A new targeting agent for the selective drug delivery of nanocarriers for treating neuroblastoma

Article abstract of DOI:10.1039/c5tb00287g

Novel targeting agents against neuroblastoma based on the meta-iodobenzylguanidine (MIBG) moiety were synthesized and biologically evaluated for nanocarrier vectorization. These compounds have been anchored on the surface of drug loaded mesoporous silica nanocarriers, resulting in the improved cellular uptake in tumoral cells. Neuroblastoma (NB) is the most frequent extracranial pediatric tumor. Advanced forms of the disease (metastatic and/or refractory) have a dismal prognosis despite the combination of chemotherapy, radiotherapy, surgery and bone narrow transplants. These treatments carry severe side effects and, in some cases, compromise the life of the patient. MIBG has been widely applied in the medical diagnosis of NB due to its affinity for tumor cells through the norepinephrine transporter (NET), which is expressed in 90% of NB tumors. The exclusive accumulation of MIBG in neuroblastoma has been widely studied; however, its properties have been never exploited as a targeting agent in nanocarrier drug delivery systems. Several structural analogues of MIBG have been prepared and attached on the surface of nanocarriers. Their selective internalization has been tested against human neuroblastoma cells, which show, in the best case, cellular uptake four times higher than that of the naked nanosystem. Furthermore, in vivo experiments showed preferential and selective accumulation and retention of the targeted nanosystem comparing with the naked and only PEGylated counterpart systems. This novel nanosystem could be easily applicable to all kinds of drug delivery nanocarriers, providing a universal tool for neuroblastoma chemotherapies that is superior to classical approaches through a novel nanosystem exclusively designed to target this terrible malignancy.

Full text of DOI:10.1039/c5tb00287g

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ARTICLE
Journal of Materials Chemistry B
VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C5TB00287G
New targeting agent in selective drug delivery
nanocarriers for treating neuroblastoma.
Cite this: DOI: 10.1039/x0xx00000x
Gonzalo Villaverde,a Alejandro Baeza,a,* Gustavo J. Melen,b Arantzazu Alfranca,c
Manuel Ramirezb and Maria Vallet-Regía,*
Received 00th January 2012,
Accepted 00th January 2012
Novel targeting agents based on meta-Iodobenzylguanidine (MIBG) moiety against neuroblastoma were
synthetized and biologically evaluated for nanocarrier vectorization. These compounds have been
anchored on the surface of drug loaded mesoporous silica nanocarriers causing improved cellular uptake
in tumoral cells. Neuroblastoma (NB) is the most frequent extracranial pediatric tumor. Advanced forms
of the disease (metastatic and/or refractory) have a dismal prognosis despite the combination of
chemotherapy, radiotherapy, surgery and bone narrow transplant. These treatments carry severe side
effects and, in some cases, compromise the patient life. MIBG has been widely applied in medical
diagnosis of NB due to its affinity for the tumor cells through the norepinephrine transporter (NET),
which is expressed in 90 % of NB. The exclusive accumulation of MIBG in neuroblastoma has been
widely studied; however, its properties have been never exploited as targeting agent in nanocarrier drug
delivery systems. Several structural analogues of MIBG have been prepared and attached on the
nanocarrier surface. Their selective internalization has been tested against human neuroblastoma cells
showing, in the best case, four times higher cellular uptake versus the naked nanosystem. Furthermore,
in vivo experiments showed preferential and selective accumulation and retention of targeted
nanosystem comparing with the naked and the just PEGylated counterpart systems. This novel
nanosystem could be easily applicable for all kind of drug delivery nanocarriers, allowing a universal tool
for neuroblastoma chemotherapies in the way to step down the classical approaches providing a
newfangled nanosystem exclusively designed against this terrible malignancy.
DOI: 10.1039/x0xx00000x
www.rsc.org/
selective active targeting agents have become into one of the
main challenges in nanomedicine.
Introduction
In recent years, the use of nanocarriers as selective drug
transporters and theranostic proposes has attached much
attention in nanomedicine applied to oncology. One of the
biggest extraꢀbenefits of using drug nanocarriers against cancer
is that they can overcome some of the major limitations of
conventional chemotherapy such as solubility issues associated
with the frequently hydrophobic nature of cytotoxic drugs,
short circulation lifetime and typical lack of selectivity.1 These
problems are already addressed through the use of high doses in
the treatment. This fact promotes the damage in healthy tissues
causing the typical side effects associated to conventional
chemotherapy.
In order to improve the selectivity of the nanomedicines
specific targeting agents have been designed along the years.2
These targeted nanosystems are able to transport the cytotoxic
compounds specifically to the target cells, and to release their
payload once there. This fact causes the destruction of the
malignant cells while minimizing the collateral damages in the
surrounding tissue.3 For this reason, the development of new
During last years, efficient ligandꢀreceptor active targeting
motifs such as RGD4,5,6 and folate,7,8,9,10 are commonly grafted
on many different kinds of nanosystems surface for
vectorization proposes. Furthermore, new specific moieties
with multiple functionality using several types of cellꢀ
penetrating
peptides,11,12,13
or
vasculature
targeting
antibodies,14,15,16 have been recently reported.
The tumoral tissue accumulation characteristics of the
nanoparticles is mainly possible due a specific passive targeting
effect, which is based on the characteristic tumoral blood vessel
architecture. These vessels present large pores and fenestrations
of diameters up to few hundred nanometers. When the
circulating nanoparticles reach a tumoral site, they pass through
these pores being accumulated during longer times within the
zone.17 Further, tumoral tissue also exhibits a poor lymphatic
drainage. Both characteristics constitute the wellꢀknown
enhanced permeation and retention effect (EPR) and is one of
the main advantages constituting the basic of the goal standard
of the use of nanoparticles in oncology.18 However, not all the
cells which form a tumoral mass are malignant due the great
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DOI: 10.1039/C5TB00287G
heterogeneity of these tissues that contain a large number of three main interactions: van der Walls interactions produced by
healthy supportive cells.19 The functionalization of the several hydrophobic moieties from NET protein with the
nanoparticle surface with molecules (targeting groups) able to aromatic ring of the norepinephrine structure; several weak
be specifically recognized by the tumoral cells constitutes the polar interactions through hydrogen bonds, and, the key one;
so called active targeting. The presence of these groups is the ionic interaction between carboxylate group from Asp75
essential for enhancing the therapeutic efficacy of the from NET pocket and the protonated amine group from
nanomedicines.
norepinephrine.27 Not by chance, MIBG is structurally close to
In this work, we are interested in developing effective norepinephrine, containing an aromatic core backbone
nanocarriers that can be applied for neuroblastoma (NB) responsible for the hydrophobic interactions and benzyl
therapy. NB has being onto one of the challenging problems in guanidine unit (pKa =12) able for proton capture which form a
pediatric oncology. This disease is the most frequent similar cationic specie than the norepinephrine amine group.
extracranial pediatric tumor which presents a dismal prognosis (Figure 1).
despite the combination of chemotherapy, radiotherapy, surgery
and bone narrow transplant.20 Thus, the development of
nanocarriers able to detect and destroy only the diseased cells
may constitute a significant improvement in the treatment
efficacy.21
As mentioned above there is a wide range of studies which
analyze the vectorization effect of generalist cancer targeting
agents in nanoparticles. Bulky targeting agents such as,
proteins, large peptides, aptamers, antibodies, etc.
