Synthesis of new water soluble β-cyclodextrin@curcumin conjugates and in vitro safety evaluation in primary cultures of rat cortical neurons

Article abstract of DOI:10.3390/ijms22063255

Self-aggregation of Curcumin (Cur) in aqueous biological environment decreases its bioavailability and in vivo therapeutic efficacy, which hampers its clinical use as candidate for reducing risk of neurodegenerative diseases. Here, we focused on the design of new Cur-β-Cyclodextrin nanoconjugates to improve the solubility and reduce cell toxicity of Cur. In this study, we described the synthesis, structural characterization, photophysical properties and neuron cell toxicity of two new water soluble β-CD/Cur nanoconjugates as new strategy for reducing risks of neurodegenerative diseases. Cur was coupled to one or two β-CD molecules via triazole rings using CuAAC click chemistry strategy to yield β-CD@Cur and (β-CD)₂ @Cur nanoconjugates, respectively. The synthesized nanoconjugates were found to be able to self-assemble in aqueous condition and form nano-aggregates of an average diameter size of around 35 and 120 nm for β-CD@Cur and (β-CD)₂ @Cur, respectively. The photophysical properties, water solubility and cell toxicity on rat embryonic cortical neurons of the designed nanoconjugates were investigated and compared to that of Cur alone. The findings revealed that both new nanoconjugates displayed better water solubility and in vitro biocompatibility than Cur alone, thus making it possible to envisage their use as future nano-systems for the prevention or risk reduction of neurodegenerative diseases.

Full text of DOI:10.3390/ijms22063255

International Journal of
Molecular Sciences
Article
Synthesis of New Water Soluble β-Cyclodextrin@Curcumin
Conjugates and In Vitro Safety Evaluation in Primary Cultures
of Rat Cortical Neurons
Amina Ben Mihoub 1,2,3,*, Samir Acherar 2,
*
, Céline Frochot 3 , Catherine Malaplate 4, Frances T. Yen 4 and
Elmira Arab-Tehrany 1,
*
1
2
3
4
LIBio Laboratory, Université de Lorraine, F-54000 Nancy, France
LCPM, CNRS, Université de Lorraine, F-54000 Nancy, France
LRGP, CNRS, Université de Lorraine, F-54000 Nancy, France; celine.frochot@univ-lorraine.fr
URAFPA, INRAE, Université de Lorraine, F-54000 Nancy, France;
catherine.malaplate-armand@univ-lorraine.fr (C.M.); frances.yen-potin@univ-lorraine.fr (F.T.Y.)
Correspondence: amina.ben-mihoub@univ-lorraine.fr (A.B.M.); samir.acherar@univ-lorraine.fr (S.A.);
elmira.arab-tehrany@univ-lorraine.fr (E.A.-T.)
*
Abstract: Self-aggregation of Curcumin (Cur) in aqueous biological environment decreases its
bioavailability and in vivo therapeutic efficacy, which hampers its clinical use as candidate for reduc-
ing risk of neurodegenerative diseases. Here, we focused on the design of new Cur- β-Cyclodextrin
nanoconjugates to improve the solubility and reduce cell toxicity of Cur. In this study, we de-
scribed the synthesis, structural characterization, photophysical properties and neuron cell toxicity
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
of two new water soluble
rodegenerative diseases. Cur was coupled to one or two
CuAAC click chemistry strategy to yield -CD@Cur and (
The synthesized nanoconjugates were found to be able to self-assemble in aqueous condition and
form nano-aggregates of an average diameter size of around 35 and 120 nm for -CD@Cur and
-CD)2@Cur, respectively. The photophysical properties, water solubility and cell toxicity on rat
β
-CD/Cur nanoconjugates as new strategy for reducing risks of neu-
-CD molecules via triazole rings using
-CD)2@Cur nanoconjugates, respectively.
ꢀꢁꢂꢃꢄꢅꢆ
β
Citation: Ben Mihoub, A.; Acherar,
S.; Frochot, C.; Malaplate, C.; Yen, F.T.;
Arab-Tehrany, E. Synthesis of New
Water Soluble β-Cyclodextrin@
Curcumin Conjugates and In Vitro
Safety Evaluation in Primary Cultures
of Rat Cortical Neurons. Int. J. Mol.
Sci. 2021, 22, 3255. https://doi.org/
10.3390/ijms22063255
β
β
β
(β
embryonic cortical neurons of the designed nanoconjugates were investigated and compared to that
of Cur alone. The findings revealed that both new nanoconjugates displayed better water solubility
and in vitro biocompatibility than Cur alone, thus making it possible to envisage their use as future
nano-systems for the prevention or risk reduction of neurodegenerative diseases.
Academic Editor: Stephen C. Bondy
Keywords: curcumin;
β-cyclodextrin; click chemistry; nanoconjugates; neurodegenerative
prevention
Received: 18 February 2021
Accepted: 19 March 2021
Published: 23 March 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, as well
as spinocerebellar ataxias, are common causes of cognitive impairment and morbidity in
older adults [
neural disorders that characterize these diseases can also be associated with inflammation,
oxidative damage and the protein aggregate formation [ ]. Epidemiological studies point
1,2]. The heterogeneous clinical presentation and physiopathology of the
3,4
towards natural antioxidant agents as promising therapeutic agents to prevent or delay the
occurrence of neurodegenerative diseases [5].
Copyright:
© 2021 by the authors.
Curcumin (Cur) is the major polyphenolic compound of Curcuma longa, an herba-
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
ceous plant endemic to South Asia [
and health benefits of Cur, including its antioxidant, anti-inflammatory, antitumor and
neuroprotective effects [ ]. In recent years, this drug has been considered as a potential
treatment for multiple neurodegenerative disorders [ ]. Moreover, multiple studies in
rodents and humans have shown that Cur crosses the blood–brain barrier (BBB) [ ]. How-
ever, despite its many biological activities, Cur is not often used in clinical applications
6]. There have been numerous studies on the bioactivity
7
8
5
Int. J. Mol. Sci. 2021, 22, 3255. https://doi.org/10.3390/ijms22063255
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 3255
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because of two main intrinsic properties: Low bioavailability due to its limited solubility in
water (11 ng/mL) and low permeability (LogP 3.28) [9].
Recently, Cur-based nano-systems, including liposomes, polymeric nanoparticles,
micelles, conjugates, solid dispersion, emulsion, peptide carriers and cyclodextrins (CDs)
have been proposed as suitable and promising systems for enhancing the overall bioavail-
ability, biodistribution and controlled release of Cur, along with reducing Cur toxic effects,
and increased transport across the BBB [
and structural properties, CDs were considered as one of the most promising biocom-
patible nanovectors for Alzheimer’s disease (AD) [13 14]. CDs ( -, -, and -CD) are a
4,7,9–12]. Because of their unique physicochemical
,
α
β
δ
family of natural glucopyranose-based cyclic oligosaccharides, with hydrophobic internal
and hydrophilic external surfaces by the exposure of hydroxyl units [10]. CDs and their
derivatives proved to form complexes with Cur [15
–
21], improving its water solubility
21]. To
by around 100 times, its bioactivity [15 18] and its anti-AD therapeutic effect [19
–
–
our knowledge, the improvement of Cur water solubility and anti-AD therapeutic effi-
cacy with CDs has been investigated, but only in cases of complex formation through
non-covalent binding.
In this current study, we explored two new nanoconjugates based on the covalent bind-
ing of Cur to either one or two β-CD molecules identified as β-CD@Cur and (β-CD)2@Cur,
respectively (Figure 1). The two nanoconjugates were synthesized using Copper-Catalyzed
Azide-Alkyne Cycloaddition (CuAAC) click chemistry. They were characterized by DLS,
NMR (1H, 13C, HMBC and HSQC), HRMS and HPLC techniques. The effect of the co-
valent binding on Cur disaggregation in aqueous conditions was investigated by spec-
troscopic measurements. Finally, cytotoxic studies were performed on primary cultures
of rat embryonic cortical neurons to evaluate the behavior of the nanoconjugates in a
biological environment.
Figure 1.
(A) Chemical structures of β-CD and Cur, and (B) schematic illustration of β-CD@Cur and
(β-CD)2@Cur nanoconjugates obtained through mono- and di-CuAAC reaction.

