Dendrimersomes: A new vesicular nano-platform for MR-molecular imaging applications

Article abstract of DOI:10.1039/c3cc49584a

A new class of nanovesicles formed by the self-assembly of amphiphilic Janus dendrimers, dendrimersomes, loaded with hydrophilic or amphiphilic magnetic resonance imaging chelates shows promising properties as a novel, efficient and versatile nanoplatform for biomedical imaging.

Full text of DOI:10.1039/c3cc49584a

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Dendrimersomes: a new vesicular nano-platform
for MR-molecular imaging applications†
Cite this: Chem. Commun., 2014,
50, 3453
Miriam Filippi,a Jonathan Martinelli,b Gilberto Mulas,ac Marisa Ferraretto,a
Eline Teirlinck,a Mauro Botta,b Lorenzo Tei*b and Enzo Terreno*ad
Received 18th December 2013,
Accepted 10th February 2014
DOI: 10.1039/c3cc49584a
www.rsc.org/chemcomm
A new class of nanovesicles formed by the self-assembly of amphiphilic stability and mechanical resistance,10 while liposomes show excel-
Janus dendrimers, dendrimersomes, loaded with hydrophilic or lent biocompatibility and marked ability to interact with natural
amphiphilic magnetic resonance imaging chelates shows promising membranes, thus providing an interesting opportunity to investigate
properties as a novel, efficient and versatile nanoplatform for many biological mechanisms at a cellular level.11 Although dendri-
biomedical imaging.
mersomes should combine all these properties,3–5 their potential in
biological scenarios is still completely unexplored. In particular,
Dendrimers are synthetic molecules with highly branched archi- their use may hold promise for molecular imaging applications.12
tectures characterized by a tree-like appearance.1 Although most Inspired by the versatility of liposomes that can encapsulate
dendrimers exhibit a roughly spherical symmetry, a new structural hydrophilic probes into the aqueous core or embed amphiphilic
class of dendrimers has been recently obtained by joining together chelates into the phospholipid bilayer, this work aims at assessing the
two chemically distinct dendritic building blocks.2 The resulting ability of dendrimersomes to load hydrophilic and/or amphiphilic
molecule is a double-faced compound with a bifunctional char- Gd-complexes as MRI probes. The clinically approved agent
acter, named Janus dendrimer, constituted by diverse peripheral Gadoteridol (GdHPDO3A) was selected as a hydrophilic probe,
groups on opposite sides. In particular, amphiphilic Janus whilst two approaches were pursued for the incorporation of
dendrimers possessing one hydrophobic and one hydrophilic amphiphilic agents into the membrane vesicles: (i) the synthesis
moiety are reminiscent of the typical structure of phospholipids, of a novel Janus dendrimer covalently conjugated to a Gd-chelate
thus favouring their self-assembly in water into supramolecular (JD1–GdDOTAMAC6), and (ii) the incorporation into the bilayer
aggregates of different morphologies, including nanosized bilayered of the lipophilic Gd-DOTAMA(C18)2 complex (Scheme 1).
vesicles named dendrimersomes.3–5 Until now, different vesicular
A large number of Janus dendrimers based on different
systems such as polymersomes and liposomes have been extensively libraries of amphiphilic molecules have been reported to form
investigated for biomedical applications, mainly as nanocarriers for self-assembled architectures of variable sizes and morphologies.3
drug delivery or as diagnostic or theranostic agents.6–9 The main
advantage associated with the use of polymersomes is their high
a Department of Molecular Biotechnology and Health Sciences,
Molecular Imaging Center, University of Torino, Via Nizza 52, 10126 Torino, Italy.
E-mail: enzo.terreno@unito.it
b
`
Dipartimento di Scienze e Innovazione Tecnologica, Universita del Piemonte
Orientale ‘‘Amedeo Avogadro’’, Viale T. Michel 11, 15121, Alessandria, Italy.