, are
commonly used. However, these agents have several possible
binding interaction points, and are usually suitable to interact
with unspecific proteins. Otherwise, small molecules with less
binding spots may go unnoticed in this media. This fact could
make the specific small moieties tolerable to body fluids
maintaining its recognizing abilities only with the desired
target. In special cases, some types of diseased cells have
intrinsic and particular receptors that make it possible to
improve the differentiation to each other. Thus, following this
philosophy and looking for selective internalization in NB cells,
metaꢀIodobenzylguanidine (MIBG) looks the most suitable
candidate for tuning the nanocarrier surface with vectorization
proposes.22 MIBG targeting properties have been applied in
conventional medical diagnosis of NB in the last decades due to
its affinity by the norepinephrine transporter (NET) which is
Figure 1. Structural equivalence between Norepinephrine, MIBG, MABG and
MAmBG analogues. attachment on MSN and NET uptake.
The possibility to perform structural modifications of MIBG in
order to study and improve its uptake selectivity for
norepinephrine transporter (NET), has been widely
discussed.28,29,30 Several groups have been incorporated all sorts
of variations in paraꢀ position of the aromatic ring and even
the aromatic ring have been replaced by a naphthyl or
expressed in 90 % of NB tumors.23
Because its high
specificity for NB and other neuroendocrine tumors; MIBG
effectiveness as tumorꢀtargeted vehicle has been also applied in
the therapeutic treatment by directed radiation using radioactive
iodine (131I).24 Additionally, nonꢀradioactive derivate has shown
anticancer activity by itself.25 The conjugation of MIBG with
antitumoral drugs not only enhances their selectivity against
tumoral cells but also improves their cytotoxic capacity through
synergic effect caused by tumor selective acidification by
enhancement of glycolytic flux.26 This synergy may convert
nonꢀradioactive MIBG analogues into important targeting agent
within the nanomedicine arena.
MIBGꢀNET interaction may able to bring the nanosystem to
NB cell internalization excluding healthy cells from the
process. NET binding pocket is naturally designed for the
recognition of small molecules which means that the size of the
targeting motifs is limited. The binding mechanism is based on
dibenzoazepine moiety without
a significative loss of
selectivity for the NET receptor.31 However, some authors
suggest that the substitution of hydrogen in position 5 of the
benzene ring is unfavourable for binding NET.32 On the other
hand, the absence of the iodine atom in metaꢀposition does not
affect the uptake/selectivity properties.
These previous structureꢀactivity relationship studies (SAR)
guided us to design, as a first approach, the synthesis of meta
ꢀ
aminobenzyl guanidine (MABG) as MIBG analogue. MABG
represents one of the simplest structurally accessible
approximations to MIBG which keep the same aromatic ring
and the guanidine recognition moiety in the same position. On
the other hand, substitution of the iodine atom with amino
group provides the grafting point to the nanoparticle surface
through amide formation (Figure 1).
2 | J. Name., 2012, 00, 1-3
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VJieowuArrtnicalelONnlainme e
DOI: 10.1039/C5TB00287G
Scheme 1. Synthesis of MAmBG analogues.
PPh3: Triphenylphosphine, DIAD: Diisopropyl azodicarboxylate, THF: Tetrahydrofuran DSS: Suberic acid bis(N-hydroxysuccinimide ester), Tol:Toluene, DCM:
Dichloromethane, DIC: N,N'-methanediylidenebis(propan-2-amine), NHS: N-hydroxysuccinimide, NEt3: Triethylamine.
The new analogue maintains the intrinsic properties from the response and selectivity of the administered nanomedicines
original MIBG and makes the structure easy functionalizable against NB cells.38,39,40
for the further grafting step on MSN surface.
In order to facilitate the monitorization of the cellular uptake
Furthermore, the use of this amine/amide metaꢀsubstitution has process, MSNs were synthesized carrying fluorescein moieties
been reported in tumor detection by Wang et al.33 with covalently attached within the silica matrix following a
promising results in terms of selective accumulation in diseased variation of Stöber41 and van Blaaderen42 method. At the same
tissues. However, this type of targeting agents have been never time, amino groups were introduced on the external surface of
applied on nanocarriers for drug delivery.
the nanoparticles through this delayed coꢀcondensation
procedure yielding the naked material MSNꢀFꢀNH2.
The XRD patterns for MSNꢀFꢀNH2 (See S.I., section B.1)
shows three of the four lowꢀangle reflections in the 2θ range 2ꢀ
8º typical of a hexagonal array as (100), (110), (200). These
measurements suggest that the pore order was not affected by
the introduction of fluorescein and the amino functionalization.
A reduction of the peak intensity observed in (210) was not
interpreted as a crystallinity lost but rather to a reduction in the
Xꢀray scattering contrast in high functionalized material.
Nitrogen absorption measurements showed a value for the total
specific surface of 1192 m2/g and the adsorption/desorption
isotherm is typical of nonꢀfilled mesoporous structure with a
pore diameter of 2.5 nm. The size distribution profile of the
nanoparticles was determined by dynamic light scattering
(DLS) showing an average diameter of 190 nm which was
confirmed by scanning electronic microscopy (SEM).
Results and discussion
Herein, we report for the first time the use of novel MIBG
analogues attached to mesoporous silica nanoparticles (MSN)
used as nanocarrier model. These nanosystems are able to
exclusively drive cytotoxic drugs to NB cells with minor
impact for healthy supportive cells which are usually present in
tumor mass. MSN have been widely used for nanomedicine
proposes due to their excellent properties as easy
functionalization, synthetic versatility, high loading drug
capacity and economically low cost starting materials, among
others.34,35,36,37 Furthermore, the external surface of these
carriers can be easy decorated with targeting motifs which
improve the selectivity and enhance the uptake rate in the
malignant cells through receptor–ligand interactions.
The attachment of MIBG based targeting compounds on MSN
loaded with cytotoxic drugs could improve both the cytotoxic
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Scheme 2. Targeting attachment on MSN. a) THF, corresponding analogue, 40ºC, overnight b) DCM, TFA, reflux, 72h.
The presence of amino groups was confirmed by recognized by the immune system leading to their clearance.
thermogravimetric analysis (around 8% in weight of organic System PEGylation increases the circulation time of
material), FTIR and crossꢀpolarization magicꢀangle spinning nanoparticles that is essential for improving EPR effect
13C CP MAS solid state NMR. These amino groups will be efficacy. However, using longer PEG chains could affect the
employed as anchoring point of the targeting agents through selectivity by shielding the targeting moiety reducing the
amide formation reactions. Additionally, the free amino groups possibilities of active uptake process.45
provided a positive charge (+40 mV) on the particle surface The first MAmBG analogue, compound (3), was synthetized
measured by zeta potential which enhanced the colloidal through two consecutive steps by treatment of MABGꢀBOC (1)
stability of these particles.
with succinic anhydride affording isolated compound (2) in
MAmBGꢀtype targeting agents were synthetized from a quantitative yield, following by transformation of the resulted
common starting material MABGꢀBOC (1), which was acid in the corresponding activated succinimide ester (3).
