PH-mediated catch and release of charged molecules with porous hollow nanocapsules

Article abstract of DOI:10.1021/ja4106946

Here, we show that the charge of the nanopores in the nanometer-thin shells of hollow porous nanocapsules can regulate the transport of charged molecules. By changing the pH of external aqueous solution, we can entrap charged molecules in nanocapsules and

Full text of DOI:10.1021/ja4106946

Communication
pubs.acs.org/JACS
pH-Mediated Catch and Release of Charged Molecules with Porous
Hollow Nanocapsules
Sergey A. Dergunov, Jeffrey Durbin, Sambit Pattanaik, and Eugene Pinkhassik*
Department of Chemistry, Saint Louis University, 3501 Laclede Avenue, St. Louis, Missouri 63103, United States
S
* Supporting Information
ABSTRACT: Here, we show that the charge of the
nanopores in the nanometer-thin shells of hollow porous
nanocapsules can regulate the transport of charged
molecules. By changing the pH of external aqueous
solution, we can entrap charged molecules in nanocapsules
and trigger the release of encapsulated content.
electively triggered uptake and release of molecules is
Suseful for creating delivery devices for drugs and imaging
agents.1 pH-mediated catch and release is particularly attractive
because it can take advantage of physiological gradients of pH.
For example, the pH values are 7.35−7.45 in the bloodstream,
5.4−6.2 in endosomes, and 4.8 in lysosomes, while extracellular
pH in the tumors, inflamed tissue, or tissue at early stages of
wound healing may range from 5.5 to 6.9.2
Previously, pH-regulated uptake and release of molecules was
reported for layer-by-layer microcapsules.3 These polymer
Figure 1. Catch and release of small charged molecules in porous
hollow nanocapsules: molecules are smaller than the pore size and can
diffuse into the capsule at acidic pH but are retained inside the cavity
by electrostatic repulsion with negatively charged pores at neutral or
capsules expanded and contracted in response to varying pH;
as a result, the gaps between polymer chains increased when
the capsules were expanded, allowing molecules to pass
through. In contracted form, nanocapsules retained entrapped
basic pH.
molecules. Other examples include pH-sensitive hydrogels that
release content at low pH.4 Bakajin et al.5 and Hummer et al.6
neutral pores would permit diffusion of molecules in and out of
showed pH-regulated transport of ions and small molecules
through membranes made of carbon nanotubes.
capsules. In neutral pH, negatively charged pores would not
release anions due to electrostatic repulsion.
We and others reported the synthesis of vesicle-templated
hollow polymer nanocapsules.7 Imprinted pores can control the
size-selective permeability and permit ultrafast transport of
species smaller than the pore size.7a,b,8
Porous shells of vesicle-templated nanocapsules are much
thinner than biological membranes and are among the thinnest
known materials, approaching graphene in thickness. To date,
there were no reports that electrostatic repulsion could
successfully prevent transport of ions through such membranes.
The ability to regulate the transport through nanometer-thin
membranes may have profound implications for the design of
nanoscale functional devices.
Carboxylic groups are particularly suitable for controlling the
charge in the physiological pH range. Typical pKa values (4.8
for acetic acid; 4.2 for benzoic acid) suggest that >99% of
carboxylic groups will be deprotonated at pH of blood (pH
∼7.4), while roughly half of carboxylic groups will be
deprotonated at pH of lysosomes (pH ∼4.8). To test the
hypothesis that the charge of nanopores can regulate the
transport, we designed capsules containing carboxylic groups in
the pore orifice (Figure 1). We hypothesized that in acidic pH,
This pH-mediated catch and release mechanism will permit
entrapping molecules in mildly acidic conditions, keeping them
entrapped under neutral or basic conditions, and releasing them
again in acidic media. The gated transport would not involve
rearrangement of the polymer shell or change the size of the
nanocapsules. Pores can be imprinted in bilayer-templated
membranes with high precision,7a,b,f,8 and this approach may
offer excellent permeability control.
