Shell-sheddable, pH-sensitive supramolecular nanoparticles based on ortho ester-modified cyclodextrin and adamantyl PEG

Article abstract of DOI:10.1021/bm500711c

We report a new type of pH-sensitive supramolecular aggregates which possess a programmable character of sequential dePEGylation and degradation. As a platform of designing and building multifunctional supramolecular nanoparticles, a family of 6-OH ortho ester-modified β-cyclodextrin (β-CD) derivatives have been synthesized via the facile reaction between β-CD and cyclic ketene acetals with different alkyl lengths. These asymmetric acid-labile β-CD derivatives formed amphiphilic supramolecules with adamantane-modified PEG through host-guest interaction in polar solvents such as ethanol. The supramolecules can self-assemble in water to form acid-labile supramolecular aggregates. The results of TEM and light scattering measurements demonstrate that the size and morphology of the aggregates are influenced by the alkyl or PEG length and the host-guest feed ratio. By carefully balancing the alkyl and PEG lengths and adjusting the host-guest ratio, well-dispersed vesicles (50-100 nm) or sphere-like nanoparticles (200-500 nm) were obtained. Zeta potential measurements reveal that these supramolecular aggregates are capable of being surface-functionalized via dynamic host-guest interaction. The supramolecular aggregates were stable at pH 8.4 for at least 12 h as proven by the ¹H NMR and LLS measurements. However, rapid dePEGylation occurred at pH 7.4 due to the hydrolysis of the ortho ester linkages locating at the interface, which resulted in aggregation of the dePEGylated hydrophobic inner cores. Upon further decreasing the pH to 6.4, the hydrophobic cores were further degraded due to the acid-accelerated hydrolysis of the ortho esters. The incubation stability of the acid-labile supramolecular aggregates in neutral buffer could be improved by incorporating hydrophobic poly(ε-caprolactone) into the core of the aggregates.

Full text of DOI:10.1021/bm500711c

Article
pubs.acs.org/Biomac
Shell-Sheddable, pH-Sensitive Supramolecular Nanoparticles Based
on Ortho Ester-Modified Cyclodextrin and Adamantyl PEG
Ran Ji, Jing Cheng, Ting Yang, Cheng−Cheng Song, Lei Li, Fu-Sheng Du,* and Zi-Chen Li*
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education,
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
*
S Supporting Information
ABSTRACT: We report a new type of pH-sensitive supra-
molecular aggregates which possess a programmable character of
sequential dePEGylation and degradation. As a platform of
designing and building multifunctional supramolecular nano-
particles, a family of 6-OH ortho ester-modified β-cyclodextrin
(
β-CD) derivatives have been synthesized via the facile reaction
between β-CD and cyclic ketene acetals with different alkyl
lengths. These asymmetric acid-labile β-CD derivatives formed
amphiphilic supramolecules with adamantane-modified PEG
through host−guest interaction in polar solvents such as ethanol.
The supramolecules can self-assemble in water to form acid-
labile supramolecular aggregates. The results of TEM and light
scattering measurements demonstrate that the size and
morphology of the aggregates are influenced by the alkyl or PEG length and the host−guest feed ratio. By carefully balancing
the alkyl and PEG lengths and adjusting the host−guest ratio, well-dispersed vesicles (50−100 nm) or sphere-like nanoparticles
(
200−500 nm) were obtained. Zeta potential measurements reveal that these supramolecular aggregates are capable of being
surface-functionalized via dynamic host−guest interaction. The supramolecular aggregates were stable at pH 8.4 for at least 12 h
as proven by the H NMR and LLS measurements. However, rapid dePEGylation occurred at pH 7.4 due to the hydrolysis of the
1
ortho ester linkages locating at the interface, which resulted in aggregation of the dePEGylated hydrophobic inner cores. Upon
further decreasing the pH to 6.4, the hydrophobic cores were further degraded due to the acid-accelerated hydrolysis of the ortho
esters. The incubation stability of the acid-labile supramolecular aggregates in neutral buffer could be improved by incorporating
hydrophobic poly(ε-caprolactone) into the core of the aggregates.
INTRODUCTION
molecular systems whose pH-sensitivity is due to the acid-
■
32−34
cleavable linkages.
Polyethylene glycol (PEG) has been widely used to modify
Supramolecules have attracted great attention because of their
expansibility in molecular architecture and reversible nature as
noncovalent materials.
cyclic oligosaccharides exhibiting excellent biocompatibility and
versatile host−guest interaction with a wide range of guest
molecules, play important roles in both scientific research and
application areas as key building components to construct
35
36
37
proteins, liposomes, polyplexes, and other polymeric
1
−6
Cyclodextrins (CDs), a family of
38
nanoparticles, in general, termed as PEGylation, to avoid
them being eliminated by the reticulo-endothelial system and
39,40
prolong their circulation time in blood.
However, over
PEGylation may cause the risk of systemic overexposure of the
drug-loaded nanoparticles and hinder their cellular uptake after
7
−9
various supramolecules and the relevant materials.
The CD-
41,42
localizing at the pathological site.
To solve this “PEG
based supramolecular nanoparticles or hydrogels have been
thoroughly exploited to develop various biomedical materi-
dilemma”, scientists have proposed a smart shedding or
dePEGylation” strategy in which the protective PEG or
“
1
0−16
als.