obtained from 3ꢀaminobenzylalcohol and N,Nꢀbis
(
tert
ꢀ
Compound (4) was obtained by direct reaction of MABGꢀBOC
butyloxycarbonyl) guanidine. The reaction follows a variation (1) with DSS in presence of NEt3. Long linked analogues were
of a Mitsunobu process previously reported for guanidination synthetized following the same methodology using
of benzyl alcohols and affords the desired analogue with good commercially available PEG derivatives with double activated
yields.43,28 Then, MABGꢀBOC (1) was functionalized through acid groups in both ends of the chain (for more synthetic
amide group formation with the desired linkers which contain information see S.I section A.1). The amounts of the MAmBG
an activated acid group at the end of their chain yielding the motif in each long linked compound were determined by UV
MAmBG derivates (Scheme 1).
spectrometry (See SI section A.2). The amount of
This methodology provides a versatile pathway to explore the benzylguanidine found in MAmBGꢀPEG2000ꢀNHS (5) and
influence of the linker chain length in the cellular MAmBGꢀPEG10000ꢀNHS (6) was 7.7% w/w and 1.2% w/w,
internalization. Two different categories of MAmBG analogues respectively. These data indicate that the reactions were
have been produced. One based on the use of short linkers resulted in around 50 % of yield in both cases, which was
between the grafting point and the targeting moiety corroborated by 1HꢀNMR. The presence of the second activated
(compounds 3 and 4, Scheme 1). And second, based on the use acid group in the other side of the chain after condensation
of longer linkers composed by polyethyleneglycol (PEG) process was checked to ensure the posterior grafting process.
chains of 2 and 10KDa of molecular weight, respectively (See S.I. section A.1).
(compounds 5 and 6, Scheme 1). Besides to separate the Deprotection of the BOC groups in all the synthetized
targeting agent far away from the surface which improves its analogues, makes the resulting compounds poorly soluble in
interaction with the NET receptor, PEG linker may enhance the most of solvents, except methanol or water. This fact, convert
circulation time of the nanocarrier inside the blood stream their isolation in a tedious process. Additionally, it was
through opsonisation abolition.44 This effect can be explained necessary to employ forced conditions refluxing the media
due steric interference from PEG chains that make the opsonins during 72h to obtain acceptable deprotection yields. Even then,
not able to be attached on the particle surface. Opsonization is unprotected MABG was isolated, however succinic derivate
the first step which allows that the nanoparticles will be compound (2) lead its undesired metaꢀpyrrolidine derivate after
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ARTICLE
DOI: 10.1039/C5TB00287G
deprotection treatment through acid catalyzed cyclization. (See
S.I, section A.1, MPyBG). Therefore, we decided to anchor the
BOC protected MAmBG analogues on the silica nanoparticles
and once there, to carried out the BOC deprotection in order to
make easier the BOC residues elimination and abolishing
possible secondary products.
Functionalization of nanoparticles always means
a new
challenge; one of the parameters more sticky in the synthesis of
new hybrid material is to control the amount of organic material
covalently immobilized on the surface. Parameters as stearic
hindrance and charges, in both, nanoparticles surface and
organic material, are key parameters for optimal
immobilization. For this reason, optimization of the ratio of the
MAmBG analogues in front of amine groups available for
reaction in the surface is a must in each case. Acid activated
MAmBG analogues were covalently grafted on MSNꢀFꢀNH2 by
addition of the corresponding compound to a dispersion of
nanoparticles in THF at 40ºC for 24 h, affording the materials
MSNꢀ(n)ꢀBOC. Posterior BOC deprotection treatment with
TFA/DCM yields the materials MSNꢀFꢀMAmBG with the
MAmBG targeting moiety available to bind with NET (Scheme
2). The amount of organic material covalently attached on the
MSN surface was determined by TGA resulting in
Figure 2. Following the synthesis of MSN-F-MAmBG-3 by 13C CP MAS-NMR solid
state spectra. a) MSN-F-NH2 b) MAmBG (2) in chloroform c) MSN-F-MAmBG-3-
BOC d) MSN-F-MAmBG-3.
The successful grafting process was confirmed by the
apparition of the typical signals of MAmBG at approximately
175 ppm (C=O) and 165 ppm (C=N), and undefined broad
signals in down field corresponding with aromatic ring from
benzylguanidine (Figure 2).
approximately 18.7
%
(MSNꢀFꢀMAmBGꢀ3ꢀBOC), 11
%
(MSNꢀFꢀMAmBGꢀ4ꢀBOC), 16.5
%
(MSNꢀFꢀMAmBGꢀ5ꢀ
On the other hand, the spectra in the case of long linked
analogues MSNꢀFꢀMAmBGꢀ5ꢀBOC and MSNꢀFꢀMAmBGꢀ6ꢀ
BOC shows a dominant signal at 72 ppm (CH2) from PEG
moiety which hide the rest of signals except the classical
APTES signals which appears in all of them (See S.I, section
B2 for more spectral data). Indeed, the PEG signal clearly
indicates the presence of the PEG chains and, as consequence,
the success of the functionalization process. Unfortunately, the
tertꢀbutyl signals from BOC protective group overlap with the
APTES moiety and others aliphatic signals (CH2 from linker
moieties) appearing as broad peaks. This fact makes impossible
to determine the deprotection grade of the guanidine groups
after the treatment with TFA by NMR. However, zeta potential
measurements showed changes in the surface charge before and
after the deprotection step, as show Table 1.
In general, the surface charge becomes more positive after BOC
deprotection which is in agreement with the presence of free
guanidine groups. Finally, the organic material determination
by TGA reveals, in all cases, that the amount of organic
material after functionalization is higher and it suffers a slight
decrease after BOC deprotection. According with the DLS
measurements, the functionalization process barely alters the
nanoparticle size which is a very important aspect in drug
delivery for antitumoral therapy (Table 1 and Figure 3a). The
preservation of the particle size and the mesoporous structure of
each material were confirmed by high resolution SEM and
TEM (Figure 3b and 3c)
BOC) and 20 % (MSNꢀFꢀMAmBGꢀ6ꢀBOC). The amount of
targeting agents incorporated in each case was determined
assuming a 1:1 equivalence in the case of the short linked
analogues (3) and (4) and 7.7% w/w and 1.2% w/w for (5) and
(6) respectively (previously calculated by UV spectrometry
determination). In all cases, it should be considered that the
blank material (MSNꢀFꢀNH2) has 8.2% w/w of organic
material. Thus, the estimation of the targeting moiety
functionalization in deprotected materials was 10.5 % and 3 %
w/w for MSNꢀFꢀMAmBGꢀ3 and MSNꢀFꢀMAmBGꢀ4
respectively, and 0.64 % and 0.14 % w/w for MSNꢀFꢀ
MAmBGꢀ5 and MSNꢀFꢀMAmBGꢀ6 as maximum values.
Obviously, the amount of MAmBG in PEGylated materials
were lower than in the short linked. This fact may be attributed
to both; the reaction conditions and the stearic hindrance of the
PEG chains. However, less amount of targeting motif does not
mean a minor selectivity, because the targeting agent may be
more exposed in this case being more accessible for the
interaction with the NET pocket.
