Formation of a lanthanoid complex shell on a nanoparticulate wax core

Article abstract of DOI:10.1016/j.ica.2020.119725

Carboxylate-rich lanthanide complexes were employed as anion counterions to the cationic surfactant cethyltrimethylammonium (CTA) and assembled to form a shell around emulsifying wax nanoparticles. The anionic complexes consisted of lanthanide ions coordinated by the ligand DOTA-tetrahomoserine (DhS). In combination with CTA the complexes formed a surfactant system (CTA)[LnDhS] (Ln = Eu, Gd) with the nonpolar tail of CTA embedded in an emulsifying wax (EW) nanoparticle core. The DhS units were then crosslinked with divinylsulfone to form a shell around the EW core. Fluorescence and NMR data on the Eu complex were consistent with the formation of a stable polymeric shell around the wax core template. These results illustrate a general method that exploits electrostatic interactions to assemble capsules around solid lipid nanoparticles. Preliminary relaxivity measurements were carried out on particles with Gd-containing shells.

Full text of DOI:10.1016/j.ica.2020.119725

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Research paper
Formation of a Lanthanoid Complex Shell on a Nanoparticulate Wax Core
Joo-Hyung Kim, Sujeethraj Koppolu, Eser Akturk, Eric Roth, Marc A.
Walters
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DOI:
Reference:
S0020-1693(19)31874-2
https://doi.org/10.1016/j.ica.2020.119725
ICA 119725
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Inorganica Chimica Acta
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Accepted Date:
1 December 2019
24 April 2020
28 April 2020
Please cite this article as: J-H. Kim, S. Koppolu, E. Akturk, E. Roth, M.A. Walters, Formation of a Lanthanoid
Complex Shell on a Nanoparticulate Wax Core, Inorganica Chimica Acta (2020), doi: https://doi.org/10.1016/
j.ica.2020.119725
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FORMATION OF A LANTHANOID COMPLEX SHELL ON A NANOPARTICULATE
WAX CORE
a
a
a
b
a,
Joo-Hyung Kim , Sujeethraj Koppolu , Eser Akturk , Eric Roth and Marc A. Walters *
a
Department of Chemistry, New York University, New York, NY 10003, USA
School of Medicine, Department of Radiology, New York University, New York, NY 10016,
b
USA
Address correspondence to:
Professor Marc A. Walters
NYU Dept. of Chemistry
1
00 Washington Square E.
marc.walters@nyu.edu
2
12-998-8477
fax: 212-995-4475



Abstract
Carboxylate-rich lanthanide complexes were employed as anion counterions to the cationic
surfactant cethyltrimethylammonium (CTA) and assembled to form a shell around emulsifying
wax nanoparticles. The anionic complexes consisted of lanthanide ions coordinated by the
ligand DOTA-tetrahomoserine (DhS). In combination with CTA the complexes formed a
surfactant system (CTA)[LnDhS] (Ln = Eu, Gd) with the nonpolar tail of CTA embedded in an
emulsifying wax (EW) nanoparticle core. The DhS units were then crosslinked with
divinylsulfone to form a shell around the EW core. Fluorescence and NMR data on the Eu
complex were consistent with the formation of a stable polymeric shell around the wax core
template. These results illustrate a general method that exploits electrostatic interactions leading
to assembly of capsules around solid lipid nanoparticles. Preliminary relaxivity measurements
were carried out on particles with Gd-containing shells.
Keywords: solid lipid nanoparticles; DOTA; cationic SLN; divinyl sulfone; hyperbranched;
theranostics
2

1
. Introduction
Bio-related coordination nanochemistry has a rich legacy in the development of diagnostic
3
+
agents. This includes a large body of work on Gd based T1 agents in, perfluorocarbons,
liposomes, fullerenes, carbon nanotubes and polymer-based hybrid nanoparticles as well as
related research on silver and gold nanoparticles.1-4 Hybrid nanoparticles (NPs) are the subject
5
of ongoing research as vehicles for simultaneous targeting, imaging, and drug delivery. For a
large fraction of hybrid NPs an organic superstructure is anchored on a noble metal core. A
previous report from this laboratory featured silver NPs with surface anchored GdDTPA
6
groups. These particles served to concentrate gadolinium complexes into the molar range,
(based on the volume of the particle) making them useful for MRI based molecular imaging.
Until recently metal NP systems had been favored for their ease of formation and rich surface
chemistry, where chemisorption on the metal surface is readily achieved with thiols, amines,
7
and carboxyl links and others. This advantage is however offset by the inflammation that
sometimes results from clinical application of these metals.
As an alternative, solid lipid nanoparticles (SLNs) of 50 - 200 nm diameter are favored by their
biocompatibility, biodegradability and the low cost of their surfactant molecule components.8-
1
0
Neutral Gd(acac) has been incorporated in the SLN hydrophobic core for use in neutron
1
1
capture therapy of the brain. In related work SLNs have been explored as containers and
structural supports for imaging agents. In particular, several research groups have shown
interest in Gd containing SLNs as MRI contrast agents. In an early report Aimé and coworkers
2
-
- 12
described particles containing [GdDTPA] and [GdDOTA] . Several later reports described
-
13-18
SLNs with [GdDTPA] on the particle surface or incorporated within the core.
In another
2
-
approach [GdDTPA] anions have been assembled by charge pairing with a cationic SLN
surface.19
3