In addition, CD-containing supramolecules or supra-
other shielding polymers can be fully or partially removed from
the nanoparticle surface upon microenvironmental change at
1
7
18−21
molecular self-assemblies responding to pH, light,
2
2
23
24
25−27
43−45
46−48
voltage, enzyme, lectins, or multistimuli
have been
the target sites.
Various stimuli including pH,
49−51
24,52,53
54,55
developed, showing potential for intelligent delivery systems.
Among them, the pH-sensitive supramolecular systems are
promising as the vehicles of nanomedicines due to the
numerous pH gradients in human body. While most of the
pH-sensitive CD-containing supramolecular systems are based
on the switchable host−guest interaction following a pH
reduction potential,
enzyme,
light,
and so
forth, have been applied to trigger the shedding processes,
resulting in the changes of morphology or surface property of
Received: May 18, 2014
Revised: July 24, 2014
Published: August 21, 2014
1
7,28−31
change,
only limited publications report the supra-
©
2014 American Chemical Society
3531
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Scheme 1. Functionalizable and PEG-Sheddable Supramolecular Nanoparticles
the nanoparticles, or enhancing release rate of the active agents.
Furthermore, dual- or multi-stimuli-responsive polymers have
attracted a growing interest to construct versatile programmed
Chemical Ltd.), 2000, and 5000 (Aldrich) were used as received.
Phosphate buffers (PB) with different pHs (5.5, 6.4, 7.4, and 8.4) were
prepared from H O, KH PO , and Na HPO , and calibrated by a pH
meter (Mettler Toledo FE20, Shanghai, China).
Synthesis of Adamantane-End Modified PEGs (Ada-PEGs).
2
2
4
2
4
5
6,57
nanomedicines,
including the dual pH-sensitive polymeric
58
prodrug with a charge-switching character. However, these
programmed systems need generally complicated syntheses or
fabrication procedures. Supramolecular chemistry could sim-
plify the molecular design and provide a useful way for the
development of programmed nanomedicines.
Adamantane-end modified PEGs (Ada-PEG 750 as G1, Ada-PEG
2
000 as G2, and Ada-PEG 5000 as G3) were synthesized by reaction
of the mPEGs with 1-adamantanecarbonyl chloride. The degree of
end-functionalization was more than 95% as estimated by the H
1
NMR spectra (Figure S1).
Ortho ester represents one of the most acid-labile motifs and
is widely used as the acid-cleavable linkage in various pH-
Synthesis of Cyclic Ketene Acetals (CKAs). 2-Ethylidene-1,3-
dioxane (EDO), 2-ethylidene-4-methyl-1,3-dioxolane (EMD), 2-ethyl-
idene-4-hexyl-1,3-dioxolane (EHD), and 2-ethylidene-4-decyl-1,3-
dioxolane (EDD) were synthesized following the published
5
9,60
sensitive delivery systems.
Recently, we have developed a
facile approach to prepare acid-labile polymers with pendent
cyclic ortho esters by modifying polyols with highly reactive
cyclic ketene acetals (CKA). Primary hydroxyl group is much
6
1,62
procedure.
Briefly, diol (0.5 mol), acrolein (0.5 mol), and PTSA
(20 mg) were dissolved in toluene (∼500 mL) in a flask with a Dean−
Stark trap. The solution was refluxed at 120 °C until ∼10 mL of water
was collected in the trap. Then, the solution was concentrated by
rotary evaporation at reduced pressure, followed by addition of
CH Cl (200 mL). After washing with 30 mL of 1% Na CO thrice
61
more active than the secondary one in the reaction. Herein,
we have prepared a family of asymmetrically ortho ester-
modified β-CD derivatives by the selective reaction of 6-OH of
β-CD and CKAs with different alkyl lengths under mild
conditions. These acid-labile β-CD derivatives can interact with
adamantane-capped PEG to form amphiphilic supramolecules,
and some of which can self-assemble into stably dispersed
nanoparticles at a mildly basic pH. The empty CD cavities on
the surface can be used for further functionalization. Of
importance, this type of supramolecular nanoparticles shows a
pH-dependent profile of sequential dePEGylation and degra-
dation in the range of physiological pHs, showing potential as
carriers of programmed nanomedicines (Scheme 1).
2
2
2
3
and 30 mL of saturated NaCl aqueous solution, the organic phase was
separated, dried over anhydrous K CO overnight, and distilled under
2
3
reduced pressure, affording vinyl acetal as a colorless liquid.
(
Ph P) RuCl (50 mg) and vinyl acetal (0.3 mol) were then charged
3
3
2
into a 50 mL flask. Under argon atmosphere, the reaction mixture was
heated to 125 °C and refluxed for 12 h with magnetic stirring until the
isomerization finished. The mixture was distilled under reduced
pressure to afford the cyclic ketene acetals. The yields of various CKAs
were in the range of 30−60%. All the CKAs and their precursors were
1
characterized by H NMR on a Bruker ARX 400 MHz spectrometer
(Figures S2−S4).
