Encapsulation and release mechanisms in coordination polymer nanoparticles

Article abstract of DOI:10.1002/chem.201302662

The interplay of guest encapsulation and release mechanisms in nanoscale metal-organic vehicles and its effect on the drug-delivery kinetics of these materials were investigated through a new multidisciplinary approach. Two rationally-designed molecular g

Full text of DOI:10.1002/chem.201302662

DOI: 10.1002/chem.201302662
Encapsulation and Release Mechanisms in Coordination Polymer
Nanoparticles
Laura Amorꢀn-Ferrꢁ,[a] Fꢁlix Busquꢁ,[a] Josꢁ Luis Bourdelande,[a] Daniel Ruiz-Molina,[b, c]
Jordi Hernando,*[a] and Fernando Novio*[b, c]
Abstract: The interplay of guest encap-
sulation and release mechanisms in
nanoscale metal–organic vehicles and
its effect on the drug-delivery kinetics
of these materials were investigated
through a new multidisciplinary ap-
proach. Two rationally-designed molec-
ular guests were synthesized, which
consist of a red-fluorescent benzophe-
noxazine dye covalently tethered to
guests into compositionally and struc-
turally equivalent coordination poly-
mer particles through distinct encapsu-
lation mechanisms: coordination and
mechanical entrapment. The two types
of particles delivered their fluorescent
cargo with remarkably different kinetic
profiles, which could be satisfactorily
modeled considering degradation- and
diffusion-controlled release processes.
This demonstrates that careful selec-
tion of the method of guest incorpora-
tion into coordination polymer nano-
particles allows selective tuning of the
rate of drug delivery from these mate-
rials and, therefore, of the time
window of action of the encapsulated
therapeutic agents.
Keywords: nanoparticles · drug de-
livery · fluorescence · metal–organic
frameworks · polymers
a
coordinating catechol group and
a protected, non-coordinating catechol
moiety. This allowed loading of the
Introduction
creasing number of reports have indeed described the suc-
cessful application of nanoscale coordination polymer parti-
cles to encapsulate and release therapeutic agents.[6] None-
theless, the use of CPPs for drug delivery is in its fledgling
stage. A detailed rationalization of guest encapsulation and
release mechanisms is still required to understand the drug-
delivery kinetics of most CPPs and, consequently, to fully
assess their potential use as nanocarriers for therapeutic pur-
poses. Although these issues have already been subject of
extensive debate for biodegradable organic polymer vehicles
as drug-delivery systems,[7–9] little attention has so far been
paid to them with regards to the emerging CPP-based mate-
rials.
Incorporation of the active molecules in coordination
polymer nanoparticles usually proceeds through two distinct
strategies: 1) binding of the drug to the polymer framework
as a CPP building block[10–14] and 2) mechanical entrapment
of the therapeutic agent within the metal–organic
matrix.[15–17] Accordingly, drug release can take place though
different mechanisms, namely slow particle degradation
through surface erosion, fast diffusion processes and/or
a combination of both. This scenario can be even more intri-
cate if undesired desorption from the particle surface occurs.
As a result, complex drug-delivery profiles are often en-
countered in CPPs that preclude unambiguous elucidation
of the relationship between encapsulation and release mech-
anisms.[5,16]
Coordination polymer particles (CPPs) have recently
emerged as a new family of metal–organic materials formed
by the self-assembly of metal ions and polydentate bridging
ligands.[1,2] Together with crystalline metal–organic frame-
works (MOFs), CPPs have been proposed for a large variety
of applications owing to the intrinsic versatility of coordina-
tion chemistry, which allows the properties of the final mate-
rials to be rationally tailored by an appropriate choice of
metals and ligands.[3] Of special interest is the use of CPPs
in medicine, which is predicted to have a broad impact in
the fields of bioimaging and drug delivery.[4–6] Since the pio-
neering work of Mirkin and co-workers in 2005,[1] an in-
[a] L. Amorꢀn-Ferrꢁ, Dr. F. Busquꢁ, Dr. J. L. Bourdelande,
Dr. J. Hernando
Departament de Quꢀmica, Universitat Autꢂnoma de Barcelona
Edifici C/n, Campus UAB, Cerdanyola del Vallꢃs, 08193 (Spain)
Fax : (+34)935811265
E-mail: jordi.hernando@uab.cat
[b] Dr. D. Ruiz-Molina, Dr. F. Novio
Institut Catalꢄ de Nanociꢃncia i Nanotecnologia (ICN2)
Edifici ICN2, Campus UAB Cerdanyola del Vallꢃs, 08193 (Spain)
Fax : (+ 34)937372648
E-mail: fernando.novio@cin2.es
[c] Dr. D. Ruiz-Molina, Dr. F. Novio
Consejo Superior de Investigaciones
Cientꢀficas (CSIC), Edificio ICN2, Campus UAB
Cerdanyola del Vallꢃs, 08193ACTHNUTRGNE(NUG Spain)
To shed more light on this issue, we have envisioned the
fabrication of morphologically equivalent CPPs bearing a flu-
orescent guest that can be either coordinated to the polymer
backbone (M1) or physically encapsulated within the parti-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302662.
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FULL PAPER
cle (M2). These two materials therefore represent excellent
benchmark systems to comparatively investigate degrada-
tion- and diffusion-controlled drug-release processes in
CPPs. A schematic representation of this approach is shown
in Figure 1. The molecular guest of choice for these studies
is a red-fluorescent benzophenoxazine dye covalently linked
to a coordinating catechol group, both in its non-protected
(1) and protected forms (2). On the other side, cobalt nano-
particles were used as carriers, with the general composition
Results and Discussion
Synthesis and characterization of fluorescent guests 1 and 2:
Scheme 1 shows the synthetic route followed to obtain 1 and
2. Briefly, the tert-butylation and subsequent allylic oxida-
tion of commercial 2-methoxy-4-methylphenol gave the
known aldehyde 3 in 97% yield,[20] which is a common inter-
mediate for both target compounds. At this point, synthetic
pathways diverged, either temporally protecting the hydrox-
yl groups of the catechol moiety as the corresponding me-
thoxymethylethers (MOM), to obtain compound 1, or per-
manently derivatizing them as the methyl ethers as found in
compound 2. Thus, known intermediate 4a was obtained
from 3 by sequential demethylation with BBr3, and protec-
tion of the corresponding catechol with methoxymethylbro-
[CoACHTUNGTRENNUNG(bix)ACHTUNGTRENNUNG(3,5-dbsq)(3,5-dbcat)], in which bix is a flexible bisi-
midazole bridging ligand and 3,5-dbsq and 3,5-dbcat repre-
sent the semiquinonate radical and catecholate forms of the
3,5-di-tert-butylcatechol, respectively.[15,16,18] Although analo-
gous CPPs containing ZnII ions and bix ligands have already
been reported and evaluated for drug-delivery applica-
tions,[15,16] the choice of [Co
ACTHNUTRGENNUG(bix)ACHTUNGTRENN(UGN 3,5-dbsq)(3,5-dbcat)] nano-
particles is justified by: 1) the high affinity of catechol
groups to coordinate to cobalt ions, which provided us with
a simple way to incorporate the fluorescent guest to the
polymer backbone in M1 without modification of the coor-
dination sphere; 2) the well-known optical properties of
(N-N)] units,[19] which must result in
[CoACHTUNGTRENNUNG(3,5-dbsq)(3,5-dbcat)HCATUNGTRENNUGN
efficient fluorescence quenching of compounds 1 and 2
while they remain in the interior of the nanoparticles and,
therefore, allow for selective detection of the released guest
molecules; and 3) the valence tautomerism exhibited by
[CoACHTUNGTRENNUNG(bix)ACHTUNGTRENNUNG
(3,5-dbsq)(3,5-dbcat)] CPPs,[18] which can be ex-
ploited to assess the morphological similarities between M1
and M2.