E-mail: lorenzo.tei@unipmn.it
c Porto Conte Ricerche S.r.l., SP 55 Porto Conte/Capo Caccia Km 8.400,
Loc Tramariglio 07041 Alghero (SS), Italy
d Center for Preclinical Imaging, University of Torino, Via Ribes 5,
10010 Colleretto Giacosa (TO), Italy. Tel: +39 0125538942/522
† Electronic supplementary information (ESI) available: Synthesis of
a Gd-
DOTAMAC6–Janus-dendrimer (JD1–GdDOTAMAC6), the technical protocol fol-
lowed for the dendrimersome assembly, DLS, stability tests in buffer, experi-
mental information about relaxivity measurements and 1H NMRD acquisition,
results about quenching of the relaxivity and internalization of fluorescent
probes. See DOI: 10.1039/c3cc49584a
Scheme 1 Janus dendrimer JD1 and its GdDOTAMAC6 conjugate structures
(top), Gd-DOTAMA(C18 2 and Gadoteridol (bottom).
)
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Among them, JD1 (Scheme 1) was chosen as it forms dendrimer-
somes with a size similar to that of liposomes (ca. 120 nm) and an
excellent polydispersity index (PDI = 0.06).3 This is based on a
pentaerythritol core with a hydrophobic moiety consisting of two
3,5-bis-dodecyl substituted benzoyl ethers and a hydrophilic segment
made of one polyester dendritic structure terminating with eight
hydroxyl groups. While JD1 was synthesized as described elsewhere,13
a novel synthetic procedure was followed to covalently conjugate a
Fig. 2 1H NMRD profiles measured at 298 K of Gadoteridol (K) and
dendrimersomes encapsulating Gadoteridol (J) (left), and dendrimersomes
GdDOTA–monoamide chelate (H4DOTA
= 1,4,7,10-tetraaza-
cyclododecane-1,4,7,10-tetraacetic acid) to the dendrimer. In
particular, one hydroxyl group of JD1 was esterified by reaction
with one equivalent of a t-butyl protected bifunctional chelating agent
bearing an activated carboxyl function (DOTAMA(tBu)3-
incorporating GdDOTAMA(C18)2 (K) and JD1–GdDOTAMAC6 (J) (right).
C6-NHS).14 The deprotection of the t-butyl esters, followed by to obtain homogeneous films that were hydrated at 50 1C with an
complexation with GdCl3, yielded the functionalized Janus isotonic solution containing Gadoteridol (250 mM) at pH 7.4. After
dendrimer ( JD1–GdDOTAMAC6; see ESI† for details).
extrusion and exhaustive dialysis, vesicles with a size ranging from
Unlike most phospholipids, which easily self-organize into 160 to 180 nm and a PDI lower than 0.2 were obtained (see ESI†).
liposomes in physiological saline buffer, JD1 forms large, micron- Dendrimersomes containing Gd-complexes embedded in the lipidic
sized aggregates under the same isotonic conditions (see ESI†). bilayer were obtained by adding 20% of JD1–GdDOTAMAC6 or
To overcome this problem, two main strategies can be followed: Gd-DOTAMA(C18)2 to the membrane formulation.18,19
(i) dendrimersomes are prepared in a 5% w/w glucose solution,
The relaxometric characteristics of these nanosized systems
which then form vesicles with a low PDI (0.095) and a mean hydro- were assessed by evaluating the magnetic field dependence of their
dynamic diameter of around 100–120 nm; (ii) charged lipids relaxivities (r1, i.e. the longitudinal relaxation enhancement of the
(DMPG or DSPE–PEG–COOH) are incorporated into the membrane water protons induced by a 1 mM solution of GdIII) at 298 K over a
architecture to prevent the aggregation by electrostatic and/or steric wide range of field strengths (from 0.00024 to 1.65 T, corresponding
repulsion.15–17 The addition of DMPG effectively allowed us to to 0.01–70 MHz proton Larmor frequencies) to obtain the so-called
obtain a homogeneous suspension of 100–130 nm sized vesicles, nuclear magnetic relaxation dispersion (NMRD) profiles. The NMRD
but only by using phospholipids in a relatively large amount profiles of Gadoteridol-loaded dendrimersomes at 298 K (Fig. 2)
(20 mol%). On the other hand, the addition of a small percentage exhibit the same shape of the free chelate, but with lower r1 values
(5%) of DSPE–PEG–COOH was effective in sterically improving over the entire range of frequency investigated. This represents a clear
the stability of the nanovesicles and preventing aggregation in indication of the occurrence of a ‘‘limiting’’ effect on the relaxivity. It is
the isotonic buffer. Moreover, it has to be noted that PEG-2000 well known that a reduced water permeability (PW) through the vesicle
is normally used for coating the surface of vesicles in order to bilayer results in a decrease in r1 for the complex.20 As the observed r1
reduce their detection by the reticuloendothelial system, thus value is only slightly lower than that observed for free Gadoteridol, we
prolonging the circulatory lifetime and improving biodistribution may conclude that the bilayer of dendrimersomes represents just a
in perfused tissues.15–17 Furthermore, the carboxylic terminal very weak ‘‘quenching’’ effect. NMRD data were analysed using the
functionality of the PEG chain can serve as an anchoring site for conventional Solomon–Bloembergen–Morgan (SBM) theory,21 suitably
attachment of molecules capable of modulating the biodistribution modified to take into account the encapsulation of the chelate in the
of the nanoparticle (e.g. targeting vectors).