Synthesis of 6-OH Modified Cyclodextrins. 6-O-(2-ethyl-4-
methyl-1,3-dioxolan)-2-yl-β-cyclodextrin (EMD-CD) was synthesized
through the facile reaction between 2-ethylidene-4-methyl-1,3-
dioxolane (EMD) and the primary hydroxyl group (6-OH) of β-
cyclodextrin (β-CD). Briefly, β-CD (4.0 g, 3.5 mmol) and PTSA (20
mg) were placed in a round flask and the trace water was removed by
azeotropic distillation with 30 mL of anhydrous toluene twice. A total
of 20 mL of newly distilled DMSO was added into the flask to
completely dissolve the residue. EMD (2.8 g, 24.5 mmol) was
predissolved in anhydrous THF for 0.5 h and dropped into the flask.
The mixture was stirred at room temperature for additional 1 h
followed by adding 0.1 mL of TEA to quench the reaction. The
reaction mixture was poured into 150 mL of diethyl ether (with 1%
TEA) to afford a white semisolid which was then dissolved in 10 mL of
dichloromethane and precipitated in 100 mL of diethyl ether (with 1%
TEA) again. The precipitate was isolated by centrifugation and dried in
EXPERIMENTAL SECTION
■
Materials. Triethylamine (TEA), p-toluenesulfonic acid (PTSA),
D O, DCl, 1,2-propanediol, 1,2-octanediol (Aldrich), 1,2-decylanediol
2
(
(
Aldrich), tris(triphenylphosphine) ruthenium(II) dichloride
(Ph P) RuCl , Alfa Aesar), 1-adamantanecarbonyl chloride (Ada-
3
3
2
CC, Aldrich), and N-(1-adamantyl)ethylenediamine (Ada-EA, TCI)
were used as received. 1-Adamantanecarboxylic acid (Ada-CA) was
prepared by hydrolysis of Ada-CC in 2% NaOH aqueous solution.
DMSO-d and CDCl (J&K Chemical Ltd.) were dried with CaH and
6
3
2
anhydrous Na CO , respectively, prior to use. DMSO and the other
2
3
solvents used in cyclodextrin modification were distilled under sodium
or CaH prior to use. β-Cyclodextrin was recrystallized from deionized
2
water at 80 °C and dried in vacuum at 40 °C. Methoxypolyethylene
glycols (mPEG) with average molecular weights of 750 (J&K
3
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1
Figure 1. H NMR spectra of β-CD and its EMD-modified derivatives in DMSO-d . Molar feed ratios of EMD β-CD are 5:1 (DS 2.1), 7:1 (DS 3.9),
6
and 10:1 (DS 6.4), respectively. Degree of substitution (DS), defined as the number of hydroxyl groups reacted with EMD per CD molecule, is
determined by comparing the intensity of peak b and peak 1. The asterisk denotes the proton signals of TEA.
vacuo, affording a white powder in 80% yield. The other CKA-
modified β-CDs at 6-OH, 6-O-(2-ethyl-1,3-dioxan)-2-yl-β-CD (EDO-
CD), 6-O-(2-ethyl-4-hexyl-1,3-dioxolan)-2-yl-β-CD (EHD-CD), and
HC
1 ⎡
1 + 13 R q + 2A C
⎤
2
g
2
=
⎢
⎣
⎥
⎦
2
R (θ)
M
w
vv
2
2
2
dn
dc
4
6-O-(2-ethyl-4-decyl-1,3-dioxolan)-2-yl-β-CD (EDD-CD) were syn-
4π n
(
)
⎛θ ⎞
4πn
λ
thesized in a similar way (yields ranging from 40 to 65%). All the
H
=
, q
=
sin⎜ ⎟
⎝
2 ⎠
1
N λ
A
CKA-modified CDs were characterized by H NMR spectra (Figures
1, S6, and S7).
where the Rayleigh ratio R (θ) was measured as the angular
dependence of the excess absolute time-averaged scattered intensity
in SLS, and N , n, dn/dc, λ, and θ was the Avogadro’s number, the
solvent refractive index, the specific refractive index increment, the
vv
Self-Assembly of Host−Guest Complex. A thin film hydration
method was applied to get dispersions of the acid-labile supra-
molecular self-assemblies. Take the host−guest system of EDD-CD
and G2 as an example. EDD-CD (9.9 mg, 4.7 μmol) and G2 (10 mg,
A
wavelength of light in vacuo, and the scattering angle, respectively. The
4.7 μmol) were dissolved in ethanol in a flask and the solution was
hydrodynamic radius R was calculated by using the Stokes−Einstein
h
equilibrated for 0.5 h at 40 °C. Then, the solvent was removed by
rotary evaporation at reduced pressure to get a transparent film at the
bottom of the flask. Phosphate buffer (pH = 8.4, 10 mM, 10 mL) was
added into the flask which was ultrasonicated for 0.5 h to afford a
transparent dispersion with a final aggregation concentration of 2.0
mg/mL. The other H-G supramolecular aggregates were prepared by
the same protocol unless otherwise mentioned. After preparation, the
dispersions were quickly frozen in liquid nitrogen and stored at −20
2
equation D = k T/6πηR , where D was obtained by extrapolating Γ/q
B
h
to zero angle. Laplace inversion program, CONTIN, was used to
obtain Γ at different angles by normalization of the line width
distribution G(Γ) obtained from DLS measurements.