Int. J. Mol. Sci. 2021, 22, 3255
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2. Results and Discussion
2.1. Synthesis of β-CD@Cur 4 and (β-CD)2@Cur 5 Nanoconjugates
For the synthesis of the nanoconjugates
combined with
To achieve this goal, Cur mono-alkyne
first synthesized as previously described [22,23] (Scheme 1).
β
-CD@Cur
4
and (
β
-CD)2@Cur
5
, Cur was
β
-CD via a triazole covalent linkage using a CuAAC click chemistry strategy.
1
, Cur di-alkyne
2
and mono-6-azido-
β
-CD
3
were
Scheme 1. Synthesis of (A) Cur mono-alkyne
1
and Cur di-alkyne
2
, (B
) mono-6-azido-β-CD
3, and (C
)
β-CD@Cur
4
and
(β-CD)2@Cur 5 nanoconjugates.
The obtained yields of Cur mono-alkyne 1 and Cur di-alkyne 2 were relatively good
(45% and 70%, respectively). These compounds were synthesized separately by mono- and
di-etherification of Cur with propargyl bromide (1.0 equiv. per propargylation) in alkaline
conditions using a previous protocol described by Raja et al. [22] with slight modifications
(Scheme 1A). Compounds
supplementary data).
1 and 2
were characterized by HPLC, 1H NMR and HRMS (see
Mono-6-azido-
previously described by our team [23] with a 49% overall yield (Scheme 1B). These two
steps consisted of mono-tosylation of -CD on the primary OH-6 function, followed by its
nucleophilic substitution by an azide group.
-CD@Cur and ( -CD)2@Cur resulting from single and double “click” conjugations
between alkyne-modified Cur ( or ) and azide-functionalized -CD were obtained
β-CD 3 was prepared in two steps from β-CD according to the protocol
β
β
4
β
5
1
2
β
3
as an orange powder and as a yellowish powder with 40% and 55% yields, respectively
(Scheme 1C).
The purity of the obtained nanoconjugates was determined by analytical HPLC and
NMR analysis. The HPLC purity of both nanoconjugates was determined using reverse
phase and based on the UV-visible detection (254 and 420 nm). As shown in Figure 2, both
β-CD@Cur
4 and (β-CD)2@Cur 5 nanoconjugates showed an excellent purity (94.2% and
95.3% for 4 and 5, respectively).

Int. J. Mol. Sci. 2021, 22, 3255
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Figure 2. Analytical HPLC chromatograms (UV detection at 420 nm) of (
CD)2@Cur nanoconjugates (C18 column, ACN/H2O containing 0.1% TFA, v/v gradients from 10/90
to 100/0 in 25 min, then 100/0 for 10 min).
A) β-CD@Cur 4 and (B) (β-
5
The purity and the successful synthesis of both nanoconjugates were characterized
with 1D and 2D NMR (1H, 13C, HSQC and HMBC) and HRMS analyses. All 1D and 2D
NMR spectra of nanoconjugates
4
and
5
are included in supplemental data (Figures S1–S8)
.
1
In the H NMR spectra of
β-CD@Cur
4
and ( -CD)2@Cur (Figures S1 and S5), the
β
5
characterization was based on the complete clearance of the propargylic CH signal at
3.59 ppm and the appearance of the characteristic signal of the triazole ring at around
8.20 ppm. The obtention of the mono-CuAAC reaction was also evidenced in NMR spectra
1
of
β-CD@Cur
4
by the presence of (i) one hydroxyl group at 9.67 ppm in the H NMR
spectrum (i.e., one phenol group in Cur unit, Figure S1) and (ii) duplicated signals in the
1H and 13C NMR spectra (i.e., H3/H3 , H4/H4 , H6/H6 , H9/H9 and H10/H10 in the 1H
0
0
0
0
0
NMR spectrum, Figure S1, and C2-C10/C2 -C10 in the 13C NMR spectrum, Figure S2) due
0
0
1
to the presence of dissymmetry in
β
-CD@Cur
-CD)2@Cur
4
. Compared to
β-CD@Cur
4
, in the H
and 13C NMR spectra of (
β
5
(Figures S5 and S6), the di-CuAAC reaction was
evidenced by the disappearance of (i) the 1H NMR signal of the hydroxyl group (i.e., no
phenol group in Cur unit, Figure S5) and (ii) the duplication of signals in 1H and 13C NMR
spectra (Figures S5 and S6). Finally, the 1H and 13C NMR signals of
assigned using 2D NMR experiments (Figures S3, S4, S6 and S8).
4 and 5 were fully
2.2. Size and Morphological
To investigate the size and morphology of the developed
CD)2@Cur nanoconjugates, DLS and TEM analyses were performed. As shown in
Figure 3, the synthesized supramolecular nanoconjugates displayed a tendency to self-
assemble in water due to the strong interaction between Cur and -CD (O-H bonds,
β-CD@Cur 4 and (β-
5
β