nanovesicles (see ESI†). The values of parameters obtained from
The dendrimersomes herein reported (Fig. 1) were obtained the data analysis for the free complex were fixed during the fit
by using the conventional film hydration method: a chloroform of the profile of the nanovesicles using only PW as the adjustable
solution of the amphiphilic molecules was dried under vacuum parameter. The value of 115 mm sÀ1 obtained for the water
permeability (Table 1) is sensibly higher than those reported
for conventional liposomes (0.3–70 mm sÀ1).22 Interestingly, the
gain in r1 due to the increase with temperature of membrane
permeability seems to be compensated by the decrease of r1
observed for free Gadoteridol (due to faster rotation), resulting in
almost unchanged r1 values for Gadoteridol-loaded dendrimer-
somes at 310 K (Fig. 3 and Fig. S5, ESI†).
NMRD profiles at 298 K of dendrimersomes incorporating
20% of JD1–GdDOTAMAC6 or self-assembled with 20% of
Gd-DOTAMA(C18)2 showed the typical shape of slowly tumbling
Gd-based systems, with a peak centred at around 20 MHz
corresponding to r1 values of 15.6 (JD1–GdDOTAMAC6) and
Fig. 1 Dendrimersomes made of JD1 dendrimers incorporating JD1–
GdDOTAMAC6 or Gd-DOTAMA(C18)2 into the membrane bilayer (left),
18.7 mMÀ1 sÀ1 (Gd-DOTAMA(C18)2) (Fig. 2, right). Data were
and encapsulating Gadoteridol in the inner core (right).
fitted only in the high-field region (43 MHz) because of the
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Table 1 Selected parameters obtained from the analysis of the 1/T1
conclusions about the role of the membrane composition in
the water permeability.
At 310 K, the relaxivity increases for both dendrimersomes
incorporating Gd-amphiphilic agents, likely as a consequence
of both tM shortening and increased water permeability
NMRD profiles at 298 K for dendrimersomes encapsulating Gadoteridol
a
or incorporating GdDOTAMA(C18 2
)
or JD1–GdDOTAMAC6
Parameter
Gadoteridol GdDOTAMA(C18 2
)
JD1–GdDOTAMAC6
20MHzr1 [mMÀ1
s
À1] 3.65
18.7
0.64 Æ 0.05
15.6
D2 [1019 sÀ2
tv [ps]
]
2.7 Æ 0.3
0.68 Æ 0.06
45.0 Æ 1.8
405 Æ 12
71 Æ 9
(Fig. S6, ESI†). Interestingly, the GdDOTAMA(C18)2 complex
16.0 Æ 1.1 23.0 Æ 1.3
65.0 Æ 7.0 550 Æ 15
exhibits a much higher relaxivity (ca. 50%) when incorporated
into dendrimersomes as compared to the case where it is
embedded in conventional liposomal bilayers (based on DPPC,
cholesterol and DSPE-PEG2000).19 This enhancement can be
mostly attributed to the higher water permeability of the bilayer
made of Janus dendrimers that limits the quenching effect on
the relaxivity of the paramagnetic units exposed towards the
inner core of the vesicles.