The confinement of complexes to the SLN surface favors water exchange which is key to
contrast enhancement in Gd-based T1 MRI contrast agents. We have designed a new approach
to the formation of such agents by assembling modified LnDOTA complexes on an SLN surface
and then crosslinking them to form a hyperbranched enclosure for the SLN. In this approach an
SLN is encapsulated in a polyanionic network of ligands paired with surfactant counterions.
Specifically, the monomer is the anionic complex, [Ln(DOTA-N,N’,N’’, N’’’-
-
-
tetrahomoserine)] ([LnDhS] ), (Ln = Eu, Gd) that is charge-paired with the cationic surfactant
+
cetyltrimethylammonium (CTA ) whose hydrophibic tail is associated with the SLN surface.
+
20-23
The CTA serves both to stabilize the SLN
and to form a double layer with the anionic
complex. The DhS ligand has four hydroxyethyl groups at the -carbons of the DOTA
carboxymethyl group. The hydroxyl end groups serve as reaction sites for crosslinking by
divinylsulfone (DVS).24-25 The synthetic approach reported here is unique in its incorporation
-
+
of [LnDhS] as a monoanionic counterion to CTA , and as a monomeric precursor to a
hyperbranched shell. (Figure 1)
+
Figure 1: CTA [Eu(DOTA-N,N’,N’’, N’’’-tetrahomoserine)]-, CTA[LnDhS], (Ln = Eu, Gd).
4

2
6
This is the first report of a crosslinked shell formation around an SLN template. A preliminary
examination of the crosslinked system with coordinated gadolinium shows an enhancement in
relaxivity relative to small precursor gadolinium complexes.
2
2
. Materials and methods
.1 Materials
Emulsifying wax (EW) was obtained from Spectrum Chemicals. Polysorbate surfactant (Brij™
L23), and α-bromo-γ-butyrolactone were purchased from Sigma Aldrich Chemicals. Europium
trichloride hexahydrate, gadolinium trichloride hexahydrate, divinyl sulfone (DVS) and
potassium carbonate were purchased from Alfa Aesar Chemicals. Cetyltrimethylammonium
chloride (CTA-Cl) was obtained from TCI America. All reagents were used as received.
2
2
.2 Syntheses
.2.1 Cyclen tetrabutyrolactone
Cyclen (1.00 g, 5.80 mmol), potassium carbonate (4.00 g, 29.02 mmol) and α-bromo-γ-
butyrolactone (6.03 g, 36.57 mmol) were refluxed for 3 days in CH CN. The filtrate was
3
collected and the solvent was removed by rotary evaporation. A solid product was collected
after several ether-wash/centrifugation cycles. The solid was re-dissolved in 15 mL of
chloroform and extracted twice with 5 mL of water. The chloroform solution was dried with
5

magnesium sulfate and the solvent was removed by rotary evaporation to give a yellow oil.
After the addition of diethyl ether the product was deposited as a yellow powder in 71 % yield.
1
H NMR (400 MHz, CDCl , d): 4.45-4.25 (m, 8 H, O-CH ), 3.95 (m, 4 H, N-CH), 3.70-3.49
3
2
1
3
(
m, 4 H, cyclen-CH ), 2.70-2.22 (m, 12 H, cyclen-CH ), 2.40-2.25 (m, 8 H, lactone-CH ); C
2 2 2
NMR (400 MHz, CDCl , d): 178.5 (C=O), 65.18 (O-CH ), 59.90 (N-CH), 47.22 (cyclen-CH ),
3
2
2
+
+
+
2
1.25 (lactone-CH ). ESI-MS m/z: 508.9 [M + H] , 531.9 [M + Na] , 546.9 [M + K] .
2
2
.2.2 CTAOH
CTA-Cl was stirred with KOH in anhydrous ethanol to prepare a stock solution of ethanolic
cetyltrimethylammonium hydroxide (CTA-OH). The byproduct, KCl(s), was removed by
filtration. The product was stored as a 1 M stock solution in ethanol.
2
.2.3 CTA[EuDOTA(N,N’,N’’’,N’’’’-tetra--homoserine)] (CTA[EuDhS])
Cyclen tetrabutyrolactone (0.13 g, 0.26 mmol) and cetyltrimethylammonium hydroxide (CTA-
OH) (1.6 mmol) were combined in 10 mL of ethanol and stirred overnight butyrolactone ring
opening to form DOTA-tetra--homoserine (DhS). Ethanol was removed by rotary evaporation
and the product (CTA) DhS was re-dissolved in 20 mL of methanol followed by EuCl .6H O
4
3
2
(0.105 gm, 0.29 mmol). The solution was stirred for 3 hours, taken to dryness by rotary
evaporation, dissolved in 25 mL of ethanol and refluxed for 1 hour. The solid product,
CTA[EuDhS], was isolated by bench top centrifugation, and dried under vacuum to give 0.032
1
g of product, 12% yield. H NMR (400 MHz, D O, d): 41.9 (s, 4H, ring CH axial, minor isomer),
2
6

2
2.0 (s, 4H, ring CH axial, major isomer). 8.1 (s, 1H, ring CH axial major isomer), 4.1 (s, 4H,
-CH (homoserine side chain)), 3.48 (m, 2H, -N-CH (CTA)), 3.0 (s, 9H, CH (N-CH from

2
3
3
CTA), 1.6 (s, 2H, -N-CH (CTA)), 1.2-1.0 (d, 26H, -CH - (CTA backbone)), 0.79 (m, 3H, -
2
2
CH (CTA terminal methyl group)), -0.5 (s, 8H, -CH (homoserine side chain)), -1.1 (s, 4H, -
3
CH (homoserine side chain)), -2.0 (s, 4H, -CH (homoserine side chain)), -3.1 (s, 4H, ring CH
equatorial, major isomer), -4.2 (s, 4 H, ring CH axial, major isomer), -4.9 (s, 4 H, ring CH
equatorial, minor isomer), -8.6 (s, 4 H, ring CH equatorial, major isomer), -12.1 (s, 4 H, CH), -
2
5.4 (s, 4 H, CH-minor isomer). ESI-MS m/z: 729.0 [M]-.
2
.2.4 CTA[GdDOTA(N,N’,N’’’,N’’’’-tetra-a-homoserine)] (CTA[Gd(DhS)])
The product, CTA[Gd(DhS)], was obtained by the method above, but with GdCl .6H O (0.29
3
2
-
mmol) substituted for EuCl .6H O. ESI-MS m/z: 734.3 [M] .
3
2
2
.2.5 Formation of [EuDhS]SLN using Brij® L23
Solid lipid nanoparticles (SLNs) were prepared in an o/w microemulsion following the methods
2
7
of Oyewumi and Mumper. Briefly, emulsifying wax, 4 mg in 60 L of chloroform, was
deposited in a glass vial by evaporating the chloroform under a flow of nitrogen. The wax was
then melted at 55 °C and maintained at that temperature while 1 mL of distilled water was added
dropwise while stirring the emulsion at 1500 rpm. A homogenous milky slurry was formed and
6
0 L (6 mol) of Brij® L23 (C H (OC H ) OH) solution (0.1 M stock soln.) were added.
1
2
25
2
4 s
+
-
After 2 min, 1200 L (60 mol) of CTA [Eu(DOTA)(N,N’,N’’’,N’’’’-tetra-a-homoserine)]
solution (50 mM stock soln.) were added. The solution was stirred until it became clear (about
7