Int. J. Mol. Sci. 2021, 22, 3255
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Host-Guest, etc.). The self-assembled
of 35.2
The self-assembled (
zeta potential of 25.7
β
-CD@Cur
4
had an average hydrodynamic diameter
±
2.1 nm in the DLS profile and zeta potential of
-CD)2@Cur showed a larger mean diameter of 120.3
0.7 mV (Figure 3B). This increase in the mean diameter may be
−
28.5
±
1.2 mV (Figure 3A).
β
5
±
1.1 nm and
−
±
accounted by the presence of the double amount of β-CD in nanoconjugate 5 compared
to 4.
Figure 3. DLS profiles in water of (
A
)
β-CD@Cur
4
and (B) (β-CD)2@Cur
5
. TEM images of (
C)
β-CD@Cur
4
and
(D) (β-CD)2@Cur 5.
As depicted in TEM images (Figure 3C,D),
β-CD@Cur
4
and (
β-CD)2@Cur
5
showed
spherical morphology with an average size of around 25 nm for
β-CD@Cur
4
(Figure 3C)
and 50 nm spheres aggregated into chains of diameter size around 100 nm (
β-CD)2@Cur
5
(Figure 3D). The size differences obtained by both DLS and TEM are possible, since DLS
and TEM are two different techniques. Indeed, DLS provides the hydrodynamic diameter
of swollen vesicles in solution [24]. TEM allows to obtain two-dimensional image of the
dried form [25]. This size difference could be also linked to the fact that we obtain relatively
high PDI values of around 0.3 for both nanoconjugates resulting in a wide distribution of
sizes and different size visualization in TEM images.
2.3. β-CD Effect on the Cur Disaggregation in Aqueous Medium
Cur is a natural hydrophobic photosensitizer having a low water solubility (i.e.,
11 ng/mL [9]). Generally, hydrophobic photosensitizers have the tendency to self-aggregate
in aqueous media due to their conjugated
π–π
electronic structure resulting in the obtention
of broad UV–vis absorption peaks with low intensity and quenching fluorescence emission.
In the case of Cur, experimental study on the Cur aggregation revealed that its aggre-
gation in water is due to
leading to a red Stockes shift by comparing UV–vis absorption and fluorescence emission
spectra [27]. It is worth noting that -CD is a natural cyclic oligosaccharide consisting of
π-stacking of phenyl rings and H-bonds with side groups [26],
β

Int. J. Mol. Sci. 2021, 22, 3255
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α
-1,4-glucopyranose units. In contrast with curcumin,
electronic structure, with the result that
UV–vis. -CD is biocompatible and biodegradable oligosaccharide known to improve the
solubility, stability and bioavailability of drugs.
The effect of -CD on the disaggregation of Cur in aqueous medium was investigated
by comparing the photophysical properties of nanoconjugates and to those of the free
β
-CD does not have a conjugated
π–
π
β-CD does not absorb and fluoresce in the
β
β
4
5
Cur in water. The UV–vis absorption, fluorescence emission and fluorescence lifetime
spectra in water of the studied molecules are shown in Figure 4. As shown in Figure 4A,
in aqueous conditions (neutral water), Cur exhibited a broad absorption band at around
427 nm with a shoulder at
with the conjugated Cur and the feruloyl unit, respectively [28,29].
∼350 nm, which arise assigned to the π−π* transitions associated
Figure 4. Photophysical properties of Cur,
absorption spectra at a single concentration of Cur (c = 12
spectra at λ = 360 nm (A = 0.2).
β
-CD@Cur
4
and (
β
-CD)2@Cur
5
in D2O at room temperature. (
A) UV–vis
µM), and (
B
) fluorescence emission and ( ) fluorescence lifetime
C
exc
360 nm
By covalent binding of Cur to
β-CD molecules, two important changes in the UV–
vis absorption spectra of Cur were observed. First, a hyperchromic effect was observed
in the UV–vis spectra of both nanoconjugates in D2O, where the absorption intensity
of Cur in water increased significantly depending on the number of
coupled to Cur (Figure 4A), indicating an increase in the solubilization of Cur inside the
nanoconjugates [28 30]. Second, the broad absorption band at 427 nm became narrower
in the case of both nanoconjugates and a significant blue shift to 365 nm occurred in the
case of the nanoconjugate (Figure 4A). Such changes might be due to the Cur -electrons
interaction with the hybridized orbitals of the surface oxygen atoms of -CD, leading to
destabilization of the *-orbitals, and a stabilization of the -orbitals causing a blue shift of
the absorption band of the coupled Cur [29].
The effect of -CD on the fluorescence intensity and lifetime of Cur in D2O was also
β-CD molecules
–
5
π
β
π
π
β
investigated. As shown in Figure 4B, Cur had a tendency to quench in aqueous media
leading to weak fluorescence intensity with a broad emission spectrum centered at 550 nm
and fluorescence lifetime of 1.6 ns (Figure 4C, Table 1). However, by coupling Cur to β-CD,