Scheme 1. Synthesis of fluorescent guests 1 and 2. (a) tBuOH, H3PO4,
808C, 10 h; (b) Br2, tBuOH, RT, 4 h; (c) BBr3, CH2Cl2, RT, 3 h;
(d) MOM-Cl, N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyr-
idine (DMAP), CH2Cl2, heat at reflux, 24 h; (e) Me2SO4, K2CO3, (n-
Bu)4NI, DMF, RT, 15 h; (f) Ph3PCHCN, toluene, heat at reflux, 18 h;
(g) H2 (2 atm), Pd/C, EtOAc 18 h; (h) LiAlH4, anhydrous THF, addition
at 08C, then RT, 15 h; (i) 3-(naphthalen-1-ylamino)propanoic acid, EDCI,
DIPEA, CH2Cl2, RT, 18 h; (j) N-ethyl-5-hydroxy-2-methyl-4-nitrosoben-
zenaminium chloride, HCl, MeOH, heat at reflux, 2 h.
Figure 1. Chemical structures of fluorescent guest compounds 1 and 2,
with which M1 and M2 coordination polymer particles were prepared to
investigate degradation- and diffusion-controlled release from CPPs.
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J. Hernando, F. Novio et al.
mide (90% overall yield).[21] Methylation of the free hydrox-
yl of compound 3 gave previously described derivative 4b
(90%).[22]
The next synthetic steps are analogous for both target
compounds. The Wittig reaction between aldehydes 4a and
4b and the stabilized phosphorane 2-(triphenylphosphoran-
Fabrication and characterization of M1 and M2 CPPs:
Adapting an experimental procedure previously published
by us,[18] coordination polymer particles M1 and M2 were
prepared by reaction of CoII ions with the ditopic ligands
1,4-bis(imidazol-1-ylmethyl)benzene and 3,5-di-tert-butylca-
techol in the presence of guest compounds 1 and 2 (Fig-
A
E
ure 3a). This led to the formation of [CoACTHNGUTERNNU(G bix)ACHTUNGTNER(NUNG 3,5-dbsq)(3,5-
(96% yield) and 5b (72% yield), as mixtures of Z- and E
isomers. Successive hydrogenation of the alkene moieties, at
high pressure of H2 under Pd/C catalyst, and nitriles, with
LiAlH4, furnished amines 7a and 7b in 61 and 51% overall
yields for both reduction reactions, respectively. After this,
troublesome formation of amides 8a (31% yield) and 8b
(35% yield) was achieved by reaction between amines 7a
and 7b and 3-(naphthalen-1-ylamino) propanoic acid, using
1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDCI) as
a coupling agent.[23] Compounds 1 and 2 were finally ob-
tained by reaction between naphthylamines 8a and 8b and
N-ethyl-5-hydroxy-2-methyl-4-nitrosobenzenaminium chlo-
ride in methanol, under acidic catalyst and heated at reflux
(45 and 35% yield for 1 and 2, respectively).[24] Importantly,
this last step did not only allow the benzophenoxazine dye
group of both fluorescent guests to be constructed, but also
concomitant cleavage of the methoxymethylethers to even-
tually obtain compound 1.
dbcat)] polymers, which readily precipitated as nanoparticles
due to their low solubility in the reaction medium. The re-
sulting CPPs were subsequently collected by centrifugation,
washed with 5:1 water/ethanol mixtures until no red fluores-
cence was observed in the supernatant solution, and then
dried. For comparison purposes, guest-free coordination
polymer nanoparticles (M0) were also prepared using this
methodology. Noticeably, very small amounts of compounds
1 and 2 were used in the preparation of materials M1 and
Once synthesized, the optical properties of compounds
1 and 2 were investigated in detail. Figure 2 plots the ab-
sorption and fluorescence emission spectra of these species
Figure 2. Absorption and fluorescence emission spectra of fluorescent
guests 1 (c) and 2 (b).
in methanol, which are mainly governed by the optical tran-
sitions corresponding to their benzophenoxazine dye unit.
As a result, compounds 1 and 2 display equivalent absorp-
tion
(lmax,1 =625,
l
max,2 =626 nm,
emax,1 =emax,2 =4.8ꢆ
104 mꢀ1 cmꢀ1) and emission bands (lmax,1 =643, lmax,2
=
645 nm), which resemble those reported for similar deriva-
tives.[25] Importantly, covalent tethering of the benzophenox-
azine unit to catechol and o-methoxyanisole groups in 1 and
2 does not quench its inherent emissive behavior, the result-
ing dyads thus presenting high fluorescence quantum yields
(Ff,1 =0.40, Ff,2 =0.41). Together with their long-wavelength
absorption and emission spectra, this makes compounds
1 and 2 ideal fluorescent reporters to monitor the guest re-
lease from CPPs as well as particle degradation.
Figure 3. (a) Schematic synthesis of CPPs doped with fluorescent guests
1 and 2; (b and c) SEM (left) and TEM (right) images of M1 (b) and M2
(c) particles. Scale bars for SEM are 1 mm and for TEM are 200 nm.
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Coordination Polymer Nanoparticles
FULL PAPER
M2 (catechol/guest molar ratio ꢁ100:1). With such low
doping loads we intended to minimize the effect of the fluo-
rescent guests on the formation of the nanoparticles, which
should allow us to unambiguously ascribe the differences
observed in their release profiles to the occurrence of dis-
tinct guest incorporation and delivery mechanisms.
Consequently, efficient quenching of dye fluorescence
through resonant energy-transfer processes is expected in
the interior of the nanoparticles, where these moieties will
be located at the near proximity of coordination complex
units regardless of whether they are directly coordinated to
the metal center or physically encapsulated within the poly-
mer network. Indeed, no red fluorescence could be mea-
sured for M1 and M2 particles in the solid state and in de-
gassed methanol, which confirms effective quenching of the
emission of the loaded guests (Figure 4b).
