer assemblies and to monitor the absorbance and drug release behavior of
DOX-loaded supramolecular assemblies. A UV-light irradiator (365 nm, 6 W, ZF-5
Lamp) and a visible-light irradiator (450 nm, 3 W, POLICE) were used to induce the
photoisomerization of Azo-PCL and the supramolecular assembly containing Azo
moieties, with a radiation distance of 5 cm. The morphologies and sizes of the
supramolecular assemblies before and after DOX was loaded were observed on an
ultrahigh-resolution JEM-2100 TEM (JEOL Corp., Japan) at an accelerating voltage of
2
00 kV. The samples were prepared by placing two drops of the micelle solutions on
the copper grid with carbon films and dried at room temperature. A laser particle
analyzer and dynamic light scattering device (DLS, BI-90Plus, Brookhaven, USA)
equipped with a 15 mV argon ion laser operating at a scattering angle of λ=660 nm were
used to measure the hydrodynamic diameter (D ) and particle size distribution (PDI) of
h
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the assemblies at a sample concentration of 0.5 mg mL . The measurements were
carried out at a deflection angle of 90° and output power of 15 mW at room
temperature.
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.4 Loading and in vitro controlled release of the drug
Twenty milligrams (0.0345 mmol) of DOX·HCl was dissolved in 6 mL of DMF,
followed by 15 μl (0.1079 mmol) of TEA to remove HCl, and the solution was stirred in
the dark environment at room temperature for 4 h. Sixty milligrams of
β-CD-g-PDMAEMA@Azo-PCL supramolecular assemblies was then dissolved in
DMF/H O (7.2 mL, v/v=1:2) with vigorous stirring for 2 h to ensure sufficient inclusion
2
complexation. The DOX solution was added dropwise to the above inclusion complex
solution without light under stirring for 6 h. Then, 3 mL of deionized water was added
-1
into the mixture at a rate of 5 μl s under rapid stirring until the solution became turbid,
indicating the formation of the micellar solution, and it was kept stirring away from
light overnight. After that, the solution was dialyzed (MWCO: 2000) against 800 mL of
deionized water for 24 h to remove free DOX and DMF. Fresh deionized water was
replaced approximately every
4
h, giving the resultant DOX-loaded
β-CD-g-PDMAEMA@Azo-PCL
micelle nanoparticles, denominated as
DOX-β-CD-g-PDMAEMA@Azo-PCL. The DOX-loaded nanoparticles were collected
and freeze-dried for further measurements, affording a brownish red solid. To quantify
the loading capacity (LC) and entrapment efficiency (EE), 4 mg of the lyophilized
DOX-loaded nanoparticles were dissolved in 200 mL of DMF. A UV-visible
spectrophotometer (UV-6100S, Maipuda, Shanghai, China) was used to monitor the
absorbance of the solution at 490 nm, and the LC and EE were calculated according to
Formulas (1) and (2), respectively:
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LC(%)=(Mass of DOX in nanoparticles/Mass of DOX-loaded nanoparticles)100% (1)
EE(%)=(Mass of DOX-loaded in nanoparticles/Mass of DOX in feed)100%
(2)
1
1
1
1
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Prior to measurements, calibration was performed with DOX standard solution in
DMF at 490 nm. The calibration equation with a high squared correlation coefficient of
2
R =0.9995 was obtained as follows:
-1
C (mg mL )=(A+0.0150)/18.7583
(3)
where C is the concentration of DOX in the solution and A is the absorbance at 490 nm.
The LC and EE values were determined to be approximately 13.2 and 66.1 %,
respectively.
To demonstrate the pH and light responsive release behavior, 4 mg of lyophilized
DOX-loaded supramolecular polymer was dispersed in 4 mL of phosphate buffered
-1
saline (PBS) with a pH of 7.4, 6.0 or 5.0 to offer a concentration of 1 mg mL , and then,
it was dialyzed (MWCO: 3500) in 200 mL of the above PBS at 37 °C with or without
UV-irradiation (365 nm, 8 W, ZF-7A lamp). At a given time interval, aliquots of 4 mL
were withdrawn from the beaker to measure the amount of DOX released in the
dialysate using a UV-vis spectrophotometer at a λex of 490 nm and replaced with an
equal volume of fresh PBS. The cumulative DOX release was estimated based on the
2
calibration curves of DOX in PBS (R =0.9999) according to the following equation:
-4
M
t
=(A+8.6647×10 )/6.8117
(4)
(5)
Cumulative DOX release (%)=M
t
/M
0
×100%
where M represents the amount of DOX released at time t and M
t
0
is the amount of
DOX loaded in the β-CD-g-PDMAEMA@Azo-PCL micelle nanoparticles.