3
0 min) and was then quickly cooled on an ice bath to form SLNs. The resulting SLN colloidal
solution was diluted by the addition of 1 mL of cold water at 4 °C, filtered through a 0.22 
pore size nylon membrane to remove large particles and stored at 4 °C.
2
.2.6 Formation of [EuDhS] SLN
CL
The pH of the [EuDhS]SLN (aq) colloidal solution was adjusted to 12.0 by the dropwise
-
addition of 1.0 M NaOH. The [EuDhS] complexes were then polymerized by the slow addition
of 25L divinylsulfone (DVS), (4.2 equivalents). The solution was stirred for 3-6 hours after
which the pH was lowered to 7.5-8 by the slow addition of 1.0 M HCl. Samples were stored as
aqueous colloidal solutions at 4°C and remained transparent for more than 8 weeks.
2
.3 Characterization
Proton NMR data, including relaxivity measurements, were obtained on a Bruker Advance 400
MHz NMR spectrometer. T1 relaxation measurements were carried out in deionized water
(
H O) solutions at 25º C with the temperature controlled by a BVT-3200. The aqueous (H O)
2
2
samples were loaded into the internal capillary tubes of a coaxial cell with D O lock solvent in
2
the exterior (5 mm o.d.) chamber. Acquisition parameters were: time domain 16K complex data
point; 6410.26 Hz sweep width; 90º pulse with a length of 9 μs; repetition time T of 45 s;
R
variable inversion time delay (τ) ranging from 100 ms to 5 s. T2 relaxation timewas determined
-
using a multi-echo Carr-Purcell-Meiboom-Gill (CPMG) sequence. Relaxivities r and r (mM
1
2
1
-1
s ) were calculated from data collected at 5 different contrast agent concentrations based on
the respective slopes of linear regressions generated from plots of the measured relaxation rates
8

3
+
(
in seconds) versus the concentration of Gd (in mM). Spectra were processed with XWIN-
NMR version 3.5.6 to obtain relaxation curves.
Electrospray ionization (ESI) mass spectrometric data were obtained using an Agilent 1100
Series Capillary LCMSD Trap XCT MS spectrometer. Inductively coupled plasma optical
emission spectroscopy (ICP-OES) chemical analyses were carried out by Galbraith
Laboratories. TEM images were acquired on a Philips CM-12 electron microscope. Samples
were prepared on carbon coated copper grids and the micrographs were recorded on a Gatan 1k
x 1k digital camera. Fluorescence data was collected on a Hitachi F-2500 fluorescence
spectrophotometer. All sample concentrations were at 10.8 mg/ml. Dynamic light scattering
measurements were carried out on a Brookhaven Instruments ZetaPALS/Zeta Potential
Analyzer and a Beckman-Coulter N4 Plus instrument.
3
3
. Results and Discussion
.1 Ligand synthesis and shell formation
In the synthesis of DOTA-tetrahomoserine (DhS) four equivalents of racemic
bromobutyrolactone were added to tetraazacyclododecane (cyclen) in refluxing CH CN with
3
K CO to form tetraazacyclododecane-N,N’,N’’,N’’’-tetrabutyrolactone. The reaction
2
3
conditions were essentially the same as those reported in the synthesis of triazacyclononane
2
8
N,N’N’’-tributyrolactone. Lactone ring-opening by base hydrolysis with CTA-OH gave a
diastereomeric cyclic octadentate DOTA-tetrahomoserine derivative. Europium trichloride
.
-
hexahydrate, EuCl 6H O was added to form the complex [EuDhS] . In the formation and
3
2
workup of the complex, three equivalents of CTA-Cl and excess CTA-OH were removed,
9

+
-
leaving the salt, CTA [EuDhS] . The four pendant hydroxyethyl groups of the ligand were later
employed to form a hyperbranched poly “EuDOTA” shell. (Figure 2)
Figure 2. Schematic illustration of CTA[EuDhS] SLN formation where EW denotes the
CL
emulsifying wax core, and DVS denotes divinylsulfone.
The lipid core of the core shell NP is emulsifying wax (EW), a multicomponent matrix that
2
9
consists of cetylstearyl alcohol and polysorbate 60 (USP28–Page 3107). Emulsifying wax
3
0
NPs have an amorphous matrix. Following the methods of Oyewumi and Mumper, EW NPs
o
were formed at 55 C in an o/w microemulsion stabilized by the surfactants Brij L23 and the
+
27
o
o
CTA ion component of the CTA[EuDhS] salt. With rapid cooling from 55 C to 4 C the
molten wax solidified to yield an SLN that incorporates the lipophilic cationic tetraalkyl
+
-
ammonium groups of CTA but not the Eu complex counterion, [EuDhS] . The combined use
+
of Brij L23 and CTA ion surfactants draws on the work of Gao et al. who described the
+
-
31
formation of stable CTA Br /Brij 35 mixed micelles.
+
We surmised that the lipophilic tails of CTA (-C H ) and Brij L23 (-C H ) would be
1
6
33
12 25
embedded in the solid lipid matrix emulsifying wax, leaving the cationic headgroup of CTA+
and the polyoxomer block of Brij L23 exposed to the aqueous bulk phase. The counterion
1
0