Fluorescence quenching is however inhibited upon guest
release and CPP degradation, which allowed us to monitor
the delivery of the particle cargo by means of highly sensi-
tive emission measurements (see below). This was demon-
strated by measuring the optical properties of M0, M1, and
M2 in non-degassed methanol, in which particle dissolution
is followed by coordination polymer degradation through
ligand exchange and concomitant oxidation of the catecho-
late and semiquinone groups. This leads to the disappear-
The formation of morphologically equivalent CPPs was
indeed revealed by scanning (SEM) and transmission
(TEM) electron microscopy images (Figure 3b and c, see
also Figure S1 in the Supporting Information). In all cases,
nanometer-sized solid particles with spherical shapes and
rather uniform and similar diameters ((195ꢂ38), (152ꢂ22),
and (185ꢂ37) nm for M0, M1, and M2, respectively) were
obtained. X-ray diffraction experiments confirmed the
amorphous character of these materials, whereas spectro-
scopic characterization upon dissolution of the nanoparticles
in degassed methanol revealed the occurrence of different
electronic absorption bands arising from their constituent
functional units (Figure 4a). Thus, an absorption band at
lꢁ625 nm was selectively found in the spectra of M1 and
M2, which corresponds to the fluorescent benzophenoxazine
moiety loaded in these materials. On the contrary, the other
absorption bands at lꢁ400, 590, and 700 nm were not only
encountered in the spectra of M1 and M2, but also observed
ance of the absorption bands associated to the [CoACTHNUTRGENNU(G bix)ACHTUNGTRENNUNG(3,5-
dbsq)(3,5-dbcat)] coordination polymers as well as pro-
nounced growth of the band at lꢁ400 nm corresponding to
the quinone species resulting from catecholate and semiqui-
none degradation (see Figure S2 in the Supporting Informa-
tion).[27] Accordingly, no energy-transfer processes are ex-
pected under such conditions and an enormous increase in
benzophenoxazine emission was indeed measured (Fig-
ure 4b). The absorption measurements in non-degassed
methanol were also used to quantify the encapsulation effi-
ciencies for the preparation of dye-doped M1 and M2 parti-
cles. Interestingly, higher values were obtained for M1
(ꢁ20%) than for M2 (ꢁ10%) under equivalent experimen-
tal conditions, which indicates that incorporation of the fluo-
rescent guest bearing a coordinating catechol moiety is sig-
nificantly more effective.
for guest-free M0. These can be ascribed to intra
metal-to-ligand/ligand-to-metal charge-transfer electronic
transitions of the [Co(bix)
(3,5-dbsq)(3,5-dbcat)] system.[26]
ACHUTGTNRENlNUG iACHTGNUTRENNUgG and and
A
ACHTUNGTRENNUNG
Noticeably, these absorption bands corresponding to the co-
ordination complex units expand all over the UV/Vis and
NIR regions, and therefore they overlap with the emission
spectrum of the benzophenoxazine dye (see Figure 2a).
Valence tautomerism of M1 and M2 CPPs: The amorphous
nature of M1 and M2 nanoparticles precludes any accurate
structural characterization by classical diffraction tech-
niques. Nevertheless, we exploited the valence tautomerism
(VT) behavior shown by [CoACTHNUTRGENNG(U bix)ACHTUNTGREN(NUNG 3,5-dbsq)(3,5-dbcat)]
CPPs[18,19] to investigate the structural similarities between
M1 and M2. These systems might interconvert reversibly be-
tween the low-spin (ls)-[CoIII
G
ACHTUNGTNER(NUNG 3,5-dbsq)(3,5-dbcat)] and
high-spin (hs)-[CoII
ACTHNUGERTN(NUNG bix)ACHTUGNTRENNNUG
lecular metal–ligand electron-transfer, a process that can be
selectively monitored by temperature-dependent measure-
ments of magnetic susceptibility.
Figure 5 plots the results obtained in those measurements
for M0, M1, and M2. In all cases, an abrupt change in effec-
tive magnetic moment (meff) is observed around 300 K,
which is consistent with valence-tautomeric interconversion
from low- to high-spin states for a large fraction of mole-
cules in the nanoparticles.[18] Importantly, the occurrence of
valence tautomerism and the actual profile of the corre-
sponding meff versus T plot is not only highly sensitive to the
composition and structure of the metal complex, but also to
Figure 4. (a) Absorption spectra of M0 (g), M1 (c), and M2 (b)
in degassed MeOH; (b) Fluorescence emission spectra recorded in de-
gassed MeOH of M1 (c) and M2 (b) and in non-degassed MeOH
of M1 (d) and M2 (g)
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J. Hernando, F. Novio et al.
these materials were found to be strikingly different. In the
case of M2, the delivery process was nearly completed after
8 h (t1/2 ꢁ1.2 h), a behavior resembling that already reported
for the release of anticancer drugs mechanically entrapped
in analogous [ZnACTHNUTRGNEUNG
(bix)] CPPs.[16] In contrast, a much slower
process was observed for M1, which required about 100 h
for completion (t1/2 ꢁ11 h).
On the basis of the non-coordinating nature of the encap-
sulated guest, the release profile of M2 at 378C was fitted
with a purely diffusion-controlled model of drug delivery. In
particular, we considered the use of Equation (1), which was
derived for drug delivery through Fickian diffusion from
spherical particles with homogenous and low-doping loads
that do not significantly swell or degrade during the release
process:[28]
~
&
Figure 5. Values of meff as a function of temperature for M0 ( ), M1 ( )
*
and M2 ( ) CPPs.
the local environment.[19] In other words, the same complex
may or may not exhibit VT, or the low-spin-to-high-spin
conversion might take place at different temperatures de-
pending on structural and environmental parameters. There-
fore, the extremely similar magnetic behavior encountered
for M0, M1, and M2 clearly indicates that they must be
formed by equivalent coordination polymers in rather com-
parable phases.
!
ꢀ
ꢁ
1
X
6
p2
1
n2
Dn2p2t
R2
Mt ¼ M1 1 ꢀ
exp ꢀ
ð1Þ
n¼1
in which Mt and M1 represent the cumulative absolute
amounts of guest released at time t and infinity, R is the
radius of the particles and D is the apparent diffusion con-
stant of the drug within the system. D is the only variable
parameter in this model; however, it is taken to remain con-
stant throughout the release process by neglecting swelling
and degradation effects on the structure of the polymeric
drug carrier.