The presence of functional groups characteristic of MAmBG
derivatives grafted on the nanoparticle surface was confirmed
by crossꢀpolarization magicꢀangle spinning 13CꢀNMR solid
state. The spectra for each group of the shorter linked analogues
were confronted with their counterparts in liquid state before
immobilization, for both protected and unprotected materials as
Figure 2 shows for MSNꢀFꢀMAmBGꢀ3. 13C CP MAS solid
state NMR spectra of short linked material MSNꢀFꢀMAmBGꢀ3,
both BOC protected and deprotected are totally recognizable in
view of the corresponding derivatives before functionalization.
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Usually, there are two differenced protein layers; the hard
corona, formed by small size proteins, which is the closer and
binder with the nanoparticle surface, and, the soft corona,
formed by higher size proteins weakly bounded.48,49 The
formation of the protein shell not only could diminish the
internalization properties of the nanoparticles but also may
hinder the targeting agent properties by protein unspecific
interactions with undesired receptors. Thus, both; previous
characterization of the nanosystem in serum multiproteic media
and internalization assays in tumor environment emulation are
essential. For this aim, the four targeted nanovehicles, as well
as nonꢀtargeted nanomaterial MSNꢀFꢀNH2 were incubated in
multiproteic serum Hyclone (36 g/L of estimated protein
content) during 1.5 hours, centrifuged, washed and reꢀdispersed
in water to be analyzed by DLS and zꢀpotential. This assay
allow to characterize the species could be formed in following
internalization experiments which carried out in the serum
media as emulation of real environment.
Table 1. Characterization of MSNꢀFꢀMAmBGꢀn nanomaterials.
MSN-F-
MAmBG-
3-BOC
MSN-F-
MAmBG-
3
MSN-F-
MAmBG-
4-BOC
MSN-F-
MAmBG-
4
MSN-F-
NH2
Z.Pot
(mV)
Size
(nm)
O.M
%
+40
190
+36.6
176.5
+29.0
190
+24.1
214
+35.6
231
8.2
15.5
11.3
18.7
18.2
(w/w)
MSN-F-
MAmBG-
5-BOC
MSN-F-
MAmBG-
5
MSN-F-
MAmBG-
6-BOC
MSN-F-
MAmBG-
6
Z.Pot
(mV)
Size
(nm)
O.M
%
+19
208
+24.2
237
+12
214
+35.3
226
16.4
12.5
20.22
15.5
(w/w)
O.M :Organic Material.
Zꢀpotential and size were analysed after the protein enriched
serum exposition. Results presented in Table 2 shows how the
presence of proteins in media clearly modifies the physicoꢀ
chemical properties of the surface in almost same magnitude
due protein corona formation in all cases.
In general, protein corona makes the nanosystems negatively
charged and bulkier than its nonꢀserum treated counterparts.
Table 2. Characterization of MSNꢀFꢀMAmBGꢀn in serum media.
MSNꢀ
FꢀNH2
MSNꢀFꢀ
MAmBGꢀ3
MSNꢀFꢀ
MAmBGꢀ4
MSNꢀFꢀ
MAmBGꢀ5
MSNꢀFꢀ
MAmBGꢀ6
Figure 3. a) Size determination by DLS measurement. b) SEM micrographs of
MSN-F-MAmBG-3 scale bars correspond to 100 nm. c) TEM micrographs of MSN-
F-MAmBG-5 scale bars correspond to 100 nm.
ꢀ23.4
+40
ꢀ22.1
ꢀ22.5
ꢀ20.8
ꢀ21.9
Finally, XRD show that pore order was maintained after both
functionalization and BOC deprotection processes in all
materials. (See S.I sections B.2 and B.3). As mentioned above,
there is a wide range of studies which analyze the vectorization
effect of cancer targeting agents in nanoparticles. The
nanosystems studied by in vitro assays, show selective avidity
and internalization behaviour for tumor cells excluding the
healthy ones. However, this effect is attenuated in replications
of biological environment as multiproteic serum through the
formation of protein corona in the nanoparticle surface.46,47 The
interaction between the surface of the nanosystem and human
body fluids is wholly influential in the targeting abilities of the
nanosystems. The formation of protein corona in their surface
changes the original physicalꢀchemical properties of the entire
system. This fact must be taken into account in order to perform
a strict evaluation of the targeting effect of the grafted
derivates.
+29.0
+35.6
+24.2
+35.3
342
190
385
190
400
231
342
237
310
226
The data afforded equals the properties of all nanosystems,
then, the possible differences of internalization to each other in
neuroblastoma cells can be only explained by ligandꢀreceptor
interaction, MAmBGꢀNET in this case.
In order to study the efficacy of the novel targeting agents to
enhance the internalization capacity of the nanocarriers, human
NB cells were incubated in the presence of each nanomaterial
conjugated with the corresponding targeting agent at a fixed
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Figure 4. a) Comparative internalization studies in NB cells between all nanosystems synthetized. b) Competitive assays for MSN-F-MAmBG-5 in NB cells with MIBG
and MABG. c) Inhibition assays with ouabain for MSN-F-MAmBG-5 (100 μg/mL) in NB cells.
concentration of 100 µg/mL during 2 hours in multiproteic MAmBG moiety grafted in the nanoparticles surface for NB
serum culture. After this time, the amounts of cells which had cells internalization proposes. In contrast, the comparison of the
uptaked nanoparticles were determined by flow cytometry better derivates of both groups (succinic linked MSNꢀFꢀ
measuring the fluorescence at 488 nm as FITC+% representing MAmBGꢀ3 from short linked group and 2 KDa PEG derivate
the percentage of cells with fluorescence. MSNꢀFꢀNH2 was MSNꢀFꢀMAmBGꢀ5 from long linked analogues), reveals
chosen as the negative targeted control, not only by the fact of obvious alternative effects besides the targeting load effect.
not being functionalized with targeting agent but also because MSNꢀFꢀMAmBGꢀ3 nanoparticles have showed less potential
is strongly positively charged (+40 mV) without serum internalization comparing with 2 KDa PEGylated system MSNꢀ
treatment. The positively charged nanoparticles have shown the FꢀMAmBGꢀ5. However the MSNꢀFꢀMAmBGꢀ3 is grafted with
greatest efficiency in cellꢀmembrane penetration and cellular more than twelve times of targeting agent. This behaviour could
internalization.50 Thus, the use of the MSNꢀFꢀNH2 as negative be connected with its more external configuration and
control; give a realistic idea of internalization capability of the disposition of the targeting moiety for interaction with NET
targeted systems competing with a nanosystem with high pocket thanks to PEG linker, making the cellular uptake process
internalization abilities.
more effective, even in presence of proteins in media. This fact
Results showed a notable internalization improvement in all improves the selective internalization for NB cells. The external
targeted materials comparing with the nonꢀtargeted control disposition effect presented by 2 KDa PEGylated material, is
MSNꢀFꢀNH2. Short linked derivates, MSNꢀFꢀMAmBGꢀ3 and hidden for 10KDa PEGylated material MSNꢀFꢀMAmBGꢀ5.