-
monomer, [EuDhS] , would then occupy the Stern layer as a mobile species that exchanges
+
between fixed-position cationic CTA headgroups anchored to the SLN surface with the
construct represented by the formula (CTA[EuDhS]SLN). Mass action by small ions, such as
-
-
Cl in high salt solutions, can displace anionic [EuDhS] complexes from the SLN surface. To
-
prevent such displacement the [EuDhS] assembly was stabilized by crosslinking at room
2
4
temperature with divinyl sulfone to form an anionic hyperbranched polymer shell enclosing
the SLN. The core-shell NP surface of (CTA)[EuDhS] SLN is represented in Figure 3.
CL
Adjacent particles are sterically shielded by the polyether -(C H O) -OH chains of Brij L23,
2
4
23
which prevents inter-particle crosslinking.
-
Further crosslinking of [EuDhS] to the lipid components cetylstearyl alcohol and polysorbate
6
0 of the SLN core is likely because a portion of the hydroxyl groups of these emulsifying wax
components occupy the SLN surface.32
-
Figure 3. Proposed model of divinyl sulfone crosslinking of [EuDhS] counterions and SLN
-
surface hydroxyls in the Stern layer formed by [EuDhS] and cations located on the SLN surface.
o
In the absence of crosslinking, samples of the nanoparticle CTA[EuDhS]SLN stored at 4 C in
aqueous media became turbid in about 2 weeks. By contrast, DVS crosslinking to form the
1
1

hybrid particle (CTA)[EuDhS] SLN stabilized the particles against core disintegration, with
CL
o
no turbidity observed over a period of 8 weeks at 4 C
3
.2 Nanoparticle characterization
Dynamic light scattering measurements gave an average hydrodynamic diameter of 65.1 ± 7.3
nm for (CTA)[EuDhS] SLN. The zeta potential of the crosslinked particle was +10.7 mV as
CL
measured by Doppler shift light scattering at pH 7.0. The particles were polydisperse by
transmission electron microscopy, but were most abundant in the 50 nm range and with nearly
spherical geometry. Smaller spherical particles were observed with diameters in the 5 – 10 nm
range while a small fraction of larger particles appeared with diameters in the range of 100 nm.
(Figure 4) Polydispersity is a common characteristic of SLNs.
Figure 4. TEM image of SLN particles. (Bar represents 100 nm)
1
2

Europium in (CTA)[EuDhS] SLN was found at the level of 5.58% of the particle mass by
CL
ICP-OES analysis. The average particle mass is determined based on particle size and the
known density of emulsifying wax, which was assumed to apply to the particles in this
investigation. The particle consists initially of emulsifying wax (EW), Brij L23, and
(CTA)[EuDhS], and divinyl sulfone. However, over time, the mass ratios of these components
undoubtedly change with the uptake of water and the loss of cetylstearyl alcohol and
polysorbate 60 during the process of dialysis to remove excess non-incorporated molecules.
Therefore we could only estimate the number of Eu ions per particle. Assuming a particle
3
density of 0.85 g/cm , the same as bulk emulsifying wax at room temperature, a particle of 65.1
nm in diameter would have a mass of 1.22 x 10-16 g. The mass of Eu per particle would then
3+
-18
4
be 6.81 x 10 g, which corresponds to 2.70 x 10 Eu ions per particle. The number of lanthanide
ions per particle is comparable to that reported for the well-known perfluorocarbon particles of
equal diameter.33
3
.3 Europium fluorescence
-
The crosslink stabilization of the capsule, [EuDhS] , was investigated by fluorescence
CL
spectroscopy using 319 nm excitation that generated fluorescence with maxima at 578, 592 and
6
17 nm. Concurrent emission at 406 nm belongs to the EW core and was ignored. To probe the
stability of the non-crosslinked (CTA)[EuDhS]SLN assembly, the sample was dialyzed (2.0
kDa cutoff dialysis membrane) against approximately 50 mL of 0.68 M sodium chloride, a
2
000-fold excess of salt. The control for this experiment entailed the dialysis of the same system
against distilled water. After 24 h the 617 nm europium emission peak of the uncrosslinked
1
3

(CTA)[EuDhS]SLN sample had decreased by 94%, while the fluorescence spectrum of the
control was unchanged. (Figure 5A) The loss of fluorescence intensity was indicative of the Cl-
-
induced displacement of anionic [EuDhS] complexes from the [EuDhS]SLN surface.
Figure 5. Fluorescence spectra in water of (A) NaCl-treated [EuDhS]SLN (red), salt-free
control [EuDhS]SLN (green), (B) NaCl-treated crosslinked [EuDhS] SLN (red) and the salt-
CL
free control [EuDhS] SLN (green).
CL
-
In the crosslinked sample, (CTA)[EuDhS] SLN, it appears that at most 19% of the [EuDhS]
CL
-
complexes were displaced by Cl as evidenced by the fluorescence intensity decrease at 617
nm.(Figure 5B) These results are consistent with the location of Eu complexes within the Stern
-
and double layer regions of the SLN and highlight the efficacy of crosslinking [EuDhS] to form
a stable shell around the cationic SLN core.
The concentration of Eu complexes in the solid lipid core must be negligible because Eu
-
-
fluorescence is absent after the displacement of Stern layer [EuDhS] by Cl ions. This
assessment, regarding the location of the Eu complex, is supported by a report that europium
complexes exhibit fluorescence of comparable intensity both in solution and in sol-gel derived
3
4
-
materials. We therefore believe that if significant amounts of [EuDhS] were located within
1
4