To implement this idea, we synthesized polymer capsules
containing multiple carboxylic groups in the pore orifice.
Carboxylic groups were imprinted using polymerizable pore-
forming templates containing multiple ester groups. The
synthesis of nanocapsules is described in detail below.
We designed a simple color scheme to observe the uptake
and release of charged molecules. Nanocapsules contain an
entrapped large blue molecule that serves a dual purpose. It acts
as a size probe showing that the nanopores do not exceed its
size in charged or neutral forms and provides a visual contrast
Received: October 19, 2013
Published: December 25, 2013
© 2013 American Chemical Society
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for a small yellow molecule during catch and release
experiments. We used Reactive Blue dye coupled with β-
cyclodextrin (RBCD, Figure 2a) as a blue probe (smallest
cross-section 1.6 nm)7b,10 and methyl orange (MO) as a small
anionic yellow dye (smallest cross-section 0.6 nm). The
sulfonic group in MO is negatively charged at both neutral
and mildly acidic pH. In our approach, capsules are blue when
they do not have pinhole defects and retain RBCD (Figure 2).
In acidic pH, carboxylic groups in the pore orifice are
protonated and the pores are electroneutral. Small anionic
MO diffuses into the nanocapsules, and capsules turn green.
When the pH is increased to neutral or slightly basic levels,
carboxylic groups in the pores become deprotonated and
negatively charged. Due to the electrostatic repulsion, anionic
molecules cannot escape from the capsules. This color scheme
shows immediately whether small yellow molecules are
entrapped and retained in nanocapsules. Lowering the pH
protonates the carboxylic groups. Following the concentration
gradient, anionic molecules can now escape from the
nanocapsules, and capsules regain their original blue color.
Nanocapsules containing RBCD were precipitated from an
aqueous suspension after the synthesis and removal of non-
entrapped dyes (left sample in Figure 2b). In the absence of
stabilizing surfactants or lipids, nanocapsules in water form
aggregates that can be easily dispersed by mild agitation or
stirring. A solution of MO at pH 4 was added to the
nanocapsules, the mixture was agitated to allow molecules to
enter the capsules, and then the pH was adjusted to 8 with
NaOH. Nanocapsules were precipitated and washed in a buffer
with mildly basic pH without loss of entrapped MO as
evidenced by green color of nanocapsules (right sample in
Figure 2b). In control experiments, capsules shown in Figure
2b (left sample) were treated with an aqueous solution of MO
at pH 4, and then the capsules were precipitated and washed
with a buffer at the same pH. No retention of MO was
observed.
In addition to MO, we conducted similar experiments with
Congo Red (CR) and 4-(phenylaza)benzoic acid (4-PABA)
with similar results. To quantify the retention, we measured the
amount of encapsulated dyes at different pH. We found that at
pH >8, 100% of entrapped molecules remained entrapped in
the capsules even after extensive washing (Supporting
Information (SI), Table S2). The amount of entrapped dyes
corresponded to the calculated volume fraction of nanocapsules
suggesting that all or nearly all of capsules were loaded with
anionic dyes at the concentration of the stock solution. At pH
7, we observed 72−81% retention of MO, while at pH 5.5 we
found 24−32% retention of MO. At lower pH, no MO
remained inside nanocapsules. These data support the central
idea of this work, regulation of permeability using electrostatic
repulsion.
To show the pH-mediated release, we acidified the aqueous
suspension of nanocapsules containing both MO and RBCD to
pH 4. A brief agitation and decantation revealed blue capsules
and a slightly yellow supernatant (Figure 2c), suggesting that
MO was released. After washing the nanocapsules with mildly
acidic buffer, the absorbance spectrum of nanocapsules was
identical to the spectrum of nanocapsules before the catch and
release experiment.