MSNꢀFꢀMAmBGꢀ4 reflected differential behaviour regarding The increase in the length chain involves a dramatic fall of
the internalization within NB cells.
internalization caused by the possible shielding of the targeting
The succinic derivate MSNꢀFꢀMAmBGꢀ3 internalization is moiety from the 10 KDa PEG combined with the low load of
notably higher than its longer linked colleague suberic acid recognition moiety in this material surface.
derivate MSNꢀFꢀMAmBGꢀ4. This behaviour is fully expected With the aim of confirming the ability of MAmBG derivates as
because the difference to each other in terms of percentage of targeting agents for nanosystems through ligandꢀreceptor
targeting unit grafted in the surface (MSNꢀFꢀMAmBGꢀ3, interaction, and, considering that, MIBG and norepinephrine
10.5% w/w versus MSNꢀFꢀMAmBGꢀ4, 3% w/w).
are active in the specific uptakeꢀ1 mechanism which takes place
Similar effect has been found for long linker derivates, where through the active energyꢀconsuming process by NET.
the 2 KDa PEGylated derivate MSNꢀFꢀMAmBGꢀ5 internalizes Inhibition and competitive in vitro experiments for abolishing
four times more than blank MSNꢀFꢀNH2 and possess 0.64 % the active mechanism of the NET internalization were carried
w/w of targeting groups in contrast with 0.14 % w/w for 10 out for the derivate MSNꢀFꢀMAmBGꢀ5.
KDa PEG derivate MSNꢀFꢀMAmBGꢀ5 which internalizes Uptakeꢀ1 can be inhibited by saturation of receptor and also
almost two times more than the nonꢀtargeted material MSNꢀFꢀ through active mechanism blocker. Internalization for the MSN
NH2.
was measured with the presence of the commercially available
Thus, comparing between derivates with proximally properties, MIBG (500 ꢁM) and the derivate MABG (500 ꢁM) in parallel
the amount of effective targeting is a key parameter. This fact, assays. These analogues can saturate the cell receptors
clearly demonstrates the effect and the influence of targeting hampering the nanoparticle uptake directed by NETꢀgrafted
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Figure 5. Confocal microscopy pictures. a) and b) Neuroblastoma cells 2h hours of incubation with 100μg/mL of MSN-F-MAmBG-5, blue DAPI for nuclei tracker,
washed with glycine/NaCl buffer for eliminating non-internalized nanoparticles in the membrane cell surface. c) Neuroblastoma cells 2h hours of incubation with
100μg/mL of MSN-F-MAmBG-5, blue DAPI for nuclei tracker, phalloidin as actin filaments marker and trypan blue for quenching non-internalized nanoparticles. d)
Neuroblastoma cells 2h hours of incubation with 100μg/mL of MSN-F-NH2, blue DAPI for nuclei tracker, phalloidin as actin filaments marker and trypan blue for
quenching non-internalized nanoparticles.
MAmBG interactions. Internalization in NB cells data were compared to the internalization results without any blocker in
confronted with afforded without any competitive NET media culture measured as mean fluorescence indexes. All
receptor in the media. FITC+% measurements reflected a poor these results suggest that the presence of MAmBG targeting
decrease of cells with internalized nanoparticles. However, the agents in the nanosystems has an important influence in the
mean fluorescence indexes (MFI) data, representing intensity of selective internalization process through NET mediated upꢀ
fluorescence per cell, showed an interesting average of 34 % taking. This selective NETꢀMAmBG interaction is part of the
and 14 % of nanoparticles internalization decrease in the NB total internalization mechanism of the whole system in NB
cells within MIBG and MABG presence respectively in MSNꢀ cells.
FꢀMAmBGꢀ5 concentrations of 50 ꢁg/mL and 100 ꢁg/mL. In order to confirm the internalization of MAmBGꢀnanoparticle
Thus, the nanocarrier internalization is clearly trending down in systems in NB cells, confocal microscopy was applied. The
excess of MIBG and MABG through saturation of NET.
internalization assay was carried out with the derivate MSNꢀFꢀ
Furthermore, additional inhibition assays have been carried out MAmBGꢀ5, which exposed the best internalization properties
abolishing the NaꢀKꢀATPase dependent internalization process in NB cells and naked nanosystem MSNꢀNH2 as control.
of MIBG through NET uptakeꢀ1. For this aim, ouabain (1 (Figure 5) The accumulation of fluorescent nanoparticles MSNꢀ
mM)22,51 was added to NB cells culture as blocker of energy FꢀMAmBGꢀ5 (Figure 5 a) b) and c)) in the NB cell cytosol is
dependent mechanism before nanoparticles treatment. clearly evident in contrast with the negative control MSNꢀNH2
Internalization of targeted nanovehicles was reduced in 28 % (Figure 5 d)), confirming the higher selectivity and avidity of
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Figure 6. In vivo homing of nanoparticles to neuroblastoma. a), Upper panels: images show fluorescence signal (F) of nanoparticles (P) 72h after i.v.
injection. Lower panels: photograps show luminescent emission (L) of tumors (T) derived from s.c. injected NB-Luc. I: saline; II: MSN-Cy7-NH2; III: MSN-
Cy7-PEG; IV: MSN-Cy7-MAmBG-5. Note co-localization of both fluorescent (nanoparticles) and luminescent (tumor) specifically in MSN-Cy7-MAmBG-5
(IV). Unspecific auto-fluorescence signal can be observed in all groups. b), Graphic shows mean + SD of fluorescence signal in each group 24, 48 and
72h after injection of nanoparticles. II, III and IV as in A. Fluorescent signal in saline-injected mice was set as background in all cases. *p<0.5 and
**p<0.1 vs. group IV.
the targeted system. The capacity to deliver cytotoxic drugs to easy visualization of the tumoral mass by luminescence
NB cells was evaluated as a proof of concept using the best measurements.
material (MSNꢀFꢀMAmBGꢀ5) and doxorubicin hydrochloride The results clearly show that the nanoparticles functionalized
as drug model. Increased concentration of drug loaded with the complete targeting system (group IV) present a
nanoparticles were administered to NB and mesenchymal cells, significative accumulation within the tumoral mass at 48 and 72
selected as negative control due to their lack of NET receptor hours after the administration, which is clearly visible by the
and their natural presence within the tumoral mass. The results strong fluorescent signal located in the tumoral region. In
show that more than 20% NB cells died at nanoparticle contrast, the naked and PEGylated particles (groups II and III,
concentration of 50 µg/mL in only
2 hours whereas respectively) barely exhibited fluorescence in this zone (Figure
mechenchymal cells were barely affected (See SI, Figure Sꢀ45). 6a). It is important to remark that the nanoparticles
Finally, in vivo experiments employing neuroblastoma murine functionalized with the MABG analogue remain in the tumoral
models have been carried out in order to test the real tissue after 72 hours whereas the naked and PEGylated ones are
effectiveness of the targeted nanocarrier which presented the rapidly cleared from the zone (Figure 6b). These findings
best performance in the previous test. In order to monitor the confirm the efficacy of this targeting agent due to improve not
tumor accumulation by IVIS imaging, the nanocarrier was only the selectivity of the nanocarriers but also enhance their
labelled with Cy7 instead of fluorescein because this retention within the diseased tissue.