Zeta Potential Measurements. Zeta potentials of the aggregates
were analyzed on a Zeta PALS analyzer (Brookhaven Instruments
Corp.) equipped with a temperature controller and a 35 mW He−Ne
solid state laser (λ = 660 nm, detection angle: 90°). The data were
measured in triplicate. The H3G2 supramolecular aggregates (0.2 mg/
mL) were prepared following the same procedure as for the LLS
measurements. To 1.0 mL of the H3G2 supramolecular dispersion, 10
μL of Ada-EA or Ada-CC (10 mg/mL in 10 mM PB) was added. The
mixture was vortexed for 30 s prior to the measurement. The molar
ratio of Ada-EA or Ada-CC to the host was 10:1.
°C, affording stock dispersions with an aggregate concentration of 2.0
mg/mL. For H3G3-1.0, the aggregate concentration was 3.5 mg/mL.
The stock dispersions were unfrozen by ultrasonication at 20 °C for
∼
5 min prior to light scattering and TEM measurements. The
freezing−unfreezing process does not influence the size and
morphology of the supramolecular aggregates.
Transmission Electron Microscopy (TEM) and Freeze-
Fracture TEM. The supramolecular aggregates used for the
preparation of TEM specimens were prepared following the same
procedure as for the LLS measurements, with an aggregate
concentration of 0.2 mg/mL. The TEM specimen was prepared by
dropping 10 μL of dispersion on one piece of copper mesh. After 60 s,
most of the liquid was removed by blotting with a filter paper, and the
remnant dispersion on the mesh was dried at room temperature for an
additional 15 min. Then, 5 μL of phosphotungstic acid aqueous
solution (1%, pH = 7.4) was dropped on the copper mesh with
aggregates to enhance contrast. After 90 s, most of the staining liquor
was removed by blotting with a filter paper, the mesh was dried at
room temperature for 12 h before TEM observation on an equipment
(JEOL JEM-100CXII) with an acceleration voltage of 100 kV.
Laser Light Scattering (LLS) Measurements. The unfrozen
stock dispersion was diluted to 0.2 mg/mL of the aggregate
concentration by adding PB (pH = 8.4, 10 mM). After incubation
for 2 h at room temperature, the diluted dispersion was filtered into a
dust-free vial through a Millipore 0.45 μm PVDF membrane prior to
LLS measurement. Both static light scattering (SLS) and dynamic light
scattering (DLS) measurements over a scattering angular range of 20−
1
50° were carried out on a commercialized equipment (BI-200SM
Goniometer, Holtsville, NY). A 100 mW solid state laser (GXC-III,
CNI, Changchun, China) operating at 532 nm was used as the light
source. A BI-TurboCo Digital correlator (Brookhaven Inc.) was used
to collect and process the data. The root mean square radius of
gyration (R ) was obtained by the following equations:
g
3
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1
For FF-TEM, the aggregates with a higher concentration (1.0 mg/
mL) were prepared in water (with ∼1% TEA) following the
aforementioned procedure. The aggregate dispersion was dropped
on a piece of copper mesh, covered quickly with another copper mesh,
and frozen quickly. A freeze-fracture apparatus (Balzers BAF400,
Germany) was used to fracture and replicate the sample at −140 °C.
After Pt/C was deposited on the sample fracture surface, the organic
part was dissolved by acetone. The Pt/C replica was reloaded on a
fresh copper mesh and observed by the aforementioned TEM
equipment.
(EMD-CDs) were characterized by H NMR spectra (Figure
). With increasing the molar feed ratio of EMD to hydroxyl
groups of β-CD from 5:1 to 10:1, the proton signals of 6-OH at
4.5 ppm gradually disappeared, while the signals of 2-OH and
-OH at ∼5.7 ppm remained almost constant. Meanwhile, the
1
∼
3
proton signals assigned to the ortho ester groups (peaks a−e)
became gradually stronger. By comparing the intensities of peak
b and peak 1, which is assigned to the C1 proton of the CD
ring, degree of substitution (DS) of the hydroxyl groups (by
ortho ester groups) can be determined and the results are
summarized in Table 1. It is seen that the DS can be tuned in
pH-Responsive DePEGylation and Degradation of the
Aggregates. Hydrolysis of the ortho ester linkage in the supra-
1
molecular aggregates was estimated directly by H NMR measure-
ment. The dispersion of H3G2-0.8 supramolecular aggregates in 1.0
mM PB (pH = 8.4, 2.0 mg/mL) was prepared by the film hydration
protocol. After incubation at 37 °C for 6 h, 2.5 mL of the dispersion
was taken out and quickly frozen in liquid nitrogen, and used as the
sample for 0 min. The remained dispersion was divided into three flask
Table 1. Reaction Condition and Results of CKAs and β-
CDs
a
b
run
CKA
temperature (°C)
feed ratio (CKA/OH)
DS
1
2
3
4
5
6
7
8
9
EMD
EMD
EMD
EMD
EMD
EDO
EDO
EHD
EDD
20
20
20
20
40
20
40
20
20
5:1
2.1
3.9
6.4
6.1
(
50 mL/flask) and stirred magnetically. The pH of the dispersion was
7:1
adjusted by adding ∼5 mL of concentrated PB (0.1 M, pH = 6.4, 7.4,
and 8.4, respectively). At specific time point, 2.5 mL of the dispersion
was taken out, quenched quickly by adding one drop of TEA, and
10:1
21:1
21:1
21:1
21:1
8:1
frozen in liquid nitrogen. After lyophilization, 0.5 mL of DMSO-d was
19.5
6.5
6
added into each sample to thoroughly dissolve the organic
compounds. The insoluble phosphate salt was removed by
15.2
4.3
centrifugation, and the hydrolysis products were analyzed by 1
NMR on the Bruker ARX 400 MHz spectrometer (Figure S13).
