Preparation of benzothiazolyl-decorated nanoliposomes

Article abstract of DOI:10.3390/molecules24081540

Amyloid β (Aβ) species are considered as potential targets for the development of diagnostics/therapeutics towards Alzheimer’s disease (AD). Nanoliposomes which are decorated with molecules having high affinity for Aβ species may be considered as potential carriers for AD theragnostics. Herein, benzothiazolyl (BTH) decorated nanoliposomes were prepared for the first time, after synthesis of a lipidic BTH derivative (lipid-BTH). The synthetic pathway included acylation of bis(2-aminophenyl) disulfide with palmitic acid or palmitoyl chloride and subsequent reduction of the oxidized dithiol derivative. The liberated thiols were able to cyclize to the corresponding benzothiazolyl derivatives only after acidification of the reaction mixture. Each step of the procedure was monitored by HPLC analysis in order to identify all the important parameters for the formation of the BTH-group. Finally, the optimal methodology was identified, and was applied for the synthesis of the lipid-BTH derivative. BTH-decorated nanoliposomes were then prepared and characterized for physicochemical properties (size distribution, surface charge, physical stability, and membrane integrity during incubation in presence of buffer and plasma proteins). Pegylated BTH-nanoliposomes were demonstrated to have high integrity in the presence of proteins (in comparison to non-peglated ones) justifying their further exploitation as potential theragnostic systems for AD.

Full text of DOI:10.3390/molecules24081540

molecules
Article
Preparation of
Benzothiazolyl-Decorated Nanoliposomes
Spyridon Mourtas 1,2,*, Panayiota Christodoulou 1, Pavlos Klepetsanis 1,2, Dimitrios Gatos 3,
Kleomenis Barlos 3,4 and Sophia G. Antimisiaris 1,2
1
Laboratory of Pharmaceutical Technology, Dept. of Pharmacy, School of Health Sciences, University of
Patras, 26510 Rio, Greece; pani_christodoulou@hotmail.com (P.C.); klepe@upatras.gr (P.K.);
S.Antimisiaris@upatras.gr (S.G.A.)
Institute of Chemical Engineering Sciences of the Foundation for Research and Technology
2
Hellas (FORTH/IEC-HT), 26504 Rio Patras, Greece
Department of Chemistry, University of Patras, 26510 Rio Patras, Greece; d.gatos@upatras.gr (D.G.);
3
barlos@cblpatras.gr (K.B.)
CBL-Patras, Patras Industrial Area, Block 1, 25018 Patras, Greece
4
*
Correspondence: s.mourtas@upatras.gr; Tel.: +30-2610-962332; Fax: +30-2610-277245
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Simona Rapposelli
Received: 16 March 2019; Accepted: 17 April 2019; Published: 18 April 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Amyloid
diagnostics/therapeutics towards Alzheimer’s disease (AD). Nanoliposomes which are decorated
with molecules having high affinity for A species may be considered as potential carriers for AD
β (Aβ) species are considered as potential targets for the development of
β
theragnostics. Herein, benzothiazolyl (BTH) decorated nanoliposomes were prepared for the first
time, after synthesis of a lipidic BTH derivative (lipid-BTH). The synthetic pathway included acylation
of bis(2-aminophenyl) disulfide with palmitic acid or palmitoyl chloride and subsequent reduction
of the oxidized dithiol derivative. The liberated thiols were able to cyclize to the corresponding
benzothiazolyl derivatives only after acidification of the reaction mixture. Each step of the procedure
was monitored by HPLC analysis in order to identify all the important parameters for the formation
of the BTH-group. Finally, the optimal methodology was identified, and was applied for the synthesis
of the lipid-BTH derivative. BTH-decorated nanoliposomes were then prepared and characterized
for physicochemical properties (size distribution, surface charge, physical stability, and membrane
integrity during incubation in presence of buffer and plasma proteins). Pegylated BTH-nanoliposomes
were demonstrated to have high integrity in the presence of proteins (in comparison to non-peglated
ones) justifying their further exploitation as potential theragnostic systems for AD.
Keywords: amyloid β; targeting; benzothiazoles; functionalization; nanoliposomes
1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous
system (CNS). Several pathological hallmarks of AD have been identified through the years, such
as decreased cholinergic neurons and acetylcholine (ACh) levels, plaques caused by aggregation
of protein fragments of amyloid-
protein, and inflammation and increased oxidative stress from reactive oxygen species (ROS) [
Although the exact cause of AD still remains unknown, several approaches aimed at inhibiting disease
progression have advanced to clinical trials [ ]. It has been shown that unbalanced A production
and clearance results in rising A monomer levels in the brain, promoting the formation of dimers and
larger oligomers. Then, the oligomers progressively aggregate to form protofibrils, fibrils, and plaques.
species are neurotoxic, and are considered as one of the major histopathological hallmarks of
β
(A
β), tangles associated with irregular phosphorylation of tau
1,2].
3–5
β
β
A
β
Molecules 2019, 24, 1540; doi:10.3390/molecules24081540
www.mdpi.com/journal/molecules