fluorescent probe is excited in the near infrared region whereas
the biological tissues are transparent to this wavelength. This
system corresponds to group IV (MSNꢀCy7ꢀMAmBGꢀ5). A
saline solution (Group I), naked nanoparticles (Group II, MSNꢀ
Cy7ꢀNH2) and PEGylated nanoparticles without targeting
agents (Group III, MSNꢀCy7ꢀPEG) were used as controls. A
xenograft murine model which presents stably expressing
luciferase neuroblastoma tumors is used in order to allow an
Experimental
Materials and Methods
Following compounds were purchased from SigmaꢀAldrich
Inc.:N,N'ꢀmethanediylidenebis(propanꢀ2ꢀamine) (DIC), Nꢀ
hydroxysuccinimide (NHS), N,Nꢀbis(tertꢀbutyloxycarbonyl)
guanidine, (3ꢀaminophenyl)methanol, Triphenylphosphine
(TPP), Diisopropyl azodicarboxylate (DIAD), Suberic acid
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bis(Nꢀhydroxysuccinimide ester (DSS), O,O′ꢀBis[2ꢀ(Nꢀ (Solution 2) was added dropwise with gentle stirring under a
Succinimidylꢀsuccinylamino)ethyl]polyethylene glycol 2KDa solution formed by 1g of CTAB, 480 mL of water and 3.5 mL
and
10KDa,
doxorubicin
hydrochloride
(European of NaOH 2M at 80 ºC . The final reaction mixture was stirred at
Pharmacopoeia), mꢀIodobenzylguanidine hemisulfate salt, 80ºC during 2 hours. Yellow solid was filtered and washed with
aminopropyltriethoxysilane (APTES), ammonium nitrate, water and EtOH.
cetyltrimethylammonium
bromide
(CTAB),
tetraethyl For template removing, solid was suspended in a solution of
orthosilicate (TEOS). All other chemicals (absolute ethanol, NH4NO3 (10 mg/mL) of EtOH/H2O in reflux during 48h.
acetone, ethyl acetate heptane, dry solvents etc.) were of the The solid was filtered, washed EtOH and dried in reduce
best quality commercially available and they have been pressure oven at 40ºC during 24h. 1.2 g of product was
employed as received.
afforded. The solid was functionalized with 6.22x10ꢀ4 mol/gr of
amine groups.52
Characterization techniques.
Synthesis of functionalized nanoparticles MSN-F-MAmBG-n
Powder Xꢀray diffraction (XRD) experiments were performed
with a Philips X’Pert diffractometer equipped with Cu Kα Compounds (3), (4), (5) and (6) were previously synthetized
radiation (wavelength 1.5406 Å). XRD patterns were collected following Scheme 1. and fully characterized. (See S.I. Section
in the 2θ range between 0.6º and 8 with a step size of 0.02º and A.1)
counting time of 5 s per step. Fourier transform infrared For the synthesis of MSNꢀFꢀMAmBGꢀn 100 mg of solid
spectroscopy (FTIR) in a Thermo Nicolet nexus equipped with starting material MSNꢀFꢀNH2 (6.22x10ꢀ4mol/gr primary amines
a Goldengate attenuated total reflectance device. The textural ready to react) were dried under reduce pressure at 80ºC placed
properties of the materials were determined by nitrogen in a Schlenk during 3 hours. The solid was lead to room
sorption porosimetry by using a Micromeritics ASAP 2010. To temperature and suspended in 20 mL of dried THF, still under
perform the N2 measurements, the samples were previously inert atmosphere, the dispersion was sonicated during 10
degassed under vacuum for 24 h at room temperature. minutes.
Thermogravimetry analysis (TGA) were performed in a Perkin NH2(solid)/acid activated derivate 1:1), 25 mg (
Elmer Pyris Diamond TG/DTA analyzer, with 5 ºC/min heating NH2(solid)/acid activated derivate 1: 0.20), 64 mg (
A
solution of 35mg
(
3
), 38 mg
(
4
5
6
)
(Ratio
) (Ratio
) (Ratio
ramps, from room temperature to 600 ºC. The hydrodynamic NH2(solid)/acid activated derivate 1: 0.10) respectively in THF
size of mesoporous nanoparticles was measured by means of a (3 mL) was added dropwise with gentle stirring. The ratios of
Zetasizer Nano ZS (Malvern Instruments) equipped with a 633 functionalization process were chosen in order to abolish
nm “red” laser. Mass spectra were acquired with a Voyager saturation of the nanoparticles surface in each case. When the
DEꢀSTR Biospectrometry MALDIꢀTOF mass spectrometer. addition was finalized the resulted dispersion was warmed to 40
Scanning electron microscopy (SEM) analyses were made on a ºC overnight.
JEOL 6400ꢀLINK AN10000 microscope (Electron Microscopy The resulted yellow solid was washed with THF (3x15mL) and
Centre, UCM). The samples underwent Au metallization EtOH (3x15) and characterized.
previous to observation. Liquid NMR experiments were made
in a Bruker AV 250MHz. Solid phase NMR MAS experiments
General proceed for nanomaterial guanidine deprotection
were carried out in a Avance 400MHz. GPC analysis were MSNꢀFꢀMAmBGꢀ3ꢀBOC, MSNꢀFꢀMAmBGꢀ4ꢀBOC, MSNꢀFꢀ
made in a Waters 2695 with Refractive Index Detector Waters MAmBGꢀ5ꢀBOC, MSNꢀFꢀMAmBGꢀ6ꢀBOC (100 mg in each
2414.
case) were dried under reduce pressure at 80ºC placed in a
Schlenk during 3 hours. The solids were lead at room
temperature and 10 mL of deprotection cocktail (TFA/DCM
Calculation procedures.
The surface area was determined using the BrunauerꢀEmmettꢀ 1:1) was added dropwise under inert atmosphere. The mixture
Teller (BET) method and the pore volume, Vpore (cm3·gꢀ1), was was sonicated during 10 minutes and raised to 65ºC with gentle
estimated from the amount of N2 adsorbed at a relative pressure stirring 72 hours.
around 0.99. The pore size distribution between 0.5 and 40 nm Solid were filtered and washed with DCM, brine and EtOH.
was calculated from the desorption branch of the isotherm by Finally, the new materials were dried under vacuum at 40 ºC
means of the BarrettꢀJoynerꢀHalenda (BJH) method. The and characterized.
mesopore size, Øpore (nm), was determined from the maximum
of the pore size distribution curve.
Internalization assay
Neuroblastoma cell line NB1691ꢀluc was seeded in 24ꢀwell
plates at 5000 cells/cm2. Cells were exposed to 100 ꢁg/ml of
MSNꢀFꢀNH2, MSNꢀFꢀMAmBGꢀ3, MSNꢀFꢀMAmBGꢀ4, MSNꢀ
Synthesis of starting amino functionalized nanoparticles MSN-F-
NH2
FITC (1mg) and APTES (2,2 µL) were solved in the minimum FꢀMAmBGꢀ5 or MSNꢀFꢀMAmBGꢀ6 for 2 hours at 37ºC, 5%
volume of EtOH, the mixture was stirred at room temperature CO2 and 95% humidity. Cells were then washed and harvested.
for 2 hours under inert atmosphere. The resulting mixture The number of FITC+ cells was evaluated by flow cytometry
(Solution1) was added under a mixture of TEOS (4.5 mL) and using the FACSCanto II flow cytometer and the FACSDiva
APTES (0.5 mL) also in inert conditions. The resulting mixture software v6.1.2 (BD Biosciences, San Jose, Ca.). Percentages
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DOI: 10.1039/C5TB00287G
of FITC+ populations are shown as the mean of two Investigación y Bienestar Animal ISCIII; procedure CBA PA
independent replicates.