tRL [ps]
tRG [ns]
S2
—
—
80 Æ 11
0.65 Æ 0.1
925 Æ 40
175 Æ 65
0.24 Æ 0.08
924 Æ 35
166 Æ 60
tM [ns]
700 Æ 30
PW [mm sÀ1
]
115 Æ 15
a
The following parameters were fixed to common values during the
fitting procedure: rGd–H = 3.0 Å, D = 2.25 Â 10À5 cm2 sÀ1, q = 1, a = 3.8 Å.
Finally, dendrimersomes encapsulating carboxyfluorescein at a
high concentration (20 mM) were also prepared (see ESI† for
details). They elicit a self-quenching effect of the fluorescence
due to the contact and the consequent non-radiative transfer of
energy among adjacent dye molecules.25,26 The quenching effect
eventually vanished when breakage of vesicles by a surfactant
(Triton-X100) led to probe release confirming the compartmentali-
zation of the fluorescent dye in the aqueous core.
By virtue of their similarity to liposomes, whose versatile
properties are being successfully exploited in various fields,
dendrimersomes also have considerable potential for effective
practical use in biomedicine. Our preliminary data indicate that the
vesicles assembled from Janus dendrimers can be considered as
new potential vectors of MRI agents, drugs and multimodal imaging
probes. However, further studies are needed to develop and fully
realize the great potential of this innovative nanosized platform.
The financial support of the ‘‘Compagnia di San Paolo’’
Fig. 3 Relaxivity measured at 21.5 MHz, 298 K and 310 K of Gadoteridol in the
free form (A) or encapsulated into dendrimersomes (B); dendrimersomes incor-
porated into the membrane bilayer JD1–GdDOTAMAC6 (C) or GdDOTAMA(C18)2
(D) compared to liposomes incorporating GdDOTAMA(C18 2
) (E) into the
lipidic bilayer.
known limitations of the SBM theory for slowly tumbling (CSP-2012 NANOPROGLY and ‘‘Validazione di molecole di tipo VHH
systems at low magnetic field strengths.21 We used our recently e Aptameri per il rilascio tumore-specifico di farmaci e la valutazione
published relaxation model for the analysis of r1 in nanovesicles contestuale della risposta mediante imaging funzionale mirato’’
incorporating paramagnetic Gd-complexes implemented using projects), University of Torino (Innovative Nanosized Theranostic
the Lipari–Szabo (LS) approach.19 The LS model was proven to be Agents), Regione Piemonte (Nano-IGT and PIIMDMT Projects),
effective to treat systems in which a local molecular rotation of a and European Union’s FP7/2007–2013 under grant agreement
chelate (characterized by a correlation time tRL) is motionally no. HEALTH-F2-2011-278850 (INMiND) is gratefully acknowl-
coupled to the global tumbling motion of the nanoparticle edged. This work was carried out within the framework of the
(tRG).23 The motional coupling is defined by the value of the COST TD1004 Action.
order parameter S2 (0 = no coupling, 1 = maximum coupling).
Data were fitted by adjusting the parameters D2, tV, tRG, tRL
,
Notes and references
tM, and S2, whereas PW was left to vary only in the range of
100–250 mm sÀ1. On the basis of the results presented in Table 1,
the lower relaxivity observed for the vesicles based on the
JD1–GdDOTAMAC6 complex with respect to those assembled
with GdDOTAMA(C18)2 is due to the faster rotational tumbling
(tRL) correlated with a poor motional coupling with the global
rotation of the nanovesicle. Likely, such a difference stems from
the flexible C6 linker that connects the coordination cage to the
Janus dendrimer in JD1–GdDOTAMAC6. The other relaxation para-
meters assume the values typical of slowly tumbling GdDOTA–
monoamide complexes.19,24 The PW values obtained are slightly
larger than the permeability determined for Gadoteridol-loaded
dendrimersomes. However, the low statistical weight of PW in
the analysis of the profiles does not allow us to draw reliable
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