2
.5 MTT assay
The cytotoxicities of free DOX, the blank and DOX-loaded supramolecular micelles
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toward
HeLa
cells
were
evaluated
by
the
3
-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay. HeLa
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cells were seeded in a 96-well plate at a density of 1×10 per well and cultured in a
DMEM medium supplemented with 10 % (v/v) fetal bovine serum (FBS), 0.1 %
penicillin-streptomycin and 5 % CO at 37 °C for 24 h. After that, the HeLa cells were
2
cultured in 200 μl of fresh DMEM medium containing the above samples and further
incubated for another 24 h. The cells were eluted with PBS thrice to remove the
metabolites, and the growth of cells ceased. Afterwards, the medium was substituted
-1
with 200 μl of fresh DMEM containing 20 μl of 5 mg mL sterile filtered MTT and
incubated for additional 4 h. Then, the supernatant was discarded, the purple formazan
crystals generated by live cells were dissolved in 150 μl of DMSO, and shaking was
performed for 10 min. At the same time, HeLa cells were planted in DMEM and
reproduced under the same conditions as a control group. The absorbance of the
solution was recorded by a universal microplate reader (Bio-Rad Laboratories (UK)
Ltd) at 570 nm, and the cell viability was calculated as follows:
Cell viability (%) = (ODsample/ODcontrol)100%
(6)
where ODcontrol and ODsample are the optical densities of the sample and control group,
respectively. The student’s t-test was used to determine the significance of any pairs of
observed differences. Differences were considered to be statistically significant at
p<0.05. The results were averaged from the data of five measurements and expressed as
the mean value±SD.
3
. Results and discussion
3
.1 Structural characterization
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Figures 1(A) and 1(B) depict the 1H and 13C NMR spectra of
1
13
β-CD-g-PDMAEMA@Azo-PCL, respectively, and the H NMR and C NMR spectra
of their precursors, host polymer β-CD-g-PDMAEMA and guest polymer Azo-PCL are
shown in Figures S1-S5 (SI). Figures S1 and S2 (SI) manifest the successful preparation
of β-CD-Br, and two –OH groups in the whole β-CD structure are proven to participate
in the reaction by calculating the integration area ratio (A) of the peak at 4.79 ppm to
the peak at 2.04 ppm (A is approximately 1:1.67, and n=7×1.67/6≈2). The host polymer
β-CD-g-PDMAEMA is synthesized via ATRP, as shown in Figure S3 (SI), and the
degree of polymerization for PDMAEMA is estimated to be approximately 33 by
1
1
1
1
1
1
1
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1
1
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comparing the integration area ratio of the -(CH
3
2
) peak in BIBB at 2.11 ppm to the
-
CH - peak in PDMAEMA at 4.04 ppm. Hence, the experimental number-averaged
2
-1
molecular weight (Mn,NMR) of β-CD-g-PDMAEMA is approximately 6620 g mol . The
syntheses of the guest polymer Azo-PCL and its precursor are magnified by Figures S4
and S5 (SI). The degree of polymerization for PCL is estimated to be approximately 65
by comparing the integration area ratio of the peak at 6.95-7.01 ppm to the peak at 2.29
ppm, offering the experimental number-averaged molecular weight (Mn,NMR) of
Azo-PCL of approximately 7,700 g mol . In H and 13C NMR spectra of the
supramolecular polymer β-CD-g-PDMAEMA@Azo-PCL in Figures 1(A) and 1(B), it
can clearly be observed that all of the characteristic proton and carbon shift signals
attributed to the host polymer β-CD-g-PDMAEMA and the guest polymer Azo-PCL can
-1
1
1
be observed, of which the peaks in the H NMR spectrum at 3.99 and 2.23 ppm are
overlapped; they are ascribed to the methylene protons attached to the -CH
structure and the methyl and methylene protons in the -N(CH /-CH
structure in the host and guest polymers, indicating
2
-CH
-CH
that
2
-OCO-
3
)
2
2
2
-CO-
the
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β-CD-g-PDMAEMA@Azo-PCL supramolecular polymer is successfully assembled by
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13
1
Figure 1 (A) H NMR, (B) C NMR and (C) 2D NOESY H NMR spectra of
β-CD-g-PDMAEMA@Azo-PCL supramolecular micelles/assemblies with
host]/[guest]=1:1 in CDCl , and (D) FT-IR spectra of (a) β-CD-g-PDMAEMA, (b)
[
3
Azo-PCL and (c) β-CD-g-PDMAEMA@Azo-PCL.
the aid of the host–guest inclusion complexation between β-CD and Azo groups [33].