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AD thereby various methodologies and approaches to target the production and/or clearance of
such amyloid-beta (Aβ) peptide species are currently considered for therapeutic and/or diagnostic
purposes [1,6–13].
Surface functionalization of biocompatible/biodegradable and stealth nanoparticles has been
extensively used as a method to increase the bioavailability of nanoparticle-associated drugs and/or
increase nanoparticle binding affinity to specific receptors due to multivalency [14–16]. Among the
known nanoparticle types, nanoliposomes have many advantages for drug delivery applications due to
their non-toxic/non-immunogenic, fully biodegradable, and structurally versatile nature [17]. Examples
of surface decorated nanoliposomes proposed for AD diagnosis/therapy include phosphatidic acid
(PA) and cardiolipin (CL) nanoliposomes which were able to target aggregated forms of A
high binding affinity (KD: 22–60 nM) [18]. Additionally, nanoliposomes decorated with curcumin
derivatives demonstrated high affinity for A 1-42 fibrils (KD: 1–5 nM) and sufficient integrity/stability
for in vivo applications [19], while anti-A monoclonal antibody (A -MAb) decorated nanoliposomes
demonstrated high affinity towards A monomers and fibrils (with KD values between 0.5 and 2 nM) [20].
β1-42 with
β
β
β
β
Also, nanoliposomes which were decorated with tetracycline derivatives [21] and non-planar curcumin
derivatives [19,22,23] were efficient in delaying the aggregation of Aβ peptide monomers.
Taking into consideration that thioflavin-T (ThT) is the most widely used (in vitro) indicator of A
aggregation displaying fluorescence enhancement and a characteristic red shift when binding to A
β
β
aggregates, the decoration of nanoliposomes with benzothiazoles (BTH), the moiety that is responsible
for the affinity of ThT towards A aggregates [24 26], may potentially be an improved method for
targeting. In fact, benzothiazoles bearing only the 2-benzothiazolyl-moiety [27 30], or more
complicated 2-benzothiazolyl-derivatives [31 36], have been prepared and tested for their affinity
β
–
A
β
–
–
towards amyloids, however none of the previous molecules were ever tested after immobilization
on nanoliposomes. Additionally, it should be pointed out that the bulky and ionic nature of ThT is a
negative parameter for its permeation across the blood–brain barrier (BBB), explaining why no benefits
were obtained from this compound in vivo [37–40].
In this context, we focused the current study on the decoration of nanoliposomes with non-charged,
less hindered BTH-groups, which have also been proven to possess affinity towards Aβ species [27–30].
In order to develop methods for efficient nanoliposome functionalization with BTH-groups, we
synthesized a BTH lipid-derivative.
2. Results and Discussion
2.1. Synthesis of Compounds
2.1.1. Optimization of BTH Formation
Since the reaction of acyl chlorides with 2-aminobenzenethiol
to produce by-products [41], we decided to use bis(2-aminophenyl) disulfide
1
in presence of a base is known
for the synthesis of a
2
novel lipid-benzothiazolyl derivative (lipid-BTH). For this, bis(2-aminophenyl) disulfide was initially
synthesized, by oxidation of 2-aminothiophenol with 35% hydrogen peroxide (H2O2) (Scheme 1).
NH2
NH2
SH
35% H2O2
1h, rt
S
S
NH2
1
2
Scheme 1. Synthesis of bis(2-aminophenyl) disulfide.
In order to identify the optimal conditions for the preparation of lipid-BTH, we reacted palmitic
acid with bis(2-aminophenyl) disulfide and N,N0-diisopropylcarbodiimide (DIC) as activator of the
carboxylic groups; or palmitoyl chloride and triethylamine (NEt3) as a HCl scavenger (Scheme 2).