Guest release mechanisms: To investigate guest release
from M1 and M2, colloidal suspensions were prepared in
phosphate-buffered saline solutions (PBS) at pH 7.4, placed
in a dialysis bag (molecular weight cut-off (MWCO):
3500 Da) at 378C, and finally dialyzed against PBS for
100 h. Relative cumulative release profiles were then mea-
sured by monitoring the fluorescence of the dialysis bath so-
lution in time. In addition, the solid material remaining in
the dialysis bag after 100 h was dissolved in methanol and
characterized by absorption spectroscopy, which allowed us
to determine the absolute release efficiency of the dialysis
experiment. Figure 6 plots the cumulative release profiles
measured for M1 and M2 under these experimental condi-
tions. Both exhibit very high release efficiencies after 100 h
(ꢁ90%) with no “burst effects” associated with undesired
desorption of guest molecules physisorbed onto the nano-
particle surface. However, the release kinetics measured for
As can be observed in Figure 6, a rather satisfactory fit of
the experimental release kinetics of M2 was obtained by
using Equation (1). Therefore, the delivery of the mechani-
cally entrapped fluorescent guest must be governed by
a
time-independent diffusion mechanism (D=6.9ꢆ
10ꢀ19 m2 sꢀ1), which indicates that the influence of degrada-
tion processes on the release kinetics is negligible in this
case even though it takes place. This is proven by Figure 7,
which displays SEM images of M2 nanoparticles suspended
in aqueous media at 378C for 0, 5, 26 and 100 h. While most
particles preserved their spherical shape after 5 h, extensive
surface erosion and an increasing amount of non-structured
material is observed in the SEM images registered at 26 and
100 h. This confirms CPP degradation, which however takes
place at a longer timescale than guest diffusion from the
nanoparticles at 378C. This is in contrast with other systems
for which clearly different delivery phases are observed that
are ascribed to the occurrence of sequential fast diffusion
and slow degradation processes.[7–9]
The release profile obtained for M1 at 378C was also ten-
tatively fitted with single-mechanism models, which in this
case should solely account for degradation-controlled deliv-
ery. However, poor agreement between the experimental
and fitted release profiles was obtained regardless of using
surface-degradation-[29] or bulk-degradation[30] models of
drug delivery. This suggests the occurrence of a more com-
plex release process, which we attempted to model by as-
suming simultaneous delivery through degradation and dif-
fusion processes. In this scenario, degradation-controlled re-
lease should apply for all guest molecules coordinated to the
polymeric backbone, whereas those that remain unbound
Figure 6. Guest release profiles of fluorescent guest molecules from M1
&
*
( ) and M2 ( ) at 378C, which were averaged over 4 independent experi-
ments. Lines correspond to fits of the experimental data as described in
the text.
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Coordination Polymer Nanoparticles
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To fit Equation (2) to the guest release profile measured
for M1 at 378C, only two variable parameters were consid-
ered: b and kd. To test the consistency of our model, D was
directly taken from the previous fit of M2 delivery kinetics,
a rather plausible constraint based on the very similar struc-
tures of the guest compounds and coordination polymer par-
ticles investigated in this work. As observed in Figure 6,
a good agreement was encountered between the experimen-
tal and fitted release profiles of M1 even under such an as-
sumption, which proves the validity of our treatment (b=
0.26, kd/ACTHNUTRGNEUNG
(C0ꢆR)=1.7ꢆ10ꢀ6 sꢀ1). From this we conclude that
most guest molecules in M1 nanoparticles (74%) are direct-
ly bound to the polymer matrix, which are therefore re-
leased by slow degradation of the material. Nevertheless,
a significant fraction of guest molecules (26%) are not coor-
dinated to cobalt ions despite presenting free catechol
groups, but they were physically encapsulated during the
formation of the particles. Accordingly, they are delivered
by a fast time-independent diffusion mechanism similar to
that encountered for M2 CPPs.
Figure 7. SEM images of M2 CPPs suspended at 378C in aqueous media
for (a) 0, (b) 5, (c) 26, and (d) 100 h. Scale bars are 500 nm.
Additional guest release experiments were performed at
608C aimed at investigating the temperature dependence of
the delivery processes in these materials (see Figure S4 in
the Supporting Information). In deep contrast to what had
been observed at 378C, no significant differences were
found between the release profiles measured for M1 and
M2 at this temperature. In both cases, complete delivery of
the fluorescence guests is observed at
but physically entrapped within the metal–organic matrix
should be preferentially delivered by fast diffusion process-
es. Based on the previous results obtained for M2 and analo-
gous [ZnACHTUNGTRENNUNG
(bix)] CPPs,[16] Equation (2) was derived to account
for such situation:
!
ꢁ5 h, revealing the occurrence of much
ꢀ
ꢁ
ꢀ
ꢀ
ꢁ ꢁꢁ
1
3
X
6
p2
1
n2
Dn2p2t
R2
kdt
ð1 ꢀ bÞC0R
faster release processes. This suggests
Mt ¼ M1 b 1 ꢀ
exp
ꢀ
þð1 ꢀ bÞ 1 ꢀ 1 ꢀ
ð2Þ
n¼1
that the degradation kinetics accelerate
enormously at 608C, which must
become at least comparable to guest
diffusion rates. As a matter of fact, we
The first term in this Equation corresponds to the Fickian
diffusion model already applied to M2, in which b is the
fraction of guest molecules that lie mechanically entrapped
within M1 particles. As previously discussed, this model as-
sumes that the diffusion-controlled release of guest mole-
cules takes place before significant degradation of the poly-
mer matrix occurs, which allows the particle radius and the
apparent guest diffusion constant to be considered time-in-
dependent. This assumption is not only supported by the be-
havior observed for M2, but also by the similar results ob-
tained when monitoring the degradation process of M1
nanoparticles at 378C in water media using SEM (see Fig-
ure S3 in the Supporting Information). The second term in
Equation (2) corresponds to an empirical model that has
been developed for degradation-controlled drug delivery
from spherical particles through surface erosion,[29] which is
indeed the degradation mechanism reported for analogous
expect the release profiles of M1 and M2 CPPs at these con-
ditions to be mainly governed by degradation processes,
which indicate that both the guest delivery kinetics and
mechanisms of these materials can be dramatically altered
by temperature control.
Conclusion
In this work we report a new rational approach to investi-
gate the relationship between guest encapsulation and re-
lease mechanisms for metal–organic nanoparticles. By ap-
propriate design of the guest compounds and particle forma-
tion conditions, two types of coordination polymer particles
were prepared that 1) are compositionally and structurally
equivalent, and 2) were loaded with the same fluorescent
guests using different encapsulation processes. As a result,
the release of their fluorescent cargo at physiological condi-
tions proceeds through distinct mechanisms that converge
upon increasing the temperature. Physically encapsulated
guest molecules are delivered by fast, time-independent dif-
fusion processes, whereas the release of coordinated guest
moieties is governed by slow particle degradation. This
[ZnACHTUNGTRENNUNG
(bix)] CPPs at physiological conditions.[16] In this expres-
sion (1ꢀb) is the fraction of guest molecules coordinated to
the metal centers in M1, kd is the surface erosion rate con-
stant, C0 is the total initial concentration of the guest in the
polymer matrix (7.2ꢆ10ꢀ4 % (w/w)) and R is the initial
radius of the nanoparticles.