The host–guest inclusion complexation between β-CD and Azo groups is proven by 2D
1
NOESY H NMR, as shown in Figure 1(C). Clearly, the proton signals of β-CD at
3
.30-3.55 ppm are associated with those of the azo protons at 6.94-7.82 ppm, giving
some cross-peaks (red circles in Figure 1(C)); this suggests that β-CD can interact with
azobenzene moieties to form a host-guest inclusion complexation system, and the
azobenzene-containing PCL moieties are entrapped in the cavity of β-CD
[8,14,22,34,35]. On the other hand, the possibility of the intermolecular and/or
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intramolecular host–guest interactions between β-CD and PDMAEMA moieties cannot
be excluded because the hydrophobic effect of PDMAEMA may exist when the
pH>pKa due to the weakened protonation, thus leading to a fast decrease of the
hydrophilicity. It can be seen from the green circle in Figure 1(C) that the proton signals
of β-CD at 3.30-3.55 ppm are correlated with those of dimethylamino(ethyl) groups of
PDMAEMA moieties at approximately 2.25 ppm, implying that β-CD can form
supramolecular assemblies by complexing with side dimethylamino(ethyl) groups of
PDMAEMA [36,37]. Therefore, it is confirmed that β-CD can interact not only with the
azobenzene moieties but also with dimethylamino(ethyl) groups of PDMAEMA to form
the host-guest supramolecular assemblies by inclusion complexation.
1
1
1
1
1
1
1
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1
1
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Figure 1(D) illustrates the FT-IR spectra of β-CD-g-PDMAEMA, Azo-PCL and
β-CD-g-PDMAEMA@Azo-PCL. The host polymer β-CD-g-PDMAEMA produces the
-1
-1
enhanced vibration peaks at 2930 and 2780 cm as well as 1730 cm ascribed to the
C-H stretching and the C=O stretching, respectively, compared with its precursors β-CD
and β-CD-Br shown in Figure S6(A) (SI). A new absorption peak emerges at 1238-1270
-1
cm , which is attributed to the C-N stretching in PDMAEMA chains. In the case of
-1
Azo-PCL, the –OH peak at 3305 cm attributed to the –OH stretching of Azo-OH
(Figure S6(B)) in associated and free states disappear. Meanwhile new absorption peaks
-1
emerge at 1728 and 1182 cm separately attributed to the C=O and C-O-C stretching
bands, manifesting the synthesis of the guest polymer through ROP. By comparing the
FTIR spectra of β-CD-g-PDMAEMA and Azo-PCL with that of
β-CD-g-PDMAEMA@Azo-PCL, it can be observed that the vibration peaks at 1728,
-1
1
243 and 1286 cm reflecting the C=O, C-N and C-O stretching still exist, whereas the
OH stretching and the aromatic skeleton vibration bands are not observed, presumably
–
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Figure 2 (A) LLS-GC traces and (B) the weight distribution curves of (a)
β-CD-g-PDMAEMA and (b) Azo-PCL.
due to the inclusion of Azo-PCL in β-CD-g-PDMAEMA through the host-guest
interactions. These findings indicate the preparation of β-CD-g-PDMAEMA@Azo-PCL.