Int. J. Mol. Sci. 2021, 22, 3255
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a significant enhancement of fluorescence intensity occurred depending on the number of
β-CD coupled to Cur with an important blue-shift in the emission maxima in the case of
nanoconjugate 5 (Figure 4B). This effect could be visually observed under a UV light of
365 nm (top right of Figure 4B). Moreover, fluorescence quantum yield and fluorescence
lifetime increased by increasing covalent binding of Cur to β-CD (Figure 4C, Table 1). This
might be due to a decrease of the aggregation and the presence of the nanoconjugates
monomers in water. Such results demonstrated that: (i) Covalent binding of Cur to
β-CD decreased its self-aggregation in aqueous conditions and (ii) the disaggregation
phenomenon was highly dependent on the number of
more there are, the better).
β-CD units linked to Cur (i.e., the
Table 1. Photophysical properties of Cur and nanoconjugates 4 and 5.
Compounds
λabs
,
(nm)
λ
(nm)
em, max
φf
τ (ns)
max
Cur
β-CD@Cur 4
(β-CD)2@Cur 5
428
426
365
~580
580
507
<<0.01
0.03
0.09
1.6
2.8
6.4
2.4. Treatment of Primary Cultured Neurons with Nanoconjugates 4 and 5
The cytotoxic effect of the synthesized nanoconjugates and on primary cultures of
rat embryo cortical neurons was investigated (the day of cell isolation and preparation of
culture was Day 0 (D0)). To assess cytotoxic effects of -CD@Cur and ( -CD)2@Cur
4
5
β
4
β
5
nanoconjugates, primary cultures of neurons were prepared followed by 24 h incubation
starting on D3 in the absence or presence of the nanoconjugates at Cur concentrations of 2,
5, 10, 15 and 20
µM. Cell viability (Figure 5) was determined by the MTT assay on D4 as
previously described [31,32].
Figure 5. Cytotoxicity of β-CD@Cur 4 and (β-CD)2@Cur 5 nanoconjugates. Primary cultures of rat
embryo cortical neurons were isolated and prepared on Day 0 (D0) in vitro, followed by treatment
on D3 with the indicated concentrations of Cur. MTT assay was performed after 24 h to assess cell
viability. Results are shown as % of viability of control cells treated with the same volume of DMSO
alone; means and SD are shown for two separate cultures each with quadruplicate determinations.
Statistical significance is shown as compared to values obtained from control cells (* p < 0.05,
** p < 0.01).
In Figure 5, Cur showed a drug concentration-dependent cytotoxicity to rat cortical
neurons cells. In fact, a significant decrease in cell viability was observed at Cur levels
starting from 10
µM; only 48% of cells remained viable after treatments with 20 µM Cur.
Such results were probably due to the fact that Cur molecules were not well dispersed

Int. J. Mol. Sci. 2021, 22, 3255
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throughout the cell medium due to its lack of water solubility [24]. However, after click-
covalent binding of Cur with
at corresponding Cur concentrations was observed. The degree of improved cell viability
was also a function of -CD molecules number coupled with Cur viability ( -CD@Cur
< ( -CD)2@Cur ). These results were consistent with the photophysical properties of
the studied molecules (Section 3.3), therefore suggesting that covalent binding of Cur to
-CD could promote its water solubility and reduce Cur toxicity in primary cultures of rat
β-CD, no significant cytotoxicity to rat cortical neurons cells
β
β
4
β
5
β
cortical neurons.
3. Materials and Methods
3.1. Chemicals
All chemicals were used without further purifications.
β-cyclodextrin (β-CD) and
curcumin (Cur) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). All
reactions involving Cur compounds were protected from the light by using aluminum foil
around the reactors and other glassware. Deionized (DI) water was processed in a Milli-Q
water purification system.
3.2. Characterization
The monitoring of the mono- and bi-propargylation of Cur was carried out using
Macharey -Nagel ALUGRAM® SIL > G/UV254 thin-layer chromatography (TLC) plates
and spots were visualized under ultraviolet (UV) light (256 nm and 355 nm). Cur-based
products purifications were performed on Agilent 1100 high-performance liquid chro-
matography (HPLC) (Agilent, Santa Clara, CA, USA) using an Agilent Pursuit 5 C18
reverse phase column (5
µ
m, 150
×
4.6 mm) eluted with acetonitrile/water (ACN/H2O,
v/v) gradients from 10/90 to 100/0 in 25 min, then 100/0 for 10 min containing 0.1% triflu-
oroacetic acid (TFA). A flow rate of 1.0 mL/min for analytical HPLC and of 12 mL/min
for preparative HPLC were used with UV detection at 254 nm and 420 nm. A Brucker
Advance 300 MHz spectrophotometer was used to perform 1D and 2D NMR spectra (i.e.,
1H, 13C, HSQC and HMBC). Deuterated DMSO with residual peaks at
δ
= 2.5 and 39.5 ppm
for 1H and 13C NMR, respectively, was chosen for NMR experiments at room tempera-
ture (T = 298 K). and J represented chemical shifts and coupling constants in parts per
δ
million (ppm) and hertz (Hz), respectively. Singlet (s), doublet (d), triplet (t), multiplet
(m), broad (br) or their combination defined the multiplicity. High-resolution mass spec-
troscopy with electrospray ionization method (ESI-HRMS) was performed using a Brucker
MicrOTOF-Q HR spectrometer. Ultraviolet-Visible (UV–vis) absorption and fluorescence
spectra were recorded on a Shimadzu UV-3600 double-beam UV–vis spectrophotometer
and a Fluorolog-3 spectrofluorometer FL3-222, respectively.
3.3. Synthesis of β-CD@Cur 4 and (β-CD)2@Cur 5 Nanoconjugates
3.3.1. Cur Mono-Alkyne 1 and Cur Di-Alkyne 2
Compounds
1 and 2 were synthesized separately as described in the literature [22]
with slight modifications by reacting Cur (200 mg, 0.543 mmol) in DMF (3 mL) with K2CO3
and propargyl bromide (1.0 equiv. per propargylation) at room temperature under N2 for
48 h. To avoid degradation of Cur, the reactions were performed in the dark. The progress
of the reactions was monitored by TLC with EtOAc/hexane (50/50). After completion of
the reactions, the solvent was removed under reduced pressure. The crude products were
purified by preparative HPLC and the collected pure fractions were lyophilized over 24 h
to yield 1 (45%) and 2 (70%) as orange powders. Compounds 1 and 2 were characterized
by HPLC, 1H NMR and HRMS (See supplementary data).
3.3.2. β-CD@Cur 4 and (β-CD)2@Cur 5 Nanoconjugates
Mono-6-azido-
β-CD 3 was synthesized from β-CD in a two steps-process. The overall
yield was 49%. The protocol has been described recently by our team [23].