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J. Hernando, F. Novio et al.
evaporated under vacuum. The crude product was purified by flash chro-
matography using hexanes and ethyl acetate (4:1, v/v) to afford 4a
(0.716 g, 100%) as a yellowish oil. 1H NMR (400 MHz, CDCl3, 258C,
TMS): d=9.87 (s, 1H), 7.55 (s, 2H), 5.31 (s, 2H), 5.23 (s, 2H), 3.66 (s,
3H), 3.52 (s, 3H), 1.45 ppm (s, 9H); 13C NMR (100 MHz, CDCl3, 258C,
TMS): d=191.5, 151.9, 150.4, 144.0, 131.5, 123.8, 114.5, 99.4, 95.4, 57.9,
56.6, 35.4, 30.3 ppm; IR (ATR): n˜ =3076.2, 2953.3, 2905.4, 2826.8, 1690.1,
1578.5 cmꢀ1; HRMS (ESI-QTOF): m/z calcd for C15H22NaO5: 305.1359;
found: 305.1356.
leads to remarkably different guest delivery profiles for the
CPPs prepared, which demonstrates that the kinetics of re-
lease can be selectively tuned up to many hours by appro-
priate choice of the mechanism of incorporation of the ther-
apeutic agent into the polymeric nanocarrier. This result
opens new venues for the future use of CPPs in medicine
owing to the feasibility of loading drugs into these carriers
by both mechanical entrapment and chemical binding to the
metal centers. The former encapsulation mechanism has
indeed already been demonstrated for anticancer drugs,[16]
whereas tethering of these molecules to coordinating ligands
could be attempted through functional groups that are read-
ily cleaved at physiological conditions (e.g., ketals[31]), thus
rendering the active form of the therapeutic agent after deg-
radation-induced release from the polymer particles. As
a result, controlling the ratio of coordinated versus physical-
ly entrapped drug molecules within CPPs would eventually
allow tailoring the release kinetics to meet the therapeutic
needs.
Synthesis of 4b: This compound was prepared according to ref. [22] with
some modifications. K2CO3 (6.95 g, 50.4 mmol) and N,N,N-tributyl-1-bu-
tanaminium iodide (270 mg, 0.73 mmol) were added to a solution of 3
(3.5 g, 16.8 mmol) in DMF (100 mL),. The reaction mixture was stirred
for 2 h at room temperature. After this time, Me2SO4 (3.2 mL,
33.6 mmol) was added drop-wise and the mixture was allowed to react
for 16 h. The resulting mixture was treated with water (100 mL) and the
aqueous layer was extracted four times with EtOAc (50 mL). The organic
extracts were dried with MgSO4 and the solvent evaporated under
vacuum to afford 4b (3.36 g, 90%) as
a
dark-green oil. 1H NMR
(400 MHz, CDCl3, 258C, TMS): d=9.91 (s, 1H), 7.48 (d, J=1.9 Hz, 1H),
7.38 (d, J=1.9 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 1.44 ppm (s, 9H).
Synthesis of 5a: (Triphenylphosphoranylidene)acetonitrile (2.070 g,
6.87 mmol) was added to a solution of 4a (1.559 g, 5.53 mmol) in toluene
(45 mL). The reaction mixture was heated at reflux for 12 h, after which
the solvent was evaporated under vacuum and the residue was purified
by flash chromatography using hexanes and ethyl acetate (6:1, v/v) to
afford a mixture of (E)- and (Z)-5a (1.621 g, 96%) as a brown oil with
a diastereomeric ratio of 2.3:1. 1H NMR (400 MHz, CDCl3, 258C, TMS):
d=7.57 (d, J=2.2 Hz, 1H), 7.47 (d, J=2.2 Hz, 1H), 7.32 (d, J=16.6 Hz,
1H), 7.16 (d, J=2.2 Hz, 1H), 7.06 (d, J=2.2 Hz, 1H), 7.03 (d, J=
12.0 Hz, 1H), 5.75 (d, J=16.6 Hz, 1H), 5.34 (d, J=12.0 Hz, 1H), 5.26 (s,
2H), 5.24 (s, 2H), 5.21 (s, 2H), 5.19 (s, 2H), 3.66 (s, 3H), 3.65 (s, 3H),
3.53 (s, 3H), 3.51 (s, 3H), 1.43 (s, 9H), 1.41 ppm (s, 9H); 13C NMR
(100 MHz, CDCl3, 258C, TMS): d=150.7, 150.5, 150.0 148.9, 148.7, 148.6,
143.9, 143.8, 128.6, 128.5, 122.5, 121.0, 118.6, 117.9, 115.0, 112.5, 99.3,
99.3, 95.5, 95.4, 94.8, 93.4, 57.9, 57.9, 56.5, 56.6, 35.5, 35.3, 30.4, 30.3 ppm;
IR (ATR): n˜ =3371.2, 2953.8, 2213.6, 1615.5, 1428.9 cmꢀ1; HRMS (ESI-
QTOF): m/z calcd for C15H22NaO5: 328.1519; found: 328.1519.
Experimental Section
Materials and characterization: All reactants and reagents were pur-
chased from Sigma–Aldrich and used as received. Solvents were pur-
chased from Scharlab and used as received. Dialysis bags were purchased
from Orange Scientific. Infrared spectra were recorded on a Bruker
Tensor 27 spectrometer equipped with a Golden Gate Single Reflection
Diamond attenuated total reflectance (ATR) accessory. High-resolution
mass analyses were performed on an ESI-QTOF Bruker Daltonics
micrOTOF-Q spectrometer. NMR spectra were recorded on a Bruker
1
ARX 400 (400 MHz for H NMR and 100 MHz for 13C NMR). The spec-
tra are given in d (ppm) using the signal of the residual non-deuterated
solvent molecules as reference. Absorption spectra were recorded on
a Hewlett Packard 8453 spectrophotometer. HPLC or spectroscopy qual-
ity solvents were used. Emission spectra were measured by means of
a custom-made spectrofluorimeter, in which a continuous wave (CW)
He–Ne Research Electro Optics (REO) laser (lexc =594 nm) was used as
excitation source and the emitted photons were detected in an Andor
ICCD camera coupled to a spectrograph. HPLC or spectroscopy quality
solvents were used. Fluorescence quantum yields were determined using
Nile Blue A in ethanol solution as reference (Ff =0.27).[32] SEM meas-
urements were registered on a HITACHI S-570 microscope (accelerating
voltage 0.5–30 kV). TEM measurements were carried out on a HITA-
CHI-7000 microscope operating at 125 kV.