Figures 2(A) and 2(B) display the LLS-GC traces and the corresponding weight
distribution curves of the host polymer β-CD-g-PDMAEMA and the guest polymer
Azo-PCL. Their apparent Mn values are approximately 12,900 g mol-1 for
-1
1
β-CD-g-PDMAEMA and 8000 g mol for Azo-PCL. The H NMR Mn value, in
particular, with respect to that of the host polymer, is lower than that by LLS-GC; this
discrepancy is probably ascribed to the large hydrodynamic volumes of the host and
guest polymer. However, it is higher than the estimated theoretical value based on a
-1
gravimetric method (4940 g mol ) due to the mass loss during preparation and
post-treatment. Both of the traces present almost a symmetric unimodal distribution
with almost no trailing. The LLS-GC weight distribution curves, however, demonstrate
that the guest polymer Azo-PCL is nearly centrally distributed, giving a relatively low
polydispersity index (PDI) of 1.20, whereas the host polymer β-CD-g-PDMAEMA
generates a bump in the section of low molecular weight, leading to a slightly wider
molecular weight distribution with a PDI of 1.39. In addition, the difference in the
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number of –OH moieties involved in the reaction in each β-CD is responsible for the
slightly higher PDI value, although there are, on average, two –OH groups in the entire
β-CD structure participating in the reaction. Anyway, the experimental results indicate
that the host and guest polymers are effectively purified and the polymerization
processes are well controlled.
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3
.2 Self-assemblies and their physiochemical properties
The formation of the supramolecular assembly and the critical micelle concentration
(CMC) were determined by a fluorescence spectrophotometer using pyrene as a
fluorescent probe. The fluorescence intensity ratios (I /I ) at 384 nm (I ) and 373 nm (I )
3
1
3
1
in the fluorescence emission spectra vs. the logarithm of the concentration (logC) was
adopted to estimate the CMC value of the supramolecular self-assemblies, which is
defined as the concentration corresponding to the intersection of the two tangents in
I
3
1
/I -logC curves [38], as tabulated in Table 1. It is clear that these supramolecular
-1
micelles possess CMC values below 111.9 mg L , showing that they have good
thermodynamic stability. As the molar ratio of the host polymer β-CD-g-PDMAEMA to
the guest polymer Azo-PCL increases from 0.5:1 to 1.5:1, the CMC value of the
Table 1 CMC and DLS data of the supramolecular assemblies with various compositions and pH.
-1
a
pHb
a
[
Host]/[guest] CMC (mg L ) D
h
(nm) PDI
D
h
(nm) PDI
0
1
.5:1
:1
111.9
68.4
52.6
213.6±20 0.121±0.008 3.4
272.0±42 0.211±0.020 7.4
310.4±44 0.405±0.030
222.3±30 0.319±0.040
290.5±25 0.197±0.017
24
1
.5:1
286.0±60 0.249±0.053 8.0
a
-1
b
Concentration: 0.5 mg mL , and [host]/[[guest]=1:1
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-
1
supramolecular self-assembly decreases from 111.9 to 52.6 mg L . This finding is not
consistent with the general theory that the greater the number of hydrophilic chain
segments, the greater the CMC value of polymer micelles [38]. The reason for this
phenomenon may be ascribed to the existence of hydrogen bonding interactions
between the host polymer molecules, β-CD-g-PDMAEMA. With increasing the content
of the host polymer, the hydrogen bond interactions are strengthened, whereas the
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(
hydrogen bond) interactions between the supramolecular polymers and water
molecules relatively decrease, leading to the decreased CMC values.
DLS is used to determine the hydrodynamic diameters (D ) of the supramolecular
h
polymer micelles and their distribution (PDI), and Table 1 summaries the DLS data of
the prepared supramolecular self-assemblies with [host]/[guest] molar ratios of 0.5:1,
1
:1 and 1.5:1. It can be found by comparing the three curves that the D values of the
h
supramolecular self-assemblies increase from 213.6±20 nm to 286.0±60 nm as the
proportion of the host polymer β-CD-g-PDMAEMA and the guest polymer Azo-PCL
rises from 0.5:1 to 1.5:1; and they have a relatively low PDI value less than 0.25 but an
increasing trend. The reason that for the increased D value with increasing content of
h
the host polymer may mainly be related with the chain conformation of the PDMAEMA
host polymers. Generally, during the formation of micelles, the Azo-PCL guest
polymers, as hydrophobic fragments, construct the micellar cores, whilst the
PDMAEMA host polymer chains, as hydrophilic segments, form the shells of the
micelles, presenting an extended chain conformation. Consequently, the higher the
proportion of the host polymer, the greater the content of the extended PDMAEMA
chain segments, and thus, the larger the particle size of the supramolecular micelles.