-
the SLN matrix the particle core would be fluorescent, which is not observed when EuDhS is
displaced by dialysis.
3
.4 NMR analyses and gadolinium relaxivity
NMR analysis of (CTA)[EuDhS] SLN is enhanced by the magnetic properties of europium
CL
7
that derive from low-lying states with J > 0 just above the diamagnetic F ground state. The
0
3
+
result is that the Eu ion serves as an intrinsic shift reagent. The expected dipolar shifts of
cyclen protons appear as a series of peaks in the upfield 0 – (-27) ppm region, and two peaks
approximately 21 and 42 ppm downfield as referenced to the adventitious water peak at 4.7
ppm in D O.(Figure 6)
2
Figure 6. Proton NMR spectrum of CTA[EuDhS] SLN in D O (bottom);
CL
2
K[EuDhS] in D O (top).
2
The dipolar shift peaks of the shell-localized europium complexes of [EuDhS] SLN are
CL
-
- 35-38
congruent with those of free [EuDhS] and to a lesser extent with [EuDOTA] .
Peaks in the
0
– 7 ppm downfield region are assigned to the homoserine sidechain, crosslinkers and SLN
-
core protons. The data from [EuDhS] ions are complex because of structural isomerization, and
1
5

3
8
overlapping peaks from the diasteriomers    and . For this reason, the
detailed assignment of peaks was not undertaken.
-
.
The congeners [GdDhS] and [GdDhS] SLN were prepared from the product of GdCl 6H O
CL
3
2
4
-
-
and DhS . The relaxivities of the Gd[DhS] and Gd[DhS] SLN, presented here as preliminary
CL
-1
-1
data, were found to be 4.16 and 7.70 s mM respectively for r , and 5.43 and 11.58 respectively
1
-
for r , with all data collected by NMR at 9.4 T (400 MHz) and 25°C.(Table 1) For Gd[DhS]
2
and Gd[DhS] SLN the respective r /r ratios, 1.31 and 1.50, are comparable to the value of 1.5
CL
2 1
-
6, 39-42
-
reported for Gd[DTPA] at 400 MHz.
Further, the r values for Gd[DhS] and
1
-1
-1
Gd[DhS] SLN are close to the high field (9.4 T) 4.25 – 5.16 mM s values reported by Li
CL
4
3
and coworkers for Gd(DO3A) tethered to a series of branched polymeric nanoparticles. Based
on the number of metal ion binding sites per particle, estimated from ICP-OES, the whole-
5
-1
-1
-3 -1
-1
particle relaxivities of Gd[DhS] SLN are r = 1.39 x 10 s mM (1.89 x 10 s mg L) and r
2
CL
1
5
-1
-1
-3 -1
-1
=
2.08 x 10 s mM (2.83 x 10 s mg L). These values must be considered as averages
because of the polydispersity of the SLN particles.
At low fields the per-Gd r relaxivity for Gd complexes tethered to nanoscale structures is
1
usually higher than those of simple Gd complexes in solution. This phenomenon has been
explained by an increase in the rotational correlation time,  , for macromolecular and
R
4
4
nanoscale species. However this advantage is lost at high field, typically above 1.5 T, where
3
+
r values tend to decrease and r values can increase by as much as a factor of ten for Gd
1
2
4
5
complexes that bind rigidly to proteins. The proximity of r values for Gd[DhS] SLN and
1
CL
-
freely rotating [GdDhS] complexes is consistent with the decoupling of the internal motion of
crosslinked GdDhS groups from the slow rotation of the SLN core or hyperbranched DhS
network that encloses the SLN. The higher r relaxivity of Gd[DhS] SLN could plausibly be
1
CL
1
6

attributed to faster water exchange in the crosslinked structure due to decreased hydrogen
bonding after hydroxyl groups of DhS are removed by ligand cross linking.
Table 1. Relaxivity (r and r ) per Gd, or per particle in H O at 9.4 T (400MHz), 25°C
1
2
2
-1
-1
-1
-1
Compound
Gd[DTPA]2-
Gd[DhS]-
Relaxivity (r , s mM ) Relaxivity (r , s mM )
1 2
4.6a
4.16
7.70
6.8a
5.43
11.58
Gd[DhS] SLN
CL
2
.70x104 (complexes
5
b
3.13x105 b (per particle)
Gd[DhS] SLN
2.08x10 (per particle)
CL
per particle)
a
References:6, 39-42
.
b
Relaxivity per particle is estimated from the Gd3+ population on the particle surface.
In the early work by Morel et al. gadolinium complexes associated with solid lipid NPs had r1
relaxivities that were similarly comparable to small freely rotating gadolinium complexes.12
-1
-1
Results from more recent studies suggest that r relaxivity values ≥ 4 – 5 s mM derive only
1
from surface localized Gd complexes. Clues about relaxivity and structure in nanoparticulate
gadolinium-based MRI contrast agents come from liposomal agents where the relaxivity for the
inner interface is apparently diminished by the low rate of water exchange between the inner
4
6
and outer regions. In these agents GdDTPA and GdDOTA complexes are preferentially
4
7
attached to the outer layer of the liposomal bilayer. It therefore appears that maximal relaxivity
in NP agents requires that GdDTPA and GdDOTA complexes be fully exposed to bulk water.
Our system fulfills this requirement. Furthermore, GdDOTA is favored as a contrast agent by
its high stability constant and inertness to substitution by metal ions that are endogenous to the
physiological environment.48-49 It is reasonable to assume that crosslinked DhS retains kinetic
1
7

and thermodynamic parity with the free DOTA ligand. With refinement to attain
monodispersity, [LnDhS] SLN could be considered for colonography where absorption into
CL
1
6
the bloodstream and slow clearance through the liver is not a factor. Similar systems with
cleavable ester linkages might prove acceptable for broader applications.
4
. Conclusions
Several reports have described the use of SLNs for MRI contrast enhancement.12-13, 16-19 For
3
+
those systems the location of the Gd coordinating groups were not a focus of the study. The
work described here highlights the spatial distribution of [EuDhS]CL relative to the SLN core,
with a preliminary look at the related relaxivity of the [GdDhS]CL units of the [LnDhS] SLN
CL
system.
+
-
We employed CTA to electrostatically position counterion anionic complexes, [Ln(DhS)] , to
form a shell around the cationic surface of the SLN core. These anionic complex monomers are
-
3+
3+
homoserine derivatives of [LnDOTA] (Ln = Eu and Gd ) with pendant hydroxyl groups that
are crosslinked by DVS to produce a hyperbranched polymer shell around an SLN core.
-
Fluorescence data show that anionic [LnDhS] complexes are not incorporated in the SLN core
but instead are restricted to the Stern layer at the cationic surface of the SLN.
The nanoparticulate [LnDhS] SLN system described here illustrates a convenient route to
CL
assemble an organic nanoparticulate contrast agent consisting of anionic LnDOTA-
tetrahomoserine, [LnDhS], monomers assembled as a crosslinked capsule, [LnDhS] , that
CL
encloses the cationic surface of an SLN. Preliminary measurements show the r relaxivity value
1
1
8