Synthesis of 5b: Synthesized from 4b using the same procedure as for
5a. Yield=72% with a diastereomeric E/Z ratio of 4.8:1. 1H NMR
(400 MHz, CDCl3, 258C, TMS): d=7.53 (d, J=2.1 Hz, 1H), 7.34 (d, J=
16.5 Hz, 1H), 7.23 (d, J=2.1 Hz, 1H), 7.05 (d, J=12.1 Hz, 1H), 7.00 (d,
J=2.0 Hz, 1H), 6.88 (d, J=2.0 Hz, 1H), 5.75 (d, J=16.5 Hz, 1H, 4.39 (d,
J=12.1 Hz, 1H), 3.92 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H),
1.39 (s, 9H), 1.37 ppm (s, 9H); 13C NMR (100 MHz, CDCl3, 258C, TMS):
d=153.7, 153.4, 151.7, 151.2, 151.0, 149.2, 144.1, 143.7, 128.5, 128.4, 122.0,
119.9, 119.8, 118.6, 110.2, 108.6, 94.5, 92.9, 60.7, 60.7, 56.0, 56.0, 35.3, 35.3,
30.4, 30.4 ppm; IR (ATR): n˜ =2952.0, 2213.3, 1615.6, 1571.5, 1415.0,
1142.9, 1067.0, 1023.7 cmꢀ1
; HRMS (ESI-QTOF): m/z calcd for
C15H19NaNO2: 268.1308, found: 268.1309.
Synthesis of 4a: This compound was prepared according to ref. [21] with
some modifications.
Synthesis of 6a: A mixture of (E)- and (Z)-5a (1.442 g, 4.8 mmol) and
10% Pd/C (5:1, substrate/catalyst) in ethyl acetate (16 mL) was stirred at
room temperature under a hydrogen atmosphere for 24 h. Next, Pd/C
was filtered off and the solvent was removed in vacuo. The residue was
purified by flash chromatography using hexanes and ethyl acetate (3:1,
v/v) to afford 6a (1.003 g, 68%) as a brown oil. 1H NMR (400 MHz,
CDCl3, 258C, TMS): d=6.90 (d, J=2.1 Hz, 1H), 6.84 (d, J=2.1 Hz, 1H),
5.18 (s, 2H), 5.16 (s, 2H), 3.64 (s, 3H), 3.51 (s, 3H), 2.88 (t, J=7.4 Hz,
2H), 2.58 (t, J=7.4 Hz, 2H), 1.41 ppm (s, 9H); 13C NMR (100 MHz,
CDCl3, 258C, TMS): d=150.6, 145.0, 143.9, 133.0, 120.6, 119.2, 114.7,
99.1, 95.6, 57.6, 56.4, 35.3, 31.7, 30.6, 19.6 ppm; IR (ATR): n˜ =2952.2,
2904.6, 2826.2, 2374.0, 1602.7, 1433.6, 1154.6, 936.8 cmꢀ1; HRMS (ESI-
QTOF): m/z calcd for C17H25NaNO4: 330.1376; found: 330.1375.
Demethylation: A solution of BBr3 (4 mL of 1m) in CH2Cl2 was added
drop-wise into a solution of 3 (0.845 g, 4 mmol) in CH2Cl2 (30 mL)
cooled down in a liquid nitrogen bath. The reaction was allowed to pro-
ceed at room temperature for 2 h. The reaction mixture was then poured
into distilled water (40 mL) and the resulting aqueous layer was extracted
twice with CH2Cl2 (30 mL). The organic extracts were dried with MgSO4
and the solvent evaporated under vacuum to afford the demethylated
compound as a yellowish solid (0.698 g, 90%). This compound was used
in the next step without further purification.
Protection of the catechol: DIPEA (2.7 mL, 15.5 mmol), DMAP (30 mg,
0.22 mmol), and methoxymethyl bromide (0.65 mL, 8.02 mmol) were
added drop-wise into a solution of the above intermediate (0.492 g,
2.54 mmol) in CH2Cl2 (8 mL) cooled down in a water bath. The solution
was heated at reflux for 8 h. The reaction mixture was treated with water
(15 mL) and the resulting aqueous layer was extracted twice with CH2Cl2
(15 mL). The organic extracts were dried with MgSO4 and the solvent
Synthesis of 6b: Synthesized from 5b using the same procedure as for
6a. Yield=67%. 1H NMR (400 MHz, CDCl3, 258C, TMS): d=6.74 (d,
J=2.1 Hz, 1H), 6.69 (d, J=2.1 Hz, 1H), 3.86 (s, 6H), 2.90 (t, J=7.4 Hz,
2H), 2.60 (t, J=7.4 Hz, 2H), 1.37 ppm (s, 9H); 13C NMR (100 MHz,
CDCl3, 258C, TMS): d=171.5, 153.7, 148.0, 144.0, 133.0, 119.7, 119.0,
17514
www.chemeurj.org
ꢅ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 17508 – 17516

Coordination Polymer Nanoparticles
FULL PAPER
111.0, 60.8, 56.0, 35.0, 32.2, 30.9 ppm; IR (ATR): n˜ =2951.4, 2866.4,
were added. This mixture was heated at reflux for 1.5 h. Then, it was
cooled to room temperature and CH2Cl2 (5 mL) and a mixture of saturat-
ed NaCl (2 mL) and 3 droplets of HCl 35% were added. The resulting
organic layer was washed twice with saturated NaHCO3 (3 mL) and once
with saturated NaCl (3 mL). Next, it was dried with MgSO4 and the sol-
vent was removed in vacuo. Crude was purified by flash chromatography
2831.8, 2245.0, 1688.2, 1580.1, 1421.9, 1346.7, 1260.0, 1067.6, 1006.1 cmꢀ1
;
HRMS (ESI-QTOF): m/z calcd for C15H21NaNO2: 270.1465; found:
270.1465.