H
8:1
4.1
The pH-responsive dePEGylation of the H3G2-0.8 supramolecular
aggregates was also monitored by LLS. Briefly, 2 mL of the H3G2-0.8
dispersion (0.2 mg/mL) in PB (1.0 mM, pH = 8.4) was filtered into a
dust-free vial through a Millipore 0.45 μm PVDF membrane. The
dispersion was measured on the aforementioned LLS equipment, and
the obtained data were used for 0 min time point. Then, 200 μL of PB
a
b
Molar feed ratio of CKA to hydroxyl groups of β-CD. Degree of
substitution (DS) of hydroxyl groups of β-CD by ortho ester groups
1
estimated by H NMR spectra.
(
0.1 M, pH = 7.4 or 8.4) was added into the dispersion, which was
the range of ∼2−6.4 by simply changing the molar feed ratio of
EMD to β-CD. However, with further increasing the feed ratio
up to 21:1, the DS did not change significantly (run 4 vs run 3
in Table 1). These results indicate that EMD selectively react
with the primary 6-OH of β-CD but not the secondary 2-OH
or 3-OH under the experimental conditions, which may be
quickly vortexed for 30 s and measured at different times with a
scattering angle of 90° at 37 °C.
For the hydrophobically modified supramolecular aggregates, that is,
the mixed aggregates of H3G3-1.0 with poly(ε-caprolactone), a
dialysis protocol was used. Briefly, H3 (2.1 mg, 1.0 μmol) and G3 (5.0
mg, 1.0 μmol) were dissolved in 1 mL of ethanol and equilibrated at
4
1
0 °C for 0.5 h. PCL (M = 5 kDa, 2.0 mg, 0.4 μmol) was dissolved in
n
ascribed to the intramolecular hydrogen bonding between the
mL of ethanol at 40 °C. These two solutions were mixed and
66
2
-OH and 3-OH of the adjacent glucose units.
equilibrated for an additional 1 h at 40 °C, followed by quickly adding
When EMD reacted with β-CD at 40 °C with a large feed
2
1
8
0 mL of PB (pH = 8.4, 1.0 mM). The mixture was ultrasonicated for
5 min and incubated at 40 °C for 4 h and dialyzed against PB (pH =
.4, 1.0 mM) for an additional 24 h at 40 °C (MWCO:1 kDa) to
ratio (21:1), both primary (6-OH) and secondary (2- and 3-
OH) hydroxyl groups were modified by the ortho ester groups.
As shown in Figure S5, the original narrow peak at ∼4.8 ppm
that is assigned to the C1 proton of the intact β-CD or the 6-
OH substituted β-CD derivatives shifted downfield to ∼4.9
ppm and became broader, which is consistent with substitution
afford H3G3-1.0-PCL aggregates with a H3 concentration of 1.0 mg/
mL and a PCL/H3 molar ratio of 0.4. The dispersion was diluted to a
H3 concentration of 0.1 mg/mL by the addition of PB (pH = 8.4, 1.0
mM) and filtered through a Millipore 0.45 μm PVDF membrane. This
diluted dispersion was used to evaluate the stability of the aggregates at
different pHs (10 mM PB, pH = 7.4, 6.4 or 5.5). The diameter of the
aggregates and scattered intensity of the dispersion were measured on
the Zeta PALS analyzer (Brookhaven Instruments Corp.) and the data
were obtained in triplicate.
67
at 2-OH or 3-OH of β-CD. The DS reached ∼20, as
estimated by comparing the signal intensities of the C1 proton
and proton b at ∼1.8 ppm (run 5 in Table 1). When another
six-membered CKA (EDO) was used to react with β-CD at a
feed ratio of 21:1, the similar phenomenon was observed with a
DS of 6.5 at 20 °C and a much higher DS (∼15) at 40 °C
(Figure S6 and Table 1).
RESULTS AND DISCUSSION
■
Click-Like Reaction between CKA and CD. The reaction
between CKAs and the hydroxyl groups of alcohols or polyols
can be recognized as a “click” reaction due to its universality,
These results are understandable because increasing temper-
ature would disrupt, at least weaken, the hydrogen bonding
between 2-OH and 3-OH, and make them reactive to the
CKAs. On the whole, β-CD can be easily modified by CKA to
afford acid-labile derivatives with cyclic ortho ester groups. The
DS can be tuned by simply changing the feed ratio or reaction
temperature. Asymmetrically ortho ester-modified β-CD
derivatives can be easily prepared at a relatively low
temperature (Scheme 2). On this basis, two CKAs with longer
alkyl chains (EHD and EDD) were applied to modify β-CD,
affording additional two acid-labile β-CD derivatives (EHD-CD
6
1,63−65
high efficiency, and facile reaction condition.
addition, the less sterically hindered primary hydroxyl groups
are much more active than the secondary ones when they react
In
6
1,63−65
with CKA.