Molecules 2019, 24, 1540
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O
NH
NH2
O
S
S
DIC (X=OH) or
NEt3 (X=Cl)
4a
4b
X
+
3
+
O
NH2
S
NH
S
S
NH2
2
2
NaBH4 / EtOH
O
AcOH
NH
S
SH
N
6 = Palm-BTH
5
Scheme 2. Synthetic method for the formation of Palm-BTH; Compound
3 was used either as palmitic
acid (X=OH) when N,N0-diisopropylcarbodiimide (DIC) was used as a condensing agent, or as
palmitoyl chloride (X=Cl) when triethylamine (NEt3) was used as a base.
The reaction of palmitic acid and bis(2-aminophenyl) disulfide in presence of DIC, formed,
as expected, the mono- 4a and bis-coupled 4b products. The reduction of the mixture (4a
by the use of NaBH4 in EtOH which gave the desired Palm-BTH derivative , after the addition of acetic
acid (AcOH) (Scheme 2). In order to identify the best reaction conditions and the conditions that are
required for the cyclization of the reduced compound , to the corresponding Palm-BTH , we followed
+4b) was done
6
5
6
the synthetic procedure by HPLC analysis (Figures 1 and 2). It was found that, the coupling reaction
between palmitic acid and bis(2-aminophenyl) disulfide gave a mixture of 4a (Figure 1; 16.0 min) and
4b (Figure 1; 20.8 min) products, as expected.
Figure 1. HPLC analysis during the reaction between bis(2-aminophenyl) disulfide and palmitic acid
in tetrahydrofuran (THF), using DIC as condensing agent; (
A) t=1d at rt; (B) t=3d at rt; Column:
LiChrospher® 100 RP-18 (5 m) LiChroCART® 250-4; Mobile phase: THF/H2O; Gradient: 50% THF to
µ
100% THF in 30 min; Flow rate: 1ml/min; Detection at 254 nm.
Although the coupling reaction was rather slow, as indicated by HPLC analysis (Figure 1), no
by-products were formed, even after 7 days of reaction between
rt (Figure 2A). The reaction mixture was then reduced with NaBH4 in EtOH. HPLC analysis of the
reduction mixture proved the existence of the reduced free thiol (Figure 2B; 11 min). It was also
proven from the HPLC results that, in the absence of AcOH, the reduced compound does not form the
desired BTH-product but isomerizes back to the oxidized 4b during the isolation process (Figure 2C).
In order to enable cyclization of , we used acetic acid (AcOH), which favors the cyclization of the
reduced free thiol group of to the desired Palm-BTH ( : Figure 2D; 17 min), after 1h stirring of the
acidified mixture at rt. This experiment revealed the importance of using an acid as catalyst, in order
to effectively enable the cyclization of 2-N-palmitoyl-aminobenzenethiol and the formation of the
2 and the activated palmitic acid at
5
5
5
5
6
5

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desired Palm-BTH
6
[
42
,43]. Herein we selected AcOH, which is an inexpensive, readily available and
easily handled reagent.
Figure 2. HPLC analysis of (
disulfide and palmitic acid in THF by using DIC as condensing agent; (
4a 4b with NaBH4; ( ) after the reduction of 4a 4b with NaBH4 without subsequent acidification of
the mixture; ( ) after the reduction of 4a 4b with NaBH4 and subsequent acidification with AcOH;
Column: LiChrospher® 100 RP-18 (5 m) LiChroCART® 250-4; Mobile phase: THF/H2O; Gradient:
A
) the final mixture of 4a
+4b after 7 days reaction at rt of bis(2-aminophenyl)
B
) during the reduction of
+
C
+
D
+
µ
50% THF to 100% THF in 30 min; Flow rate: 1mL/min; Detection at 254nm.
In another effort, the reaction of 2-aminobenzenethiol with palmitoyl chloride in presence of
NEt3, formed, in addition to the desired bis-coupled product 4b (Figure 3A: 20.8 min), an unexpected
by-product (Figure 3A; 14.5 min). Further reduction of the product mixture with NaBH4 and addition
of AcOH gave the expected Paml-BTH, after the reduction and cyclization of 4b, but the by-product
was not affected, being present in the final reaction mixture (Figure 3B). Although the by-product was
not further analyzed, this is an obvious drawback of this specific method for synthesis of Palm-BTH.
Figure 3. HPLC analysis of (
and palmitoyl chloride in presence of NEt3; (
acidification with AcOH; Column: LiChrospher® 100 RP-18 (5
THF/H2O; Gradient: 50% THF to 100% THF in 30 min; Flow rate: 1mL/min; Detection at 254 nm.
A
) the reaction mixture product between bis(2-aminophenyl) disulfide
) the reaction mixture after reduction with NaBH4 and
m) LiChroCART® 250-4; Mobile phase:
B
µ