Int. J. Mol. Sci. 2021, 22, 3255
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To a solution of Cur mono-alkyne
1
or Cur di-alkyne
2
(0.08 mmol, 1.0 equiv.) and
3
(1.0 equiv. per alkyne function) in H2O/DMF (1/4, v/v, 5 mL) was added successively
CuSO4 5H2O (0.1 equiv. per alkyne function) and Na ascorbate (0.5 equiv. per alkyne
·
function). The reaction mixtures were maintained at room temperature under vigorous
stirring in the dark for 24 h. The solvent was evaporated under reduced pressure. Prepar-
ative HPLC was used to purify the crude product and the collected pure fractions were
lyophilized over 24 h to afford -CD)2@Cur
powder and a yellowish powder, respectively.
-CD@Cur δ 2.85–4.45 (m, 54H, H2, H3, H4, H5, H6,
: 1H NMR (300 MHz, DMSO-d6):
β-CD@Cur
4
(40%) and (
β
5 (55%) as an orange
β
4
OH6, H7-OMe and H70-OMe), 4.48–5.26 (m, 13H, H1, OH2, OH3, H1 and H11), 5.26–6.08
(3, 12H, OH2 and OH3), 6.75 (d, J = 8.4 Hz, 1H, H90), 6.76 (d, J = 15.9 Hz, 1H, H30), 6.81 (d,
J = 15.6 Hz, 1H, H3), 6.83 (d, J = 8.4 Hz, 1H, H9), 7.14 (d, J = 8.4 Hz, 1H, H10), 7.21 (br s,
1H, H60), 7.29 (d, J = 8.4 Hz, 1H, H100), 7.32 (br s, 1H, H6), 7.56 (d, J = 15.9 Hz, 1H, H40),
7.58 (d, J = 15.6 Hz, 1H, H4), 8.19 (s, 1H, H13), 9.68 (br s, 1H, H80-OH). 13C NMR (75 MHz,
DMSO-d6):
δ
50.4 (C11), 55.6 (C7-OMe and C70-OMe), 59.1 (C6), 59.9 (C6), 61.5 (C1), 70.0
(C2, C3 and C5), 72.1 (C2, C3 and C5), 72.4 (C2, C3 and C5), 73.0 (C2, C3 and C5), 81.5 (C4),
82.1 (C4), 83.4 (C4), 100.9 (C1), 101.9 (C1), 111.4 (C6), 113.2 (C60), 115.7 (C9 and C90), 121.1
(C30), 12.3 (C3), 122.7 (C100), 123.2 (C10), 125.8 (C13), 126.3 (C50), 128.0 (C5), 140.1 (C40),
141.0 (C4), 142.2 (C12), 148.0 (C80), 149.2 (C7), 149.4 (C70), 149.7 (C8), 182.7 (C2 and C20).
HRMS (ESI) for C66H91N3O40: [M + H]+ calculated 1566.5257, found 1566.5531, [M + Na]+
calculated 1588.5076, found 1588.5293 and [M + 2H]2+ calculated 783.7667, found 783.7844.
1
(
β-CD)2@Cur
5
: H NMR (300 MHz, DMSO-d6):
δ
3.04–3.75 (m, 93H, H2, H3, H4, H5,
H6 and OH6), 3.78 (s, 6H, H7-OMe), 3.95–4.07 (m, 3H, OH6), 4.47–5.62 (m, 48H, H1, OH2,
OH3, H1 and H11), 7.00 (d, J = 15.3 Hz, 2H, H3), 7.20 (d, J = 8.4 Hz, 2H, H9), 7.31 (d, J = 8.4
Hz, 2H, H10), 7.37 (s, 2H, H6), 7.61 (d, J = 15.3 Hz, 2H, H4), 8.17 (s, 2H, H13). 13C NMR
(75 MHz, DMSO-d6):
δ 50.4 (C11), 55.7 (C7-OMe), 59.0 (C6), 59.9 (C6), 61.5 (C1), 70.0 (C2,
C3 and C5), 71.7 (C2, C3 and C5), 72.1 (C2, C3 and C5), 72.4 (C2, C3 and C5), 73.0 (C2, C3
and C5), 81.1 (C4), 81.5 (C4), 82.1 (C4), 83.4 (C4), 101.3 (C1), 102.0 (C1), 111.3 (C6), 112.9
(C9), 118.8 (C3), 124.1 (C10), 125.8 (C13), 127.0 (C5), 142.0 (C12), 144.7 (C4), 149.1 (C8), 150.5
(C7), 193.1 (C2). HRMS (ESI) for C111H162N6O74: [M + 2H]2+ calculated 1382.4627, found
1382.5022, [M + 2Na]2+ calculated 1404.4446, found 1404.4793 and [M + 2K]2+ calculated
1420.4186, found 1420.4437.
3.4. Nanoconjugates Size and ζ-Potential Measurements
The size, polydispersity index (PDI) and ζ-potential of the synthesized nanoconju-
gates were measured following their dilution with ultrapure water (1:200) using DLS
(Zetasizer Nano ZS, Malvern, UK). Nanoconjugates were examined by standard capillary
electrophoresis cell. The size and ζ
-potential were measured at room temperature (25 ◦C)
using a 0.01 absorbance under a scattering angle and a refractive index of 173◦ and 1.59,
respectively.
3.5. Nanoconjugates Morphological Study
The morphology of the synthesized nanoconjugates in water was monitored via TEM
using a negative staining method. In brief, the nanoconjugates were first diluted in distilled
water. A 2% ammonium molybdate solution was added with a ratio of 1:1. The mixtures
were reserved at room temperature for 3 min. Then, one drop of the nanoconjugates
suspension was deposited and dried on a grid type EMS CF200-Cu Formvar carbon-coated
copper grid (200 mesh, 3 mm diameter HF 36). The nanoconjugates morphology was
studied with a Philips CM20 TEM equipped with an Olympus TEM CCD camera at 200 kV.
3.6. β-CD Effect on Cur Disaggregation in Aqueous Medium
The influence of the presence of
CD)2@Cur nanoconjugates in water at room temperature was studied by comparing their
UV–vis absorption, fluorescence emission and fluorescence lifetime spectra with those of
β-CD on the solubility of Cur in β-CD@Cur 4 and (β-
5