In further control experiments, we showed that dyes did not
adsorb on the surface of vesicle-templated capsules at any pH
used in this work (SI, Figures S9 and S10). Also, dyes did not
adsorb onto a polymer containing carboxylic groups. We
conclude that MO, CR, and 4-PABA were indeed retained
inside the hollow nanocapsules by the electrostatic repulsion
while the carboxylic groups in the pore orifice were
Figure 2. pH-regulated uptake and release of small charged molecules
in porous hollow nanocapsules. (a) Size probes: blue: Reactive Blue 2/
β-cyclodextrin conjugate (RBCD, 1.6 nm));7b,10 yellow: methyl orange
(MO, 0.6 nm). RBCD remains entrapped in nanocapsules. In acidic
conditions, electroneutral pores allow MO diffuse in and out of
nanocapsules. In neutral and basic media, negatively charged
nanopores keep negatively charged MO inside nanocapsules via
electrostatic repulsion. (b) Precipitated nanocapsules with entrapped
RBCD are treated with an acidic solution of MO, followed by increase
in pH and precipitation of nanocapsules. Removal of non-entrapped
MO by decantation and washing yields green nanocapsules, showing
that both RBCD and MO are now entrapped. (c) Addition of acid to
the basic suspension of green nanocapsules with co-entrapped RBCD
and MO causes the release of MO, resulting in blue nanocapsules
containing RBCD and slightly yellow supernatant.
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Figure 3. Electron micrograph and DLS of nanocapsules. (a,b) TEM images and DLS of polymer nanocapsules at pH 4.3 and 7.1, respectively,
corresponding to samples shown in Figure 2b. (c) SEM image of polymer nanocapsules.
Figure 4. Directed assembly of nanometer-thin polymer capsule shells with uniform functionalized nanopores. First step: a self-assembled bilayer is
loaded with hydrophobic monomers with cross-linkers and pore-forming template. The template consists of polymerizable moieties (blue) to anchor
the template covalently to the polymer matrix, degradable linkers (red) to create a functionalized nanopore, and a bulky hydrophobic unit (violet) to
define the pore size. Second step: polymerization produces a cross-linked shell with copolymerized templates in the bilayer interior, followed by the
removal of the scaffold and chemical degradation of the template to yield nanometer-thin films containing nanopores decorated with multiple
functional groups.
deprotonated. When the carboxylic groups became protonated,
the pores permitted transport of charged molecules. Additional
experiments with attempted entrapment of glucose and
glucosamine, representing a neutral molecule and a cation,
respectively, showed no evidence of entrapment (SI, Figures
S4−S7). These observations ruled out potential expansion or
contraption of pores at different pH and further supported the
regulation of transport by electrostatic interactions.
Electron micrographs and data from dynamic light scattering
(DLS) analysis demonstrated the preservation of the shape and
integrity of the nanocapsules (Figure 3). Transmission electron
microscopy (TEM) images and DLS data show that the size
and shape of polymer nanocapsules in acidic conditions (Figure
3a) are identical to those in neutral medium (Figure 3b).
Average diameter remained 210 nm with polydispersity index
values <0.25. Scanning electron microscopy (SEM) images
shows clusters of nanocapsules with the same size (Figure 3c).
These results suggest that the nanocapsules with nanometer-
thin walls are stable at different pH. Exposure of nanocapsules
to solutions with different concentrations of small and large
osmolytes (up to 2700 mOsm/kg) showed no evidence of
rapture of nanocapsules.
removal of the lipid scaffold, pore-forming templates were
hydrolyzed in alkaline medium to form nanopores with
carboxylic groups (Figure 4). Previous studies showed that
the monomer:cross-linker and monomer:lipid ratios affect the
permeability of nanocapsules.7d,9 In control experiments, we
confirmed that the shells of nanocapsules prepared without
pore-forming templates were impermeable to the molecules
used here (SI, Figure S8 and Table S5).