In order to reveal if there is a difference
between the reactivity of the primary hydroxyl group (6-OH)
and the secondary hydroxyl groups (2-OH and 3-OH) of
cyclodextrins toward CKA, β-CD was applied to react with
EMD at different feed ratio at 20 °C, and the reaction products
3
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Scheme 2. Reaction between CKAs and β-CDs
an inclusion complex; therefore, there were little preformed
amphiphilic supramolecules in the thin film. In ethanol or
methanol with a strong polarity, the host−guest interaction
between H3 and G2 is much stronger than in THF or CHCl ,
3
which favors the preformation of the amphiphilic supra-
6
8,69
molecules in the film.
By using the same procedure with ethanol as the organic
solvent, the aggregation behaviors of the other H-G pairs in a
molar ratio of 1:1 were investigated and the results are shown
in Table S1. Both alkyl length of the hosts and PEG length of
the guests influence the aggregation of the H-G supramolecules.
In case of G1, all three of the H-G pairs formed macroscopic
precipitates probably due to the short PEG chain, which is not
long enough to effectively stabilize the supramolecular
aggregates. For the longer G2 and G3, the stably dispersed
nanoparticles were only formed by pairs of H2G2, H3G2, and
H3G3; the other three pairs did not show obvious aggregation
due to the high degree of hydrophilic−lipophilic balance.
H3G2 supramolecular nanoparticles have a vesicular morphol-
ogy with a small diameter (∼50−80 nm), while H3G3 formed
sphere-like nanoparticles with a diameter of ∼100−220 nm
(
Figure 2 and Table 2). H2G2 supramolecular aggregates may
possess a loose structure, as indicated by the large diameter
/R
and EDD-CD) with a DS in the range of 4−4.5 (Figure S7 and
Table 1).
(∼350−550 nm) and R
application of the aggregates as carriers for nanomedicine
(Figure S8 and Table S1).
(∼1.8), which limits the
g
h
Self-Assembly of Supramolecules. All of the CKA-CDs
except run 1 shown in Table 1 are not soluble in water because
of their hydrophobicity and cannot form stably dispersed
nanoparticles in aqueous media directly by themselves.
However, they are able to form amphiphilic supramolecules
with adamatane-modified PEG (Ada-PEG) through host−guest
interaction between the adamantyl group and CD ring. These
acid-labile supramolecules may self-assemble into nanoparticles
of various morphologies and sizes. Here, we selected three
asymmetrically modified CKA-CDs with different alkyl chain
lengths as the hosts (H1, H2, and H3) and three Ada-PEG of
different PEG lengths as the guest molecules (G1, G2, and G3)
and studied the aggregation profiles of the supramolecules
formed by various H-G pairs (Scheme 3). In order to find an
Besides the alkyl or PEG chain length, the ratio of host to
guest exerts a significant effect on the size and morphology of
the H-G supramolecular aggregates (Figure 2 and Table 2).
The aggregates gradually evolved from small vesicles to large
solid-like nanospheres as the H3/G2 molar ratio increased.
With increasing the H3/G2 ratio from 1:1 to 1:0.9, both size of
the vesicles and thickness of the vesicular membrane increased,
implying that more H3 molecules were incorporated into the
membrane. Further increase in H3/G2 ratio resulted in the
disappearance of the supramolecular vesicles, and instead the
formation of sphere-like loose aggregates at the ratio of 1:0.8
and more compact solid-like particles at the ratio of 1:0.7,
respectively. The morphologies of H3G2 aggregates were also
supported by their different loading capacities for water-soluble
model compound, 6-carboxyfluorescein (6-CF; Table S3).
While H3G2-1.0 and H3G2-0.9 vesicles were able to effectively
load 6-CF in their hydrophilic interior, H3G2-0.7 nanoparticle
showed little capability of loading 6-CF due to the solid-like
inner structure. The loading capacity of H3G2−0.8 aggregate
was smaller than that of the H3G2-1.0 or H3G2-0.9 vesicle but
much larger than that of the H3G2-0.7 nanoparticle, implying
the loose structure of H3G2-0.8 aggregate. These results are
illustrated in Scheme 4. In the case of H3G3 supramolecular
aggregates, the particle size decreased with increasing H3/G3
molar ratio from 1:1 to 1:0.7, no vesicular structure was formed
Scheme 3. Host and Guest Molecules Used in the
Supramolecular Self-Assembly
(
Table S2).
appropriate method to get stably dispersed supramolecular
nanoparticles, H3-G2 pair in a molar ratio of 1:1 was first
studied by using the film hydration protocol. When THF or
Surface Modification of the Supramolecular Aggre-
gates. To demonstrate whether the surface CD cavities can be
used for further functionalization of the supramolecular
aggregates, Ada-EA and Ada-CC as the charged model guests
were added into the dispersions of H3G2 aggregates with
different H3/G2 feed ratios, and zeta potentials of the
aggregates were measured at different times. As shown in
Figure 3, in the absence of the charged guests, all the aggregates
have similar zeta potentials of ∼-10 mV. After 5 min of adding
10-fold of Ada-EA (compared to H3), zeta potential of the
aggregates increased gradually from −8.6 mV for H3G2-1.0 to
CHCl was used as a solvent to prepare supramolecular thin
3
film, it is difficult to obtain well-dispersed nanoparticles upon
ultrasonication in phosphate buffer. However, if supramolecular
thin film was prepared by using ethanol or methanol as organic
solvent, hydration of the film under ultrasonication afforded
well dispersed supramolecular nanoparticles. These results can
be attributed to the difference in polarity of the used solvents.