Int. J. Mol. Sci. 2021, 22, 3255
10 of 13
free Cur in the same conditions. Briefly, each product was dissolved in D2O. Fluorescence
(Φf) quantum yield was determined using Quinine (Quin) solution in acetonitrile as
fluorescence standard (Φf = 0.55) [33].
3.7. Cell Cytotoxicity Studies
3.7.1. Primary Culture of Cortical Neurons
Cell culture studies were carried out at the Bioavailability–Bioactivity (Bio-DA, Lor-
raine, France) platform. Primary cultures of cortical neurons were prepared from rat
fetuses (day 16–17 of gestation) as previously reported [31,32]. Cells were plated at
10 × 104 cells/cm2 onto plates and cultured in neuronal culture medium M2 [DMEM-
F12 (Invitrogen, Illkirch, France) medium containing 0.5 µM insulin, 60 µM putrescine,
30 nM sodium selenite, 100 µM transferrin, 10 nM progesterone and 0.1% (w/v) ovalbumin
(Sigma-Aldrich (Saint-Quentin Fallavier, France)]. Neurons were incubated at 35 ◦C in 6%
CO2 with day (D) 0 being the day of the preparation of the cells. β-CD, Cur, β-CD@Cur 4
and (
β
-CD)2@Cur
5
were first dissolved in DMSO (Final volume of DMSO
≤
0.25%), and
the solutions were diluted to the desired concentrations in M2 culture medium. On Day 3,
the neuronal culture medium was treated with each compound separately at a final Cur
concentration of 2–20 µ
M and incubated for 24 h at 35 ◦C in 6% CO2. Control cells were
treated with the same concentrations of DMSO alone that are not toxic for cells.
3.7.2. MTT Test
The neuronal viability of the different molecules was assessed using the 3-(4,5 dimethy-
lthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich (Saint-Quentin Fallav-
ier, France)) reduction activity assay. MTT solution (4 mg/mL, 37 ◦C) was added to each
well and the plates were then incubated for 1 h at 35 ◦C in 6% CO2. Media was removed
and replaced with DMSO followed by incubation while shaking at room temperature for
10 min. The absorbance of formazan was measured at wavelength of 570 nm [(Fluostar
Galaxy, Biolise software) (BMG Lab technologies, Offenberg, Germany)]. Cell viability
was expressed as percentage of living cells compared to values obtained from control
untreated cells.
3.7.3. Statistical Analysis
Experiments were performed in triplicate, and results are shown as the mean
unless otherwise indicated. A variance analysis was done by ANOVA supplemented with
±
SD,
a Scheffe’s post hoc test and a p-value greater than 0.05 was regarded as significant.
4. Conclusions
Two novel nanoconjugates denoted
β-CD@Cur and (β-CD)2@Cur self-assembled
in water to form nanoparticles with an average diameter size of around 35 and 120 nm,
respectively. These nanoconjugates were successfully synthesized using CuAAC click
chemistry strategy via a triazole covalent link between Cur and β-CD molecules. The
chemical structure and purity of the synthesized nanoconjugates was checked by NMR
spectroscopy (1H, 13C, HSQC and HMBC), ESI-HRMS and HPLC analyses. Furthermore,
the
effect on rat cortical neurons were investigated.
The findings indicated that -CD covalent binding with Cur led to an improvement
β-CD effect on the disaggregation of Cur in aqueous medium and cell cytotoxicity
β
of its solubility and photophysical properties in aqueous conditions preventing cytotoxic
effects. Firstly, UV–vis absorption and fluorescence emission spectra in D2O revealed
that, compared to free Cur, water solubility of the nanoconjugates increased with the
number of
restoration of fluorescence. Secondly, the addition of
toxicity of Cur illustrated by around 90% and 80% rat cortical neurons viability for (
CD)2@Cur and -CD@Cur nanoconjugate, respectively, at 20 M concentration, compared
to only 48% viability with free Cur at the same concentration. These results showed the
β-CD resulting in an improvement of the absorption intensity and in the
β-CD reduced significantly the
β-
β
µ

Int. J. Mol. Sci. 2021, 22, 3255
11 of 13
potential interest of these nanoconjugates as possible new Cur derivatives for application
in neurodegenerative diseases prevention. The next step will be to determine the efficacy
of curcumin’s neuroprotective effects of these two nanoconjugates, including protection
against Aβ cell toxicity, and antioxidative properties using cell and animal models, with
the goal towards developing prevention strategies to reduce risk of neurodegenerative
diseases.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-0
067/22/6/3255/s1, Figure S1: 1H NMR spectrum (300 MHz, DMSO-d6, 298 K) of
nanoconjugate, Figure S2: J mod 13C NMR spectrum (75 MHz, DMSO-d6, 298 K) of
nanoconjugate, Figure S3: 2D HSQC NMR spectrum (300 MHz, DMSO-d6, 298 K) of
β
-CD@Cur
-CD@Cur
-CD@Cur
4
4
4
β
β
nanoconjugate, Figure S4: 2D HMBC NMR spectrum (300 MHz, DMSO-d6, 298 K) of
nanoconjugate, Figure S5: 1H NMR spectrum (300 MHz, DMSO-d6, 298K) of (
-CD)2@Cur
-CD)2@Cur
β-CD@Cur
4
β
5
5
5
5
nanoconjugate, Figure S6: J mod 13C NMR spectrum (75 MHz, DMSO-d6, 298 K) of (
nanoconjugate, Figure S7: 2D HSQC NMR spectrum (300 MHz, DMSO-d6, 298 K) of (
nanoconjugate, Figure S8: 2D HMBC NMR spectrum (300 MHz, DMSO-d6, 298 K) of (
nanoconjugate.
β
β-CD)2@Cur
β-CD)2@Cur
Author Contributions: Conceptualization, A.B.M., S.A. and E.A.-T.; methodology, A.B.M., S.A., C.M.
and E.A.-T.; software, A.B.M.; validation, A.B.M., S.A., C.F., C.M., F.T.Y. and E.A.-T.; formal analysis,
A.B.M., S.A., C.M. and E.A.-T.; investigation, A.B.M.; resources, A.B.M., S.A., F.T.Y. and E.A.-T.; data
curation, A.B.M., S.A. and F.T.Y.; writing—original draft preparation, A.B.M. and S.A.; writing—
review and editing, A.B.M., S.A., C.F., F.T.Y. and E.A.-T.; visualization, A.B.M.; supervision, S.A.,
C.F., C.M. and E.A.-T.; project administration, S.A., C.F., C.M., F.T.Y. and E.A.-T.; funding acquisition,
A.B.M., S.A., C.F., C.M., F.T.Y. and E.A.-T. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by “Impact Biomolecules” project of the “Lorraine Université
d’Excellence” (in the context of the «Investissements d’avenir» program implemented by the French
National Research Agency (ANR)).
Institutional Review Board Statement: The animals were housed in government-certified animal
facilities in accordance with the European Communities Council Directive (EU 2010/63) for the use
and care of laboratory animals. Since the procedure for preparation of rat embryo cortical neurons
involved tissue sampling after animal sacrifice using authorized methodology (overdose of isoflurane
gas anesthesia), ethical review and approval were waived for this study (directive 2013-118).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or supplementary material.
Acknowledgments: The authors gratefully acknowledge Olivier Fabre for running NMR experi-
ments, Solenne Fleutot and Thomas Girardet for TEM experiments and, Marion Huguet and Claire
Soligot for isolation of Rat Embryonic Neural Cells.
Conflicts of Interest: The authors declare no competing financial interest.
Abbreviations
Cur
Curcumin
β-CD
β-Cyclodextrin
CuAAC Copper-catalyzed azide–alkyne cycloaddition
BBB
AD
Blood Brain Barrier
Alzheimer’s disease
References
1.
Erkkinen, M.G.; Kim, M.-O.; Geschwind, M.D. Clinical Neurology and Epidemiology of the Major Neurodegenerative Diseases.
Cold Spring Harb. Perspect. Biol. 2018, 10, a033118. [CrossRef]
2.
Bertram, L.; Tanzi, R.E. The genetic epidemiology of neurodegenerative disease. J. Clin. Investig. 2005, 115, 1449–1457. [CrossRef]