Synthesis of 7a: A solution of 6a (695 mg, 2.2 mmol) in anhydrous Et2O
(2 mL) was added drop-wise to
a suspension of LiAlH4 (298 mg,
using CH2Cl2 and MeOH (10:1, v/v) to afford
1 (87 mg, 45%) as
7.9 mmol) in anhydrous Et2O (2 mL) cooled down in a water bath. The
reaction mixture was then stirred at room temperature for 14 h under an
inert atmosphere. Next, the reaction mixture was cooled down to 08C
and quenched with NaOH 1m (15 mL). The resulting aqueous layer was
extracted with Et2O (15 mL) and CHCl3 (15 mL). The combined organic
extracts were dried with MgSO4 and the solvent removed in vacuo to
afford 7a (627 mg, 89%) as a yellowish oil. This product was used with-
a bluish-violet solid. 1H NMR (400 MHz, [D4]MeOD, 258C, TMS): d=
8.73 (d, J=8.1 Hz, 1H), 8.22 (d, J=8.1 Hz, 1H), 7.82 (t, J=7.5 Hz, 1H),
7.71 (t, J=7.5 Hz, 1H), 7.51 (s, 1H), 6.90 (s, 1H), 6.70 (s, 1H), 6.40 (s,
1H), 6.38 (s, 1H), 3.95 (t, J=6.2 Hz, 2H), 3.49 (q, J=7.2 Hz, 3H), 3.21
(m, 2H), 2.75 (t, J=6.2 Hz, 2H), 2.35 (m, 2H), 2.29 (s, 3H), 1.67 (qt, J=
7.2 Hz, 2H), 1.46 ppm (s, 9H); 13C NMR (100 MHz, [D4]MeOD, 258C,
TMS): d=172.9 158.2, 156.9, 152.5, 149.3, 145.7, 143.3, 136.8, 133.9, 132.9,
132.6, 132.5, 132.4, 132.3, 130.7, 129.0, 125.5, 124.5, 123.6, 118.2, 113.5,
94.5, 94.1, 41.9, 40.3, 39.8, 35.8, 34.0, 32.5, 30.1, 17.8, 14.2 ppm; IR
1
out further purification. H NMR (400 MHz, CDCl3 258C, TMS): d=6.85
(d, J=2.0 Hz, 1H), 6.80 (d, J=2.0 Hz, 1H), 5.17 (s, 2H), 5.16 (s, 2H),
3.64 (s, 3H), 3.50 (s, 3H), 2.73 (t, J=7.6 Hz, 2H), 2.58 (t, J=7.6 Hz, 2H),
1.74 (qt, J=7.6 Hz, 2H), 1.40 ppm (s, 9H); 13C NMR (100 MHz, CDCl3,
258C, TMS): d=150.2, 143.9, 143.3, 137.2, 120.6, 114.7, 99.1, 95.5, 57.6,
56.4, 42.1, 35.7, 35.2, 33.4, 30.7 ppm; IR (ATR): n˜ =3362.8, 2949.4, 1578.6,
(ATR): n˜ =3213.7, 3076.2, 2921.8, 2852.5, 1640.1, 1587.6, 1540.9, 1433.8,
1307.7, 1160.8 cmꢀ1; HRMS (ESI-QTOF): m/z calcd for C35H41N4O4
:
+
581.3122; found: 581.3124.
1431.9, 1076.7, 961.7 cmꢀ1
;
HRMS (ESI-QTOF): m/z calcd for
Synthesis of 2: Synthesized from 8b using the same procedure as for 1.
Yield=35%. 1H NMR (250 MHz, [D4]MeOD, 258C, TMS): d=8.70 (d,
J=8.1 Hz, 1H), 8.20 (d, J=8.1 Hz, 1H), 7.81 (t, J=7.6 Hz, 1H), 7.70 (t,
J=7.6 Hz, 1H), 7.48 (s, 1H), 6.91 (s, 1H), 6.70 (s, 1H), 6.51 (s, 1H), 6.49
(s, 1H), 4.56 (s, 2H), 3.97 (t, J=5.9 Hz, 2H), 3.74 (s, 3H), 3.70 (s, 3H),
3.49 (q, J=6.2 Hz, 2H), 3.22 (t, 2H, J=6.8 Hz, 2H), 2.77 (t, J=5.9 Hz,
2H), 2.39 (t, J=6.8 Hz, 2H), 2.28 (s, 3H), 1.68 (qt, J=6.8 Hz, 2H), 1.43–
1.23 ppm (m, 12H); 13C NMR (63 MHz, [D4]MeOD, 258C, TMS): d=
173.0, 158.2, 156.8, 154.3, 152.5, 149.2, 147.8, 143.6, 140.2, 137.4, 133.9,
132.9, 132.6, 132.2, 130.7, 129.0, 125.5, 124.5, 123.6, 119.4, 114.7, 112.1,
94.5, 60.7, 56.2, 41.9, 40.2, 39.8, 35.7, 34.9, 34.2, 33.0, 32.4, 31.9, 31.1 ppm;
IR (ATR): n˜ =2920.8, 2851.6, 1640.4, 1588.1, 1541.4, 1451.0, 1310.0,
C17H29NNaO4: 334.1989; found: 334.1979.
Synthesis of 7b: Synthesized from 6b using the same procedure as for
7a. Yield=76%. 1H NMR (400 MHz, [D4]MeOD, 258C, TMS): d=6.74
(d, J=2.0 Hz, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 2.70 (t, J=7.5 Hz, 2H),
2.58 (t, J=7.5 Hz, 2H), 1.79 (qt, J=7.5 Hz, 2H), 1.34 ppm (s, 9H);
13C NMR (100 MHz, [D4]MeOD, 258C, TMS): d=153.5, 146.9, 142.7,
136.8, 118.7, 111.3, 59.8, 55.3, 40.8, 34.8, 33.9, 33.3, 30.2 ppm; IR (ATR):
n˜ =3452.3, 2936.2, 1578.1, 1421.9, 1321.1, 1262.1, 1144.8, 1066.5,
1008.1 cmꢀ1; HRMS (ESI-QTOF): m/z calcd for C15H25NNaO2: 252.1958;
found: 252.1963.
Synthesis of 8a: A solution of 7a (956 mg, 3 mmol) in anhydrous CH2Cl2
(10 mL) was added to a solution of 3-(naphthalen-1-ylamino)propanoic
acid (646 mg, 3 mmol), 1-hydroxybenzotriazole (HOBt; 589 mg,
4.3 mmol), EDCI (760 mg, 3.9 mmol) and DIPEA (1.6 mL, 9.1 mmol) in
20 mL of anhydrous CH2Cl2,. The reaction mixture was stirred at room
temperature for 17 h. Then, it was washed twice with a solution of satu-
rated NaHCO3 (10 mL) and once with a solution of saturated NaCl
(10 mL). The organic layer was dried with MgSO4 and solvent was evapo-
rated under vacuum. The crude product was purified by flash chromatog-
raphy using hexanes and ethyl acetate (1:1, v/v) to afford 8a (482 mg,
31%) as a brown oil. 1H NMR (400 MHz, CDCl3, 258C, TMS): d=7.81
(d, J=8.1 Hz, 1H), 7.74 (d, J=7.6 Hz, 1H), 7.38–7.23 (m, 4H), 6.79 (d,
J=1.9 Hz, 1H), 6.4 (d, J=1.9 Hz, 1H), 6.58 (d, J=7.6 Hz, 1H), 6.04 (s,
1H), 5.16 (s, 2H), 5.11 (s, 2H), 3.63 (s, 3H), 3.54 (t, J=6.02 Hz, 2H),
3.46 (s, 3H), 3.24 (dd, J=13.1 Hz, J=6.7 Hz, 2H), 2.50 (m, 4H), 1.73 (qt,
J=7.6 Hz, 2H), 1.39 ppm (s, 9H); 13C NMR (100 MHz, CDCl3, 258C,
TMS): d=171.9, 150.1, 143.4, 143.1, 136.23, 134.4, 128.6, 126.5, 125.9,
124.9, 123.9, 120.4, 117.8, 114.5, 104.5, 99.0, 95.4, 57.6, 56.4, 40.4, 39.3,
35.3, 35.2, 33.3, 31.2, 30.7 ppm; IR (ATR): n˜ =3304.4, 2949.4, 1638.2,
1580.4, 1526.7, 1199.4, 1035.5, 961.9 cmꢀ1; HRMS (ESI-QTOF): m/z calcd
for C30H40N2NaO5: 531.2829; found: 531.2834.