In the less polar THF or CHCl , H3 and G2 could hardly form
3
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Figure 2. TEM images of the H-G Supramolecular aggregates. (a) H3G2-1.0 vesicles (inset: FF-TEM image), (b) H3G2-0.9 vesicles, (c) H3G2-0.8
nanoparticles, (d) H3G2-0.7 nanoparticles, (e) H3G3-1.0 nanoparticles, and (f) H3G3-1.0-PCL nanoparticles. For H3G2 supramolecules,
concentration of the aggregates was 0.2 mg/mL; for H3G3 systems, concentration of H3 in the aggregates was 0.1 mg/mL. pH = 8.4 (1.0 mM PB);
stained with phosphotungstic acid (1%, pH = 7.4).
Table 2. Characterization of H3G2 and H3G3 Aggregates
a
b
c
c
aggregate
nhost/nguest
R
(nm)
Rg (nm)
Rh (nm)
Rg/Rh
morphology
H3G2-1.0
H3G2-0.9
H3G2-0.8
H3G2-0.7
H3G3-1.0
H3G3-1.0-PCL
1.0/1.0
1.0/0.9
1.0/0.8
1.0/0.7
1.0/1.0
20−35
30−60
90−110
150−250
50−110
30−120
48
44
1.09
1.01
0.99
0.86
vesicle
73
72
vesicle
125
278
127
322
nanoparticle
nanoparticle
nanoparticle
nanoparticle
d
d
d
118
136
0.87
1.0/1.0
b
79
115
0.69
a
c
Molar ratio of host to guest. Average radius measured by TEM. Average hydrodynamic radius (R ) and the radius of gyration (R ) measured by
LLS, 0.2 mg/mL in phosphate buffer (pH = 8.4, 10 mM), 37 °C. Averaged data of two measurements.
h
g
d
Scheme 4. Morphological Change of the H3G2
Supramolecular Aggregates with Increasing the H/G Molar
Ratio
25 mV). This phenomenon can be attributed to the dynamic
mechanism of the host−guest interaction.
pH-Responsive DePEGylation and Degradation of the
Aggregates. The effect of pH on H3G2-0.8 supramolecular
aggregates with incubation time was first studied by using LLS.
As shown in Figure 4a, both R of the aggregates and scattered
h
intensity of the dispersion did not change obviously in 12 h at
pH 8.4, indicating that the aggregates were stable. By contrast,
at pH 7.4, both Rh and intensity increased greatly from
beginning of the incubation, and the apparent R reached a
h
value larger than 1000 nm after 150 min. TEM images reveal
that large clusters composed of smaller spherical nanoparticles
adhered together were formed after incubation for 12 h at pH
1
2.5 mV for H3G2-0.7. By contrast, when the negatively
7
.4 (Figure S11). The hydrolysis behaviors of the aggregates
charged Ada-CC (10-fold to H3) was added, zeta potential of
the aggregates decreased gradually from −12.4 mV for H3G2-
1
were further investigated by H NMR spectroscopy at different
pHs (Figure S13). The kinetic curves of hydrolysis of the ortho
1.0 to −35.3 mV for H3G2-0.7. For the same aggregate, for
1
ester calculated from the H NMR spectra are shown in Figure
example, H3G2-0.7, adding Ada-EA increased zeta potential
from −9 to 12.5 mV, while the addition of Ada-CC made zeta
potential dropped from −9 to −36 mV. These results indicate
that the supramolecular aggregates are capable of being
functionalized via host−guest interaction; the degree of
functionalization increases with increasing the H3/G2 feed
ratio because there would be more empty cavities at a relatively
high H3/G2 ratio. Interestingly, when the mixture of H3G2
aggregates and Ada-CC was incubated for a longer time (1 h),
the difference in zeta potential of the aggregates diminished and
all the four samples showed the similar zeta potentials (−20−
4
b. At pH 8.4, hydrolysis rate of the ortho ester was very slow
with a hydrolysis degree less than 5% in 10 h. At pH 7.4, the
ortho ester hydrolyzed much faster at the initial stage, with a
hydrolysis degree of ∼10% within 1 h; however, the kinetic
curve almost leveled off in the following ∼9 h. When pH of the
buffer decreased to 6.4, the kinetic curve became S-shaped. At
the initial stage, the ortho ester hydrolyzed faster than at pH
7.4, reaching a hydrolysis degree of ∼11% in 30 min. After a
period with a very slow hydrolysis rate (30−300 min), the
hydrolysis rate of the ortho ester increased again.