Figure 3 displays TEM micro-images of the supramolecular self-assemblies having a
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Figure 3 TEM microimages of the supramolecular self-assembly with
-1
[
host]/[guest]=1:1 (0.5 mg mL ): (a) the original micelle and (b) the irradiated
counterpart with 365 nm ultraviolet light. The inset in Figure 3(a) shows the TEM
image of the DOX-loaded supramolecular micelle.
-1
[
host]/[guest] molar ratio of 1:1 at a concentration of 0.5 mg mL . As shown in Figure
3
(a), the supramolecular polymers spontaneously form a spherical core-shell micellar
structure in aqueous solution through the host-guest inclusion complexation interactions,
with the darker part in the center constituting the PCL core of the micelles and the
lighter part in the periphery forming the PDMAEMA shell of the micelles. The
self-assemblies have a uniform size distribution and an average diameter of ca. 167 nm.
However, the particle size measured by DLS is generally much larger than that
measured by TEM because the former offers hydrodynamic diameters in aqueous
solution whereas the latter gives the particle size in a dried state. The extended or
swelled micelles make the particle size of micelles increase significantly. In contrast, the
drying process may be accompanied by the shrinkage of micelle particles, resulting in a
smaller particle size.
3.3 Light and pH dual stimuli responsiveness
The supramolecular assembly containing azobenzene and tertiary amine groups is
anticipated to exhibit photosensitive and pH sensitive responses. The light-responsive
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feature can be proven by TEM images of the supramolecular assemblies
([host]/[guest]=1:1) after irradiation with 365 nm ultraviolet light, as illustrated in
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Figure 3(b). Clearly, one can observe that after UV irradiation, the regular spherical
supramolecular micelles in Figure 3(a) change into irregular hierarchical assemblies and
even some aggregates with various sizes, which is consistent with the results reported
elsewhere [14,35,39,40]. The reason for this phenomenon is that the azobenzene units in
the guest polymers are in a trans-configuration before UV light irradiation, which can
enter the β-CD cavity through the host-guest interactions, self-assemble and form
supramolecular micelles presenting spherical core-shell structures. Under ultraviolet
light, azobenzene units undergo isomerization transformation from the trans- to
cis-configuration, which does not match the cavity structure of β-CD. As a result, the
Azo-PCL moieties depart from the cavity of β-CD, and the supramolecular assemblies
dissociate into the host and guest parts. Considering the hydrophobicity of the
cis-azobenzene-containing PCL chains and the hydrophilicity of PDMAEMA fragments,
the wrecked host-guest system reconstructs new hydrophilic-hydrophobic balances,
forming irregular hierarchical assemblies or aggregates with the hydrophobic Azo-PCL
wrapped by the hydrophilic β-CD-g-PDMAEMA.
A UV-visible spectrophotometer is further used to investigate the light and pH
responses of supramolecular polymer (micelle) solutions. Figure 4(a) shows the UV-vis
spectra of a guest polymer, Azo-PCL, dissolved in DMF under UV light (365 nm) and
visible light (450 nm) irradiation at different times. The characteristic absorption peaks
*
*
at 350 and 440 nm are attributed to the π-π leap of trans-azobenzene and the n-π leap
of cis-azobenzene, respectively [41]. Clearly, with the extension of the ultraviolet
illumination time, the absorbance of Azo-PCL at 350 nm significantly decreases, while
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Figure 4 UV-vis spectra of (a and b) Azo-PCL (0.5 mg mL ) and (c and d)
-1
supramolecular self-assemblies (0.1 mg mL , [host]/[guest]=1:1) under (a and c) 365
nm UV and (b and d) 450 nm visible irradiation.
the value at 440 nm slightly increases, indicating that azobenzene changes from the
trans- to cis-configuration under ultraviolet irradiation. In contrast, with the extension of
visible light time, the absorbance of Azo-PCL at 350 nm significantly increases, while
the value at 440 nm slightly decreases, suggesting that azobenzene reversibly changes
from the cis- to trans-configuration under visible light irradiation. Therefore, the
supramolecular self-assemblies containing azobenzene units can reversibly
self-assemble/dissociate by virtue of the trans→cis and cis→trans isomerization
transition under alternating ultraviolet and visible light. On this basis, the absorption
peak change at 350 nm was adopted to examine the trans→cis and cis→trans
isomerization transition of the supramolecular assemblies with the [host]/[guest] ratio of
1
:1, as depicted in Figures 4(c) and 4(d). With the extension of the ultraviolet irradiation
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