The pore-forming templates were synthesized by exhaustive
esterification of glucose using either p-vinylbenzoic acid or
methacrylic anhydride (Scheme 1). Typically, glucose and
Scheme 1. Synthesis of Polymerizable and Degradable Pore-
Forming Templates
Nanocapsules containing imprinted pores with carboxylic
groups were synthesized by a directed assembly approach using
lipid vesicles (liposomes) as temporary self-assembling
scaffolds.7a−c Hydrophobic monomers with cross-linkers and
pore-forming templates were loaded into the hydrophobic
interior of the bilayer (Figure 4). The pore-forming templates
were pentaesters of glucose containing polymerizable styrene or
methacrylate moieties. These groups copolymerized with
monomers and cross-linkers forming a polymer shell inside
the bilayer. Following the UV-induced polymerization and
esterifying agent were mixed with a base and a catalyst on an ice
bath, and the reaction mixture was allowed to warm to room
temperature and stirred for 24 h. Synthesis of compound 2 was
reported previously.11
We used template 1 for the synthesis of polystyrene capsules
and template 2 for the synthesis of polyacrylate capsules using
monomers and cross-linkers shown in Figure 3.
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Notes
We used quantitative IR measurements on polystyrene
nanocapsules prepared with template 1 to evaluate the average
number of carboxylic groups imprinted in each pore. Carboxylic
groups on the pore orifice were converted to amide groups by
sequential reactions with oxalyl chloride and butylamine. The
conversion was done to differentiate the carbonyl groups from
the starting ester and resulting amide. The IR absorption band
of the carboxylic acid is too close to the absorption band of the
ester for confirming that the hydrolysis occurred quantitatively.
Also, the intensity of the absorption of the carboxylic group
depends on hydrogen bonding, adding uncertainty to
quantitative measurements. After the hydrolysis of the pore-
forming templates and conversion of the remaining carboxylic
groups to the amides, no absorption band for the ester or
carboxylic group was found in the IR spectrum (SI, Figure
S11). In the IR spectra of capsules before the hydrolysis of the
pore-forming templates, the carbonyl band arises from five ester
groups per template. After the hydrolysis and conversion of the
carboxylic acids to the amides, the carbonyl band appears only
due to those carboxylic groups that became part of the polymer
shell. Nonreacted vinylbenzoic moieties from the template are
removed from the capsules during the hydrolysis. By comparing
absorbance of the ester carbonyl in the starting capsules with
the absorbance of the amide carbonyl in the functionalized
capsules, we found that nanopores in newly prepared
nanocapsules contained 3.3 0.3 carboxylic groups on average.
This number accounted for the intrinsic molar absorptivity of
the ester and amide determined from standard calibration
curves. Considering that the synthesis of the capsule shell with
embedded templates occurred within the bilayer, essentially, a
two-dimensional solvent,12 incorporation of the majority of the
functional groups from the template into the cross-linked shell
is a favorable outcome.
In summary, we report a new mechanism for selective uptake
and triggered release of charged molecules using porous hollow
nanocapsules. The catch and release mechanism is based on
electrostatic repulsion and controlling the charge of the
nanopores by the pH of the external solution.
It is likely that the release characteristics of nanocapsules can
be programmed by varying the number of carboxylic groups per
pore and number of pores per capsule. These variations may
result in fine-tuning of the pH threshold and/or kinetics of the
release of encapsulated cargo. Coupled with previously reported
ability to control the size of imprinted pores, this approach can
offer a versatile method for controlling the permeability of
nanothin porous materials.
Other nanometer-thin materials, such as graphene, may likely
control permeability using electrostatic repulsion. These results
are likely to have broader implications beyond hollow
nanocapsules. Regulating permeability in the nanometer-thin
membranes can lead to new functional nanodevices and
advanced separation systems.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Science Foundation
(CHE-1316680, CHE-1012951, and CHE-0933363) and Saint
Louis University Presidential Research Fund.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedures and data on uptake and
release; synthesis of nanocapsules and pore-forming templaes.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
epinkhas@slu.edu
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