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Figure 3. Zeta potentials of the supramolecular aggregates in the absence or presence of Ada-EA or Ada-AC. Concentration of aggregate, 0.2 mg/
mL; concentration of Ada-EA or Ada-AC, 0.1 mg/mL; pH = 8.4 (10 mM PB).
61
and even unstable in neutral aqueous buffer. In addition, the
ortho ester linkages in the supramolecular aggregates can be
divided roughly into two types; most of them are inside the
hydrophobic core and the others locate at or near the relatively
hydrophilic interface between the core and the PEG shell. The
incubation stability of the aggregates at pH 8.4 is guaranteed by
the PEG shell due to the relative stability of ortho ester. At pH
7
.4, the ortho ester linkages locating at the interface quickly
hydrolyzed, causing dePEGylation and the subsequent
aggregation of the dePEGylated hydrophobic inner cores.
The remained ortho esters (∼90%) inside the cores (or their
clusters) would be much stable due to the hydrophobic
microenvironment and hydrolyzed very slowly at neutral pH. It
is well-known that, besides pH and molecular structure,
hydrolysis kinetics of ortho ester is also greatly influenced by
70,71
the hydrophilic/hydrophobic balance of microenvironment.
Upon further decreasing the pH to 6.4, the hydrophobic cores
or their clusters can be further degraded due to the acid-
accelerated hydrolysis of the ortho esters. Using nile red (NR)
as a hydrophobic fluorescent probe, we studied the pH-
dependent releasing behaviors of NR from H3G2-0.8 nano-
particles (Figure S14). The normalized intensity of NR did not
significantly decreased (<10% in 24 h) at pH 8.4, which
indicates that the nanoparticles were stable. With decreasing
pH, the intensity decreased greatly. The lower the pH, the
faster release of NR.
As an ideal pH-sensitive programmable delivery system
targeting the remote tumor tissues, the nanoparticles should be
stable enough at pH 7.4 to ensure a long circulation in blood.
Unfortunately, as aforementioned, H3G2-0.8 nanoparticles
were unstable even at pH 7.4. In the case of H3G3-1.0
nanoparticles, which contain the longer PEG chains as the
hydrophilic shell, the incubation stability was still poor, with
only a slight improvement as compared to the H3G2-0.8
nanoparticles (Figure S15). In order to further improve the
stability at pH 7.4, the mixed supramolecular nanoparticles
were prepared by incorporating hydrophobic poly(ε-caprolac-
tone) (PCL) into the core of the H3G3-1.0 aggregates. As
shown in Figure 5, the incubation stability of the mixed
nanoparticles (H3G3-1.0-PCL) in neutral buffer was greatly
improved, with only a little decrease in the scattered intensity
and a negligible change in size of the nanoparticles in 16 h. At
Figure 4. (a) Scattered light intensity and hydrodynamic radius (Rh)
of H3G2-0.8 aggregates at different incubation time at pH 7.4 and 8.4,
respectively: detected angle, 90°; concentration of aggregates, 0.2 mg/
mL. (b) Remaining ortho ester (percentage) in the aggregates at
different pH and incubation time: PB concentration, 10 mM; 37 °C.
These results can be rationally demonstrated in Scheme 5. As
we reported previously, the ortho ester linkage used in this
work is relatively stable in the weakly basic phosphate buffer
(
pH = 8.4) but extremely labile to a mildly acidic condition,
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Scheme 5. pH-Responsive Sequential DePEGylation and Degradation of the Supramolecular Aggregates
morphology of which are influenced by the host−guest feed
ratio, changing from smaller vesicles to larger solid-like
spherical nanoparticles. These supramolecular aggregates are
stable at pH 8.4 but show a pH-dependent profile of the
sequential dePEGylation and degradation. Ease of fabrication,
capability of further functionalization via kinetic host−guest
interaction, and pH-sensitivity enables this type of supra-
molecular aggregate, promising candidates for programmable
drug delivery systems targeting the diseased sites with tiny pH
gradients.
ASSOCIATED CONTENT
■
*
S
Supporting Information
H NMR spectra of the guest molecules (G1−G3), CKAs, and
1
their precursors; more CKA-modified CD derivatives, and the
aggregates H3G2-0.8 incubated at different pH and time; more
TEM images and LLS results. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*
Phone: +86-10-62757155. Fax: +86-10-62751708. E-mail:
fsdu@pku.edu.cn.
Phone: +86-10-62755543. Fax: +86-10-62751708. E-mail:
*
zcli@pku.edu.cn.
Notes
The authors declare no competing financial interest.
Figure 5. Changes in (a) diameter and (b) scattered intensity of the
H3G3-1.0-PCL supramolecular aggregates at different incubation
times and various pHs: detected angle, 90°; concentration of H3, 0.1
mg/mL; PCL/H3 molar ratio, 0.4; PB (10 mM); 37 °C.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (21174002 and 21090351) and
the National Science Fund for Distinguished Young Scholars of
China (21225416).
the lower pHs, the size of the nanoparticles increased and the
scattered intensity of the dispersion decreased gradually due to
the acid-triggered dePEGylation and the subsequent formation
of hydrophobic clusters that easily precipitated. The lower the
pH, the faster the dePEGylation occurred.
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