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2.1.2. Synthesis of Lipid-BTH
The previous observations led us to use a lipid-COOH derivative and to couple it with
bis(2-aminophenyl) disulfide, in order to prepare a lipid-BTH derivative. For this, we synthesized
the lipid-COOH derivative 7, according to Scheme 3. In brief, (2,2-dimethyl-1,3-dioxolan-4-yl)methanol
was reacted with NaH in methanol (MeOH) and then benzyl bromide (Bz-Br) was added. The derived
product was further hydrolyzed in presence of DCM/H2O/TFA (10:1:2) to 3-(benzyloxy)propane-1,2-diol,
which was further coupled with palmitic acid in presence of DIC and 4-dimethylaminopyridine (DMAP).
The O,O0-bis-acylated product was hydrogenalyzed in presence of Palladium on Carbon catalyst (Pd-C)
and finally reacted with succinic anhydride/DMAP to form the desired lipid-COOH derivative 7 (Scheme 3).
O
HO
O
O
HO
DCM/H2O/TFA
O
1. NaH
O
Bz
O
2. Bz-Br
HO
Bz
4-((benzyloxy)methyl)-2,2-
dimethyl-1,3-dioxolane
3-(benzyloxy)propane-
1,2-diol
(2,2-dimethyl-1,3-
dioxolan-4-yl)methanol
RCO2H
DIC / DMAP
O
O
O
O
O
O
R
O
O
O
O
R
O
O
R
O
O
H2 / Pd-C
Bz-H
R
O
O
R
R
O
DMAP
O
OH
HO
Bz
O
7 = Lipid-COOH
Scheme 3. Synthetic method for the formation of Lipid-COOH; R=CH3-(CH2)14-.
Subsequently, we applied our previous findings for the synthesis of lipid-BTH
performed the coupling reaction of lipid-COOH with bis(2-aminophenyl) disulfide
of DIC, and the derived mixture was further reduced with NaBH4 in EtOH. This was acidified with
AcOH (according to our previous findings) to form the desired lipid-BTH (Scheme 4). The later
8. Thus, we first
7
2
in presence
8
reaction was monitored by HPLC and our previous findings were confirmed. The derived lipid-BTH
was isolated as a white solid and was recrystallized from anhydrous EtOH (and/or AcCN) (total yield:
55%). The product was characterized by HPLC (Figure 4A; 22 min), ESI-MS (Figure 4B; (M+H)+: 758.56
(calc.), 758.54 (found)), 1H-NMR, 13C-NMR (as described in detail in materials and methods).
2.2. Nanoliposome Preparation
Following the synthesis of lipid-BTH, we were interested in the preparation of stable (in
terms of size distribution, stability, and integrity) nanoliposomes, which could be potentially used
for in vivo applications. Thus, the lipid-BTH was incorporated in nanoliposome preparations
consisted of six different combinations of 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC),
Cholesterol (Chol), as well as 1,2-dipalmitoylsn-glycerol-3-phosphatidyl glycerol (DPPG) and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium
salt) (DSPE-PEG2000-OMe (in some cases), by the thin film hydration method. DPPC and Chol were
selected in order to prepare nanoliposomes with rigid membranes, and DPPG for formation of