25_2011ꢀv2).
Competition and Inhibition assays
Conclusions
Following the same set up as for the internalization assay 50
ꢁg/ml and 100 ꢁg/ml MSNꢀFꢀMAmBGꢀ5 uptake was
challenged in the presence of 500 ꢁM MIBG or derivative
MABG. Cells were preꢀincubated during 15 min with either
competitor. After a medium changed containing both MSNꢀFꢀ
AmBGꢀ5 and MIBG or MABG the incubation proceeded for 2
more hours. The inhibition assay with 1mM ouabain was
performed as published elsewhere.22 In both cases, the number
of FITC+ cells was evaluated by flow cytometry as in the
previous experiment. Mean fluorescence indexes and
percentage of FITC+ populations are shown as the mean of two
independent replicates.
In conclusion, different novel targeting agents based on MIBG
structure have been synthesized and grafted on the surface of
mesoporous silica nanoparticles, employed as carrier model.
This type of structure has been chosen due to the wellꢀknown
affinity of MIBG for the norepinephrine transporter which is
highly expressed by neuroblastoma cells. The efficacy of each
targeting agent to induce an enhanced nanocarrier cellular
uptake have been tested using neuroblastoma cell line NB1691ꢀ
luc, showing in the best case an increase of up to four times.
This system was able to work in a serum multiꢀproteic media,
keeping the targeting properties of the grafted agent coexisting
with protein corona effect. Furthermore, drug loaded targeted
nanoparticles showed high exclusive cytotoxic effect in NB
cells. Finally, the in vivo efficacy of these carriers was tested
using murine neuroblastoma models. The results indicate that
the surface functionalization with these targeting agents
provokes a significative higher tumoral tissue accumulation and
retention in comparison with the nonꢀdecorated particles. This
type of selective targeting agents could be easily applied to
different nanoparticle platforms paving the way for the
application of novel nanomedicines for the treatment of
neuroblastoma.
Confocal Imaging
For confocal analysis cells were seeded on glass chamber slides
(LabTek II, Nunc, Rochester, NY) and incubated with 100
ꢁg/ml MSNꢀFꢀAmBGꢀ5 or MSNꢀFꢀNH2 for
2
hours.
Internalization was stopped by washing cells with iceꢀcold PBS
followed by two acid washes with 250mM NaCl, 100mM
glycine pH 2.5 to remove any adsorbed particles. Cells were
then fixed in 4% PFA, stained with Texas RedꢀX phalloidin
(Molecular Probes, Eugene, OR) and mounted in DAPIꢀ
containing Fluoroshield (Sigma, St. Louis, MO). Images were
capture via a 63×/1.4 oilꢀimmersion objective on the Leica SP5
confocal microscope and the Leica LAS AF software.
Acknowledgements
This work was supported by the Ministerio de Economía y
Competitividad, through project MAT2012ꢀ35556, CSO2010ꢀ
11384ꢀE (Agening Network of Excellence) and CIBERꢀBBN
and ECO Foundation through project Smart4NB. We also thank
the Xꢀray Diffraction C.A.I. and the National Electron
Microscopy Center, UCM.
In vivo homing/biodistribution assays.
Human neuroblastoma NBꢀ1691 cells (kindly provided by Dr.
Antonio Pérez, Hospital La Paz, Madrid) were transduced with
pHRSINꢀCSꢀLucꢀIRESꢀemGFPꢀWdlNotI lentiviral vector
(kindly provided by Dr. Antonio Rodríguez, Universidad
Autónoma de Madrid) in order to generate a stably expressing
luciferase neuroblastoma cell line (NBꢀLuc). 2.5x106 NBꢀLuc
cells were subcutaneously injected into the left flank of
Notes and references
a
Dpto. Química Inorgánica y Bioinorgánica. Instituto de Investigación
Sanitaria Hospital, 12 de Octubre i+12.UCM. Centro de Investigación
Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina
(CIBERꢀBBN). Madrid, Spain.
NOD.CgꢀPrkdcscid
Il2rgtm1Wjl/SzJ
(NSG;
Jackson)
l of saline
immunodeficient mice. Three weeks later, 200
ꢀ
b
Dpto. de Hematología y Oncología Pediátricas, Hospital Infantil
(group I) or 1mg of either Cy7ꢀlabeled bare nanoparticles
(group II), PEGꢀcovered nanoparticles (group III) or MIBGꢀ
PEGꢀcovered nanoparticles (group IV), were inoculated i.v.
into these mice. In vivo biodistribution of Cy7ꢀlabeled
nanoparticles was monitored through fluorescent image
acquisition in an IVIS Lumina XRMS (Perkin Elmer) at 24, 48
and 72h after nanoparticles injection. The presence and
localization of NBꢀLucꢀderived tumors was confirmed through
the detection of luminescent signal after i.p. injection of
1.25mg/mice DꢀLuciferin (Perkin Elmer). Image analysis was
performed with Living Image® 4.4 software (Perkin Elmer).
Animal studies were in accordance with the guidelines of the
EU on animal care (2010/63/EU) and approved by the
institutional ethics committee (Comité de Ética de la
Universitario Niño Jesús, Madrid, Spain.
Servicio de Inmunología. Hospital Universitario de La Princesa. Diego
de León, 62. 28006 Madrid, Spain.
c
Electronic Supplementary Information (ESI) available:
Experimental proceeds for synthesis of the organic precursors.
Aditional spectra information; FTIR, NMR, XRD, BET, size
distributions. UV determination of targeting units for pegylated
derivates. See DOI: 10.1039/b000000x/
1
2
3
S. Marchesan and M. Prato, Acs Med. Chem. Lett., 2013, 4, 147–149.
N. Ž. Knežević and J.ꢀO. Durand, Chempluschem, 2015, 80, 26–36.
I. Brigger, C. Dubernet and P. Couvreur, Adv. Drug Deliv. Rev.
2012, 64, 24–36.
,
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 11

Journal of Materials Chemistry B
Page 12 of 13
VJieowuArrtnicalelONnlainme e
ARTICLE
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4
5
6
7
8
9
I.ꢀJ. Fang, I. I. Slowing, K. C.ꢀW. Wu, V. S.ꢀY. Lin and B. G. 31 D. Hadrich, F. Berthold, E. Steckhan and H. Bönisch, J. Med. Chem.
Trewyn, Chemistry, 2012, 18, 7787–92. 1999, 42, 3101–8.
J. Sun, D.ꢀH. Kim, Y. Guo, Z. Teng, Y. Li, L. Zheng, Z. Zhang, A. C. 32 G. Vaidyanathan, S. Shankar, D. J. Affleck, K. Alston, J. Norman, P.
,
Larson and G. Lu, J. Mater. Chem. B, 2015,
G.ꢀF. Luo, W.ꢀH. Chen, Y. Liu, J. Zhang, S.ꢀX. Cheng, R.ꢀX. Zhuo
and X.ꢀZ. Zhang, J. Mater. Chem. B, 2013, , 5723–5732.
3
, 1049–1058.
Welsh, H. LeGrand and M. R. Zalutsky, Bioorg. Med. Chem., 2004,
12, 1649–56.
1
33 W. Wang, J. Shohet, M. Mawad and S. Ke, US Pat. App. 13/638,060,
Z. Tang, D. Li, H. Sun, X. Guo, Y. Chen and S. Zhou, Biomaterials
2014, 35, 8015–27.