-
-1
-1
of [Gd(DhS)] in the shell of (CTA)[Gd(DhS)] SLN, to be 7.70 s mM , which could be
CL
suitable for certain clinical imaging applications.
Acknowledgements
We thank Dr. Zahi A. Fayad and Ms. Jojo Read and Dr. Iwao Teraoka for assistance in the
acquisition of dynamic light scattering data. This work was supported by the National Science
Foundation Chemistry Research Instrumentation and Facilities program (Grant CHE-0840277),
and the NYU Research Challenge Fund.
Abbreviations: CTA - cetyltrimethylammonium
DhS - DOTA-tetrahomoserine
DOTA - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate
DTPA - diethylenetriaminepentaacetic acid
DVS - divinylsulfone
EW - emulsifying wax
SLN - solid lipid nanoparticle
References
1
1
2
.
Na, H. B.; Hyeon, T., Nanostructured T1 MRI contrast agents. J. Mater. Chem. 2009,
9 (35), 6267-6273.
Wallace, K. D.; Hughes, M. S.; Marsh, J. N.; Caruthers, S. D.; Lanza, G. M.; Wickline,
.
S. A. In From in vivo ultrasound and MRI imaging to therapy: contrast agents based on target-
specific nanoparticles, Wiley-VCH Verlag GmbH & Co. KGaA: 2009; pp 19-50.
3
.
Accardo, A.; Tesauro, D.; Aloj, L.; Pedone, C.; Morelli, G., Supramolecular aggregates
containing lipophilic Gd(III) complexes as contrast agents in MRI. Coord. Chem. Rev. 2009,
2
4
53 (Copyright (C) 2013 American Chemical Society (ACS). All Rights Reserved.), 2193-2213.
. Aime, S.; Delli, C. D.; Geninatti, C. S.; Gianolio, E.; Terreno, E., Pushing the Sensitivity
Envelope of Lanthanide-Based Magnetic Resonance Imaging (MRI) Contrast Agents for
Molecular Imaging Applications. Acc. Chem. Res. 2009, 42 (Copyright (C) 2013 American
Chemical Society (ACS). All Rights Reserved.), 822-831.
5
.
Walters, M., The Exploration of Biomedical Multimodality in Small Solid Core
Nanoparticles. In Hybrid Nanomaterials: Synthesis, Characterization, and Applications,
Interscience Book ed.; Chauhan, B. P. S., Ed. Wiley: 2011; p 300.
6
.
Siddiqui, T. S.; Jani, A.; Williams, F.; Muller, R. N.; Elst, L.; Laurent, S.; Yao, F.;
Wadghiri, Y. Z.; Walters, M. A., Lanthanide complexes on Ag nanoparticles: Designing
contrast agents for magnetic resonance imaging. J. Colloid Interface Sci. 2009, 337 (1), 88-96.
1
9

7
.
Vigderman, L.; Zubarev, E. R., Therapeutic platforms based on gold nanoparticles and
their covalent conjugates with drug molecules. Adv. Drug Delivery Rev. 2013, 65 (Copyright
C) 2013 American Chemical Society (ACS). All Rights Reserved.), 663-676.
Lammers, T.; Aimé, S.; Hennink, W. E.; Storm, G.; Kiessling, F., Theranostic
Nanomedicines. Acc. Chem. Res. 2011, 44 (10), 1029-1038.
Andreozzi, E.; Seo, J. W.; Ferrara, K.; Louie, A., Novel method to label solid lipid
nanoparticles with 64Cu for positron emission tomography imaging. Bioconjugate Chemistry
(
8
.
9
.
2
1
011, 22 (4), 808-818.
0. Müller, R. H.; Mader, K.; Gohla, S., Solid lipid nanoparticles (SLN) for controlled drug
delivery - a review of the state of the art. Eur. J. Pharm. Biopharm. 2000, 50 (1), 161-177.
1. Oyewumi, M. O.; Mumper, R. J., Engineering tumor-targeted gadolinium hexanedione
nanoparticles for potential application in neutron capture therapy. Bioconjugate Chemistry 2002,
1
1
1
3 (6), 1328-1335.
2. Morel, S.; Terreno, E.; Ugazio, E.; Aimé, S.; Gasco, M. R., NMR relaxometric
investigations of solid lipid nanoparticles (SLN) containing gadolinium(III) complexes. Eur. J.
Pharm. Biopharm. 1998, 45 (2), 157-163.
1
3.
Zhu, D. H.; White, R. D.; Hardy, P. A.; Weerapreeyakul, N.; Sutthanut, K.; Jay, M.,
Biocompatible nanotemplate-engineered nanoparticles containing gadolinium: Stability and
relaxivity of a potential MRI contrast agent. J. Nanosci. Nanotechnol. 2006, 6 (4), 996-1003.
1
4.
Zhu, D.; Lu, X. L.; Hardy, P. A.; Leggas, M.; Jay, M., Nanotemplate-engineered
nanoparticles containing gadolinium for magnetic resonance imaging of tumors. Investigative
Radiology 2008, 43 (2), 129-140.
1
5.
Bui, T.; Stevenson, J.; Hoekman, J.; Zhang, S. R.; Maravilla, K.; Ho, R. J. Y., Novel Gd
Nanoparticles Enhance Vascular Contrast for High-Resolution Magnetic Resonance Imaging.
Plos One 2010, 5 (9).
1
6.
nanoparticle-based magnetic resonance colonography. Magn Reson Med 2011, 65 (3), 673-9.
7. Rolla, G. A.; Botta, M.; Tei, L.; Cabella, C.; Ghiani, S.; Brioschi, C.; Maiocchi, A.,
Sun, J.; Zheng, W.; Zhang, H.; Wu, T.; Yuan, H.; Yang, X.; Zhang, S., Development of
1
Paramagnetic solid lipid nanoparticles as a novel platform for the development of molecular
MRI probes. Chemistry 2013, 19 (34), 11189-93.
1
8.
Ghiani, S.; Capozza, M.; Cabella, C.; Coppo, A.; Miragoli, L.; Brioschi, C.; Bonafe, R.;
Maiocchi, A., In vivo tumor targeting and biodistribution evaluation of paramagnetic solid lipid
nanoparticles for magnetic resonance imaging. Nanomedicine (N. Y., NY, U. S.) 2017, 13 (2),
6
1
93-700.
9. Chen, Z.-j.; Zhang, N., Preliminary study on cationic solid lipid nanoparticles/Gd-DTPA
complex as molecular imaging agent. Zhongguo Xinyao Zazhi 2009, 18 (15), 1443-1447.
0. Eccleston, G. M., Functions of mixed emulsifiers and emulsifying waxes in
dermatological lotions and creams. Colloid Surface A 1997, 123, 169-182.
1. Kim, H. R.; Kim, I. K.; Bae, K. H.; Lee, S. H.; Lee, Y.; Park, T. G., Cationic solid lipid
2
2
nanoparticles reconstituted from low density lipoprotein components for delivery of siRNA.
Mol. Pharmaceut. 2008, 5 (4), 622-631.
2
2.
Tabatt, K.; Sameti, M.; Olbrich, C.; Muller, R. H.; Lehr, C. M., Effect of cationic lipid
and matrix lipid composition on solid lipid nanoparticle-mediated gene transfer. Eur. J. Pharm.
Biopharm. 2004, 57 (2), 155-162.
2
3.
Siddiqui, A.; Patwardhan, G. A.; Liu, Y. Y.; Nazzal, S., Mixed backbone antisense
glucosylceramide synthase oligonucleotide (MBO-asGCS) loaded solid lipid nanoparticles: In
vitro characterization and reversal of multidrug resistance in NCI/ADR-RES cells. Int. J. Pharm.
2
010, 400 (1-2), 251-259.
2
0