1160.9, 1133.6, 1006.6 cmꢀ1
C37H44N4NaO4: 609.3435; found: 609.3435.
Synthesis of M0: To solution of di-tert-butylcathecol (107.2 mg,
0.48 mmol) and 1,4-bis(imidazol-1-ylmethyl)benzene (59.6 mg,
0.25 mmol) in EtOH (5 mL), an aqueous solution of [Co-
(CH3COO)2]·4H2O (1 mL, 61.7 mg, 0.24 mmol) was added drop-wise.
The mixture was stirred for 10 min and then the formation of nanoparti-
cles was induced by fast addition of miliQ H2O (25 mL). The excess
ligand was removed by centrifugation and the nanoparticles were washed
three times with H2O.
; HRMS (ESI-QTOF): m/z calcd for
a
AHCTUNGTRENNUNG
Synthesis of M1: An aqueous solution (4 mL) of [CoACTHNUTRGNE(UNG CH3COO)2]·4H2O
(121.4 mg, 0.49 mmol) were added drop-wise to a solution of 1 (5.5 mg,
9.5 mmol), di-tert-butylcathecol (211.5 mg, 0.95 mmol) and 1,4-bis(imida-
zol-1-ylmethyl)benzene (117.3 mg, 0.49 mmol) in EtOH (20 mL). The
mixture was stirred for 10 min and then the formation of nanoparticles
was induced by fast addition of miliQ H2O (100 mL). Ligand excess was
removed by centrifugation and the nanoparticles were washed with a mix-
ture of EtOH/H2O (v/v 1:5) until no red fluorescence was observed from
the supernatant solution.
Synthesis of M2: An aqueous solution (2 mL) of [CoACTHNUTRGNE(UNG CH3COO)2]·4H2O
Synthesis of 8b: Synthesized from 7b by using the same procedure as for
8a. Yield=35%. 1H NMR (400 MHz, [D4]MeOD, 258C, TMS): d=7.93
(d, J=8.1 Hz, 1H), 7.69 (d, J=7.9 Hz, 1H), 7.41–7.23 (m, 3H), 7.14 (d,
J=8.1 Hz, 1H), 6.69 (d, J=2.9 Hz, 1H), 6.69 (s, J=2.9 Hz, 1H), 6.65 (s,
1H), 6.60 (d, J=4.3 Hz, 2H), 3.76 (s, 3H), 3.69 (s, 3H), 3.54 (t, J=
6.60 Hz, 2H), 3.17 (t, J=7.00 Hz, 3H), 2.59 (t, J=6.60 Hz, 2H), 2.53–
2.45 (m, 4H), 1.73 (qt, J=7.6 Hz, 2H), 1.31 ppm (s, 9H); 13C NMR
(101 MHz, [D4]MeOD, 258C, TMS): d=174.6, 154.3, 147.7, 144.7, 143.5,
137.6, 135.8, 129.3, 127.6, 126.6, 125.4, 125.1, 121.7, 119.5, 118.1, 112.1,
105.1, 60.7, 56.1, 41.6, 40.0, 36.3, 35.8, 34.2, 32.3, 31.1 ppm; IR (ATR): n˜ =
2919.5, 2478.6, 2065.58, 1627.1, 1577.7, 1450.4, 1420.8, 1143.8,
(68.9 mg, 0.28 mmol) was added drop-wise to a solution of 2 (3.1 mg,
5.1 mmol), di-tert-butylcathecol (120 mg, 0.53 mmol) and 1,4-bis(imidazol-
1-ylmethyl)benzene (65 mg, 0.27 mmol) in EtOH (10 mL). The mixture
was stirred for 10 min and then the formation of the nanoparticles was in-
duced by fast addition of miliQ H2O (50 mL). The excess ligand was re-
moved by centrifugation and the nanoparticles were washed with a mix-
ture of EtOH/H2O (v/v 1:5) until no red fluorescence was observed from
the supernatant solution.
Guest release experiments: A dialysis bag (MWCO: 3500) containing M1
or M2 (ca. ꢁ3 mgmLꢀ1) dispersed in phosphate buffered saline solution
(PBS; pH 7.4) was placed into a solution of PBS (150 mL, pH 7.4; dialy-
sate) at 378C under light stirring. To determine the increase in the con-
centration of 1 or 2 diffused through the dialysis bag, aliquots of the ex-
ternal PBS solution (0.5 mL) were taken from the dialysate at prefixed
times and diluted in MeOH (2 mL), and each aliquot was analyzed by
fluorescence spectroscopy. The solid material remaining in the dialysis
1067.9 cmꢀ1
;
HRMS (ESI-QTOF): m/z calcd for C28H36N2NaO3:
449.2799; found: 449.2804.
Synthesis of 1: To a solution of N-ethyl-5-hydroxy-2-methyl-4-nitrosoben-
zenaminium chloride (72 mg, 0.4 mmol) in MeOH (1 mL) cooled down
in a water bath and under an inert atmosphere, a solution of 8a (170 mg,
0.33 mmol) in degassed MeOH (1 mL) and a 3 droplets of HCl 35%
Chem. Eur. J. 2013, 19, 17508 – 17516
ꢅ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
17515

J. Hernando, F. Novio et al.
bag after 100 h was dissolved in methanol and characterized by using ab-
sorption spectroscopy.
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Acknowledgements
We acknowledge the financial support of the “Ministerio de Economꢀa y
Competividad” (MINECO) through projects MAT2012–38318-C03–02,
MAT2012–38319-C02–01, CTQ2012–30853 and CTQ2010–15380. L.A.-F.
thanks the “Universitat Autꢂnoma de Barcelona” for a pre-doctoral
grant. F.N. thanks the “Ministerio de Economꢀa y Competitividad” for
a JdC (JCI-2011–09239) post-doctoral grant.
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Received: July 9, 2013
Published online: November 20, 2013
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