Int. J. Mol. Sci. 2021, 22, 3255
12 of 13
3.
Torres-Ramos, Y.; Montoya-Estrada, A.; Cisneros, B.; Tercero-Pérez, K.; León-Reyes, G.; Leyva-García, N.; Hernández-Hernández,
O.; Magaña, J.J. Oxidative Stress in Spinocerebellar Ataxia Type 7 Is Associated with Disease Severity. Cerebellum 2018, 17, 601–609.
[CrossRef] [PubMed]
4.
5.
Grabrucker, A.M.; Ruozi, B.; Belletti, D.; Pederzoli, F.; Forni, F.; Vandelli, M.A.; Tosi, G. Nanoparticle transport across the blood
brain barrier. Tissue Barriers 2016, 4, e1153568. [CrossRef] [PubMed]
Voulgaropoulou, S.D.; Van Amelsvoort, T.; Prickaerts, J.; Vingerhoets, C. The effect of curcumin on cognition in Alzheimer’s
disease and healthy aging: A systematic review of pre-clinical and clinical studies. Brain Res. 2019, 1725, 146476. [CrossRef]
[PubMed]
6.
7.
Mathew, A.; Fukuda, T.; Nagaoka, Y.; Hasumura, T.; Morimoto, H.; Yoshida, Y.; Maekawa, T.; Venugopal, K.; Kumar, D.S.
Curcumin Loaded-PLGA Nanoparticles Conjugated with Tet-1 Peptide for Potential Use in Alzheimer’s Disease. PLoS ONE 2012
7, e32616. [CrossRef]
,
Giacomeli, R.; Izoton, J.C.; dos Santos, R.B.; Boeira, S.P.; Jesse, C.R.; Haas, S.E. Neuroprotective effects of curcumin lipid-core
nanocapsules in a model Alzheimer’s disease induced by
146325. [CrossRef]
β-amyloid 1-42 peptide in aged female mice. Brain Res. 2019, 1721,
8.
9.
Maiti, P.; Dunbar, G.L. Use of Curcumin, a Natural Polyphenol for Targeting Molecular Pathways in Treating Age-Related
Neurodegenerative Diseases. Int. J. Mol. Sci. 2018, 19, 1637. [CrossRef] [PubMed]
Jampílek, J.; Král’ová, K.; Novák, P.; Novák, M. Nanobiotechnology in Neurodegenerative Diseases; Rai, M., Yadav, A., Eds.; Springer
Nature: Cham, Switzerland, 2019; Volume 4, pp. 65–138. [CrossRef]
10. Del Prado-Audelo, M.L.; Caballero-Florán, I.H.; Meza-Toledo, J.A.; Mendoza-Muñoz, N.; González-Torres, M.; Florán, B.; Cortés,
H.; Leyva-Gómez, G. Formulations of Curcumin Nanoparticles for Brain Diseases. Biomolecules 2019, 9, 56. [CrossRef]
11. Sun, M.; Su, X.; Ding, B.; He, X.; Liu, X.; Yu, A.; Lou, H.; Zhai, G. Advances in nanotechnology-based delivery systems for
curcumin. Nanomedicine 2012, 7, 1085–1100. [CrossRef]
12. Del Prado-Audelo, M.; Magaña, J.; Mejía-Contreras, B.; Borbolla-Jiménez, F.; Giraldo-Gomez, D.; Piña-Barba, M.; Quintanar-
Guerrero, D.; Leyva-Gómez, G. In vitro cell uptake evaluation of curcumin-loaded PCL/F68 nanoparticles for potential application
in neuronal diseases. J. Drug Deliv. Sci. Technol. 2019, 52, 905–914. [CrossRef]
13. Camilleri, P.; Haskins, N.J.; Hewlett, D.R. β-Cyclodextrin interacts with the Alzheimer amyloid β-A4 peptide. FEBS Lett. 1994,
341, 256–258. [CrossRef]
14. Yao, J.; Ho, D.; Calingasan, N.Y.; Pipalia, N.H.; Lin, M.T.; Beal, M.F. Neuroprotection by cyclodextrin in cell and mouse models of
Alzheimer disease. J. Exp. Med. 2012, 209, 2501–2513. [CrossRef] [PubMed]
15. Dandawate, P.R.; Vyas, A.; Ahmad, A.; Banerjee, S.; Deshpande, J.; Swamy, K.V.; Jamadar, A.; Dumhe-Klaire, A.C.; Padhye, S.;
Sarkar, F.H. Inclusion Complex of Novel Curcumin Analogue CDF and β-Cyclodextrin (1:2) and Its Enhanced In Vivo Anticancer
Activity Against Pancreatic Cancer. Pharm. Res. 2012, 29, 1775–1786. [CrossRef]
16. Tomren, M.; Masson, M.; Loftsson, T.; Tønnesen, H.H. Studies on curcumin and curcuminoids: XXXI. Symmetric and asymmetric
curcuminoids: Stability, activity and complexation with cyclodextrin. Int. J. Pharm. 2007, 338, 27–34. [CrossRef]
17. Yallapu, M.M.; Jaggi, M.; Chauhan, S.C. β-Cyclodextrin-curcumin self-assembly enhances curcumin delivery in prostate cancer
cells. Colloids Surf. B Biointerfaces 2010, 79, 113–125. [CrossRef]
18. Hegge, A.B.; Vukicevic, M.; Bruzell, E.; Kristensen, S.; Tønnesen, H. Solid dispersions for preparation of phototoxic supersaturated
solutions for antimicrobial photodynamic therapy (aPDT): Studies on curcumin and curcuminoides L. Eur. J. Pharm. Biopharm.
2013, 83, 95–105. [CrossRef] [PubMed]
19. Quitschke, W.W.; Steinhauff, N.; Rooney, J. The effect of cyclodextrin-solubilized curcuminoids on amyloid plaques in Alzheimer
transgenic mice: Brain uptake and metabolism after intravenous and subcutaneous injection. Alzheimer’s Res. Ther. 2013, 5, 16.
[CrossRef]
20. Cheng, K.K.; Yeung, C.F.; Ho, S.W.; Chow, S.F.; Chow, A.H.L.; Baum, L. Highly Stabilized Curcumin Nanoparticles Tested in an In
Vitro Blood–Brain Barrier Model and in Alzheimer’s Disease Tg2576 Mice. AAPS J. 2012, 15, 324–336. [CrossRef]
21. Ramdani, L.; Bourboulou, R.; Belkouch, M.; Jebors, S.; Tauran, Y.; Parizot, C.; Suwinska, K.; Coleman, A.W.; Duyckaerts, C.; Lazar,
A.N. Multifunctional Curcumin-Nanocarriers Based on Host-Guest Interactions for Alzheimer Disease Diagnostic. J. Nanomed.
Nanotechnol. 2015, 6, 2. [CrossRef]
22. Raja, K.; Alonso, A.; Banerjee, P.; Dolai, S.; Corbo, C.; Averick, S.; Mogha, A.; Debnath, S. Curcumin Derivatives. WO
2011106691A2. 2011. Available online: https://patentimages.storage.googleapis.com/44/6a/79/e7561b802ebbd8/WO20111
06691A2.pdf (accessed on 23 March 2021).
23. Ben Mihoub, A.; Youssef, Z.; Colombeau, L.; Juan-Hureaux, V.; Arnoux, P.; Frochot, C.; Vanderesse, R.; Acherar, S. Inclusion
complex vs. conjugation of hydrophobic photosensitizers with β-cyclodextrin: Improved disaggregation and photodynamic
therapy efficacy against glioblastoma cells. Mater. Sci. Eng. C 2020, 109, 110604. [CrossRef]
24. Sun, T.; Zhang, H.; Kong, L.; Qiao, H.; Li, Y.; Xin, F.; Hao, A. Controlled transformation from nanorods to vesicles induced by
cyclomaltoheptaoses (β-cyclodextrins). Carbohydr. Res. 2011, 346, 285–293. [CrossRef]
25. Provder, T. Challenges in particle size distribution measurement past, present and for the 21st century. Prog. Org. Coatings 1997
,
32, 143–153. [CrossRef]
26. Hamaguchi, T.; Ono, K.; Yamada, M. REVIEW: Curcumin and Alzheimer’s Disease. CNS Neurosci. Ther. 2010, 16, 285–297.
[CrossRef]