2
4.
Liu, S. Y.; Ma, Y. H.; Armes, S. P., Direct verification of the core-shell structure of shell
cross-linked micelles in the solid state using X-ray photoelectron spectroscopy. Langmuir 2002,
1
2
2
2
8 (21), 7780-7784.
5.
Read, E. S.; Armes, S. P., Recent advances in shell cross-linked micelles. Chem. Comm.
007, (29), 3021-3035.
6.
Walters, M. A. Lanthanoid complex capsule and particle contrast agents, methods of
making and using thereof. WO2012151539A2, 2012.
7. Oyewumi, M. O.; Mumper, R. J., Gadolinium-loaded nanoparticles engineered from
microemulsion templates. Drug Dev. Ind. Pharm. 2002, 28 (3), 317-328.
8. Lazar, I.; Egri, A.; Kiraly, R.; Baranyai, Z.; Ivanyi, T.; Brucher, E., Synthesis,
conformation and equilibrium study of new piperazine and azamacrocyclic ligands with N-
tetrahydro-2-oxofuran-3-yl) and N-[(carboxy)(2-hydroxyethyl)methyl] pendant arms. Eur. J.
Org. Chem. 2002, (2), 351-360.
9. Dong, X.; Mumper, R. J., The metabolism of fatty alcohols in lipid nanoparticles by
alcohol dehydrogenase. Drug Dev. Ind. Pharm. 2006, 32 (8), 973-980.
2
2
(
2
3
2
3
0.
002, 22 (2), 129-150.
1. Gao, H. C.; Zhu, R. X.; Yang, X. Y.; Mao, S. Z.; Zhao, S.; Yu, J. Y.; Du, Y. R.,
Ulrich, A. S., Biophysical aspects of using liposomes as delivery vehicles. Biosci. Rep.
Properties of polyethylene glycol(23) lauryl ether with cetyltrimethylammonium bromide in
mixed aqueous solutions studied by self-diffusion coefficient NMR. J. Colloid Interface Sci.
2
3
4
3
004, 273 (2), 626-631.
2.
Eccleston, G. M., Multiple-Phase Oil-in-Water Emulsions. J Soc Cosmet Chem 1990,
1 (1), 1-22.
3.
Tran, T. D.; Caruthers, S. D.; Hughes, M.; Marsh, J. N.; Cyrus, T.; Winter, P. M.;
Neubauer, A. M.; Wickline, S. A.; Lanza, G. M., Clinical applications of perfluorocarbon
nanoparticles for molecular imaging and targeted therapeutics. Int. J. Nanomed. 2007, 2 (4),
5
3
15-526.
4. Matthews, L. R.; Knobbe, E. T., Luminescence Behavior of Europium Complexes in
Sol-Gel Derived Host Materials. Chem. Mater. 1993, 5 (12), 1697-1700.
5. Aimé, S.; Botta, M.; Fasano, M.; Marques, M. P. M.; Geraldes, C. F. G. C.; Pubanz, D.;
3
Merbach, A. E., Conformational and coordination equilibria on DOTA complexes of lanthanide
metal ions in aqueous solution studied by H-1-NMR spectroscopy. Inorg. Chem. 1997, 36 (10),
2
3
059-2068.
6. Desreux, J. F., Nuclear Magnetic-Resonance Spectroscopy of Lanthanide Complexes
with a Tetraacetic Tetraaza Macrocycle - Unusual Conformation Properties. Inorg. Chem. 1980,
1
3
9 (5), 1319-1324.
7. Aimé, S.; Botta, M.; Ermondi, G., Nmr-Study of Solution Structures and Dynamics of
Lanthanide(Iii) Complexes of Dota. Inorg. Chem. 1992, 31 (21), 4291-4299.
8. Woods, M.; Aime, S.; Botta, M.; Howard, J. A. K.; Moloney, J. M.; Navet, M.; Parker,
3
D.; Port, M.; Rousseaux, O., Correlation of water exchange rate with isomeric composition in
diastereoisomeric gadolinium complexes of tetra(carboxyethyl)dota and related macrocyclic
ligands. J. Am. Chem. Soc. 2000, 122 (40), 9781-9792.
3
9.
Cage, B.; Russek, S. E.; Shoemaker, R.; Barker, A. J.; Stoldt, C.; Ramachandaran, V.;
Dalal, N. S., The utility of the single-molecule magnet Fe-8 as a magnetic resonance imaging
contrast agent over a broad range of concentration. Polyhedron 2007, 26 (12), 2413-2419.
4
0.
micelle MRI contrast agent with changeable relaxivity. J. Controlled Release 2006, 114 (3),
25-333.
Nakamura, E.; Makino, K.; Okano, T.; Yamamoto, T.; Yokoyama, M., A polymeric
3
2
1