Molecules 2019, 24, 1540
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negatively charged nanoliposomes [44]; DSPE-PEG2000-OMe was used in order to have a PEG coating
on the nanoliposome surface, which is known to prolong nanoliposome blood circulation, improve
their distribution in tissues, and increase their physical stability (by providing a strong interbilayer
repulsion that can overcome the attractive van der Waals forces). A PEG concentration of 8 mole%
(to total lipid) was selected in accordance with previous results [44,45].
O
O
O
CH2
CH
NH2
S
O
S
+
NH2
OH
H2C
O
O
2
O
7: lipid-COOH
1. DIC
2. NaBH4 / EtOH
3. AcOH
O
O
CH2
CH
O
O
S
H2C
O
O
8: lipid-BTH
N
Scheme 4. Synthetic method for the formation of lipid-BTH.
Figure 4.
(A µm)
) HPLC analysis of purified lipid-BTH.; Column: LiChrospher® 100 RP-18 (5
LiChroCART® 250-4; Mobile phase: THF/H2O; Gradient: 50% THF to 100% THF in 30 min; Flow rate:
1ml/min; Detection at 254nm; (B) ESI-MS of lipid-BTH.
The exact compositions of the nanoliposomes, as well as their physicochemical properties are
presented in Table 1. LIP1 and LIP2 were composed of DPPC/Chol (1:1) and 10% or 20% molar lipid-BTH
(for LIP1 and LIP2, respectively). LIP3 and LIP4 were composed of DPPC/DPPG/Chol (9:1:10) with
10% lipid-BTH (LIP3) or 20% lipid-BTH (LIP4). In other nanoliposome types, PEG was inserted in the
nanoliposome membranes, and DPPC/Chol/DSPE-PEG2000-OMe (1:1:0.08 mole/mole) nanoliposomes
were prepared with 10% lipid-BTH (LIP5) or 20% lipid-BTH (LIP6). As seen in Table 1, there is a slight
increment in vesicle size as a function of the percent of lipid-BTH incorporation in the membranes,
of all the types of nanoliposomes prepared (LIP1 versus LIP2; LIP3 versus LIP4; LIP5 versus LIP6).
In all cases the polydispersity index values were relatively low (ranging from 0.148 to 0.186), except
for the DPPC/Chol/Lipid-BTH nanoliposomes with 20mol% BTH, which had a PDI value around

Molecules 2019, 24, 1540
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0.318, suggesting that these nanoliposomes have an increased tendency to aggregate (compared to all
the other nanoliposome types). The -potential of DPPC/Chol and DPPC/Chol/DSPE-PEG2000-OMe
BTH-decorated vesicles ranges between 2.27 and 4.85, as expected, since all the lipids in their
ζ
−
−
composition are zwitero-ionic; DPPG-containing nanoliposomes have negative ζ-potential values
between −9.61 and −12.2 mV, since DPPG is a negatively charged lipid.
Table 1. Nanoliposome types, molar ratios of composition, and physicochemical characteristics.
The vesicle mean diameter (nm), Polydispersity index and -potential (mV) values reported, are the
ζ
mean values from at least 5 measurements of 3 different preparations, in each case. The % percentage
(10% or 20%) of Lipid-BTH is expressed corresponding to DPPC content.
Mean Hydrodynamic
Diameter (nm)
Polydispersity
Index (PDI)
Nanoliposome Type
Molar Ratio
ζ-Potential (mV)
DPPC/Chol/Lipid-BTH (10%)—LIP1
DPPC/Chol/Lipid-BTH (20%)—LIP2
DPPC/DPPG/Chol/Lipid-BTH
(10%)—LIP3
DPPC/DPPG/Chol/Lipid-BTH
(20%)—LIP4
DPPC/Chol/DSPE-PEG2000
-OMe(8%)/Lipid-BTH (10%)—LIP5
DPPC/Chol/DSPE-PEG2000
-OMe(8%)/Lipid-BTH (20%)—LIP6
1:1:0.1
1:1:0.2
84.98 ± 0.22
0.148 ± 0.009
−4.85 ± 0.223
−4.13 ± 0.138
107.01 ± 0.38
0.318 ± 0.003
9:1:10:0.1
9:1:10:0.2
1:1:0.08:0.1
1:1:0.08:0.2
81.84 ± 0.28
94.91 ± 1.30
102.4 ± 0.25
108.7 ± 1.22
0.186 ± 0.019
0.186 ± 0.015
0.170 ± 0.005
0.168 ± 0.003
−9.61 ± 0.156
−12.2 ± 0.141
−3.13 ± 0.235
−2.27 ± 0.165
The physical stability (stability of mean hydr. diameter and
ζ
-potential) of the different
◦
nanoliposome preparations during