Int. J. Mol. Sci. 2021, 22, 3255
13 of 13
27. Kee, T.W.; Adhikary, R.; Carlson, P.J.; Mukherjee, P.; Petrich, J.W. Femtosecond Fluorescence Upconversion Investigations on the
Excited-State Photophysics of Curcumin. Aust. J. Chem. 2011, 64, 23–30. [CrossRef]
28. Mandal, S.; Banerjee, C.; Ghosh, S.; Kuchlyan, J.; Sarkar, N. Modulation of the Photophysical Properties of Curcumin in Nonionic
Surfactant (Tween-20) Forming Micelles and Niosomes: A Comparative Study of Different Microenvironments. J. Phys. Chem. B
2013, 117, 6957–6968. [CrossRef]
29. Gonçalves, J.L.; Valandro, S.R.; Poli, A.L.; Schmitt, C.C. Influence of clay minerals on curcumin properties: Stability and singlet
oxygen generation. J. Mol. Struct. 2017, 1143, 1–7. [CrossRef]
30. Baglole, K.N.; Boland, P.G.; Wagner, B.D. Fluorescence enhancement of curcumin upon inclusion into parent and modified
cyclodextrins. J. Photochem. Photobiol. A Chem. 2005, 173, 230–237. [CrossRef]
31. Malaplate, C.; Poerio, A.; Huguet, M.; Soligot, C.; Passeri, E.; Kahn, C.J.F.; Linder, M.; Arab-Tehrany, E.; Yen, F.T. Neurotrophic
Effect of Fish-Lecithin Based Nanoliposomes on Cortical Neurons. Mar. Drugs 2019, 17, 406. [CrossRef]
32. Colin, J.; Allouche, A.; Chauveau, F.; Corbier, C.; Pauron-Gregory, L.; Lanhers, M.-C.; Claudepierre, T.; Yen, F.T.; Oster, T.;
Malaplate-Armand, C. Improved Neuroprotection Provided by Drug Combination in Neurons Exposed to Cell-Derived Soluble
Amyloid-β Peptide. J. Alzheimer’s Dis. 2016, 52, 975–987. [CrossRef]
33. Brouwer, A.M. Standards for photoluminescence quantum yield measurements in solution (IUPAC Technical Report). Pure Appl.
Chem. 2011, 83, 2213–2228. [CrossRef]