4
1.
Sun, G. Y.; Feng, J. H.; Jing, F. Y.; Pei, F. K.; Liu, M. L., Synthesis and evaluation of
novel polysaccharide-Gd-DTPA compounds as contrast agent for MRI. J. Magn. Magn. Mater.
2
4
003, 265 (2), 123-129.
2. Vaccaro, M.; Accardo, A.; D'Errico, G.; Schillen, K.; Radulescu, A.; Tesauro, D.;
Morelli, G.; Paduano, L., Peptides and gd complexes containing colloidal assemblies as tumor-
specific contrast agents in MRI: Physicochemical characterization. Biophys. J. 2007, 93 (5),
1
4
736-1746.
3. Li, Y.; Laurent, S.; Esser, L.; Vander Elst, L.; Muller, R. N.; Lowe, A. B.; Boyer, C.;
Davis, T. P., The precise molecular location of gadolinium atoms has a significant influence on
the efficacy of nanoparticulate MRI positive contrast agents. Polym. Chem. 2014, 5 (7), 2592-
2
4
601.
4.
Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B., Gadolinium(III) Chelates as
MRI Contrast Agents: Structure, Dynamics, and Applications. Chem. Rev. 1999, 99 (9), 2293-
2
4
352.
5.
Caravan, P.; Farrar, C. T.; Frullano, L.; Uppal, R., Influence of molecular parameters
and increasing magnetic field strength on relaxivity of gadolinium- and manganese-based T1
contrast agents. Contrast Media Mol Imaging 2009, 4 (2), 89-100.
4
6.
Muller, R. N.; Laurent, S.; Elst, L. V.; Thirifays, C., Paramagnetic liposomes: Inner
versus outer membrane relaxivity of DPPC liposomes incorporating lipophilic gadolinium
complexes. Langmuir 2008, 24 (8), 4347-4351.
4
7.
Tumour Imaging. Int. J. Mol. Sci. 2010, 11 (4), 1759-1776.
8. Viola-Villegas, N.; Doyle, R. P., The coordination chemistry of 1,4,7,10-
Kamaly, N.; Miller, A. D., Paramagnetic Liposome Nanoparticles for Cellular and
4
tetraazacyclododecane-N,N ',N '',N '''-tetraacetic acid (H(4)DOTA): Structural overview and
analyses on structure-stability relationships. Coord. Chem. Rev. 2009, 253 (13-14), 1906-1925.
4
9.
transmetallation of a gadolinium complex. Contrast Media & Molecular Imaging 2010, 5 (6),
05-308.
Laurent, S.; Vander Elst, L.; Henoumont, C.; Muller, R. N., How to measure the
3
Author Statement File
The project described in the submitted manuscript was conceived by Prof. Walters. The initial
monomer ligand syntheses were carried out by Eser Akturk, an undergraduate in the Walters
group. After his graduation the syntheses were optimized by Joo-Hyung, a graduate student in
the group. Sujeethraj Koppolu, another graduate student in the group, was responsible for
nanoparticle formation. Lastly Eric Roth an electron microscopist played a pivotal role in
particle characterization.
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may
be considered as potential competing interests:
2
2



Anionic metal complexes are assembled with the aid of cationic surfactants
The surfactant tails are embedded in a solid lipid nanoparticle core which
serves to arrange the metal complexes above the nanoparticle surface
The metal complexes are then crosslinked and the resulting hyperbranched
metallopolymer network encloses the solid lipid nanoparticle
The resulting nanoparticle containing gadolinium can, in principle, be used
for certain types of high contrast MRI imaging where cell uptake is
proscribed


Relaxivity of Gd(Dhs) complex
-
1
-1
[
Gd] (mM)
T1 (s)
1/T (s )
T2 (s)
1/T (s )
1
2
1
2
3
4
5
0.117298
0.076783
0.058906
0.045826
0.04028
8.525295
13.02372
16.9762
0.08774 11.39731
0.05842 17.11743
0.04375 22.85714
0.03457 28.92682
0.03039 32.90556
21.82167
24.82597
T1
3
2
2
1
1
0
5
0
5
0
5
0
2
3
0
1
2
3
4
5
6
[Gd] (mM)

T
2
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
0
2
4
6
[Gd] (mM)
2
4

relaxivity of CTA[GdDhS)]CL SLN Final
sample # [c] (mM) T1 (s) 1/T (s )
-1
-1
[c] (mM)
3.83
T2 (s)
1/T (s )
1
2
1
2
3
4
5
4.38
1.46
0.026123 38.28044
0.065467 15.27487
0.085826 11.65148
0.149785 6.676236
0.020112 49.72156
1.276667
0.638333
0.319167
0.1915
0.05279
0.06828
0.12146
0.13814
18.94298
14.64558
8.233163
7.239033
0.73
0.365
0.219
0.16753
5.96908
T1
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
0
1
2
3
4
5
[c] (mM)
T2
6
5
4
3
2
1
0
0
0
0
0
0
0
0
1
2
3
4
5
[c] (mM)
2
5

2
2