,
2011.
34 M. ValletꢀRegi, A. Rámila, R. P. del Real and J. PérezꢀPariente,
C. Chen, J. Ke, X. E. Zhou, W. Yi, J. S. Brunzelle, J. Li, E.ꢀL. Yong,
H. E. Xu and K. Melcher, Nature, 2013, 500, 486–9.
Chem. Mater., 2001, 13, 308–311.
35 A. Baeza, M. Colilla and M. ValletꢀRegí, Expert Opin. Drug Deliv.
,
,
J. Wang, P. P. Gao, X. X. Yang, T. T. Wang, J. Wang and C. Z.
2014, 1–19.
Huang, J. Mater. Chem. B, 2014,
2, 4379.
36 V. Mamaeva, C. Sahlgren and M. Lindén, Adv. Drug Deliv. Rev.
2013, 65, 689–702.
10 Q. Zhang, X. Wang, P.ꢀZ. Li, K. T. Nguyen, X.ꢀJ. Wang, Z. Luo, H.
Zhang, N. S. Tan and Y. Zhao, Adv. Funct. Mater., 2014, 24, 2450– 37 M. ValletꢀRegí and E. RuizꢀHernández, Adv. Mater., 2011, 23, 5177–
2461. 218.
11 C. H. Wu, Y. P. Chen, S. H. Wu, Y. Hung, C. Y. Mou and R. P. 38 Z. Li, J. C. Barnes, A. Bosoy, J. F. Stoddart and J. I. Zink, Chem.
Cheng, ACS Appl. Mater. Interfaces, 2013, , 12244–12248. Soc. Rev., 2012, 41, 2590–605.
12 J. Li, F. Liu, Q. Shao, Y. Min, M. Costa, E. K. L. Yeow and B. Xing, 39 C. Sanchez, C. Boissiere, S. Cassaignon, C. Chaneac, O. Durupthy,
5
Adv. Healthc. Mater., 2014,
3
, 1230–1239.
M. Faustini, D. Grosso, L. Nicole, D. Portehault, F. Ribot, L. Rozes
and C. Sassoye, 2014.
13 Y. P. Chen, C. T. Chen, Y. Hung, C. M. Chou, T. P. Liu, M. R.
Liang, C. T. Chen and C. Y. Mou, J. Am. Chem. Soc., 2013, 135
,
40 A. Baeza, E. Guisasola, A. TorresꢀPardo, J. M. GonzálezꢀCalbet, G.
J. Melen, M. Ramirez and M. ValletꢀRegí, Adv. Funct. Mater., 2014,
24, 4625–4633.
1516–1523.
14 F. Chen, H. Hong, Y. Zhang, H. F. Valdovinos, S. Shi, G. S. Kwon,
C. P. Theuer, T. E. Barnhart and W. Cai, ACS Nano, 2013,
7, 9027– 41 W. E. R. N. E. R. Stober, 1968, 69, 62–69.
9039.
42 A. Van Blaaderen and A. Vrij, 1992, 81, 2921–2931.
15 F. Chen, T. R. Nayak, S. Goel, H. F. Valdovinos, H. Hong, C. P. 43 D. S. Dodd and A. P. Kozikowski, 1994, 35, 977–980.
Theuer, T. E. Barnhart and W. Cai, Mol. Pharm., 2014, 105 44 H. Meng, M. Xue, T. Xia, Z. Ji, D. Y. Tarn, J. I. Zink and A. E. Nel,
16 S. Veeranarayanan, A. C. Poulose, M. S. Mohamed, S. H. Varghese, ACS Nano, 2011, , 4131–44.
Y. Nagaoka, Y. Yoshida, T. Maekawa and D. S. Kumar, Small, 2012, 45 Q. He, J. Zhang, J. Shi, Z. Zhu, L. Zhang, W. Bu, L. Guo and Y.
.
5
8, 3476–3489.
Chen, Biomaterials, 2010, 31, 1085–1092.
17 F. Danhier, O. Feron and V. Preat, J. Control. Release, 2010, 148
,
46 A. Salvati, A. S. Pitek, M. P. Monopoli, K. Prapainop, F. B.
Bombelli, D. R. Hristov, P. M. Kelly, C. Åberg, E. Mahon and K. a
135–146.
18 S. Sengupta and A. Kulkarni, ACS Nano, 2013,
7
, 2878–2882.
Dawson, Nat. Nanotechnol., 2013,
8
, 137–43.
, 79–80.
19 D. Hanahan and R. a Weinberg, Cell, 2011, 144, 646–74.
20 J. M. Maris, N. Engl. J. Med., 2010, 362, 2202–2211.
21 E. Ruoslahti, S. N. Bhatia and M. J. Sailor, J. Cell Biol., 2010, 188
759–768.
47 R. Gaspar, Nat. Nanotechnol., 2013,
8
48 P. Verderio, S. Avvakumova, G. Alessio, M. Bellini, M. Colombo, E.
Galbiati, S. Mazzucchelli, J. P. Avila, B. Santini and D. Prosperi,
,
Adv. Healthc. Mater., 2014, 3, 957–76.
22 R. J. Mairs, M. N. Gaze and A. Barrett, 1991, 295, 293–295.
49 J. Townson, Y. Lin and J. Agola, J. Am. Chem. Soc., 2013, 135,
23 K. K. Matthay, R. E. George and A. L. Yu, Clin. Cancer Res., 2012,
18, 2740–53.
16030–16033.
50 A. Verma and F. Stellacci, Small, 2010, 6, 12–21.
24 J. P. Howard, J. M. Maris, L. S. Kersun, J. P. Huberty, S.ꢀC. Cheng, 51 K. A. Streby, N. Shah, M. A. Ranalli, A. Kunkler and T. P. Cripe,
R. a Hawkins and K. K. Matthay, Pediatr. Blood Cancer, 2005, 44
,
2015, 5–11.
232–9.
52 Y. Zhang and Y. Chen, IET Nanobiotechnol., 2012, 6, 76–80.
25 S. Ekelund, P. Nygren and R. Larsson, Biochem. Pharmacol., 2001,
61, 1183–93.
26 a Kuin, M. Aalders, M. Lamfers, D. J. van Zuidam, M. Essers, J. H.
Beijnen and L. a Smets, Br. J. Cancer, 1999, 79, 793–801.
27 A. Schlessinger, E. Geier, H. Fan, J. J. Irwin, B. K. Shoichet, K. M.
Giacomini and A. Sali, Proc. Natl. Acad. Sci. U. S. A., 2011, 108
,
15810–5.
28 G. Vaidyanathan, S. Shankar and M. R. Zalutsky, 2001, 786–797.
29 G. Vaidyanathan, S. Shankar, D. J. Affleck, P. C. Welsh, S. K. Slade
and M. R. Zalutsky, 2001, 798–806.
30 G. Vaidyanathan, P. C. Welsh, K. C. Vitorello, S. Snyder, H. S.
Friedman and M. R. Zalutsky, Eur. J. Nucl. Med. Mol. Imaging
2004, 31, 1362–70.
,
12 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5TB00287G
Novel MIBG analogues as targeting agents for neuroblastoma nanomedicines.
254x190mm (96 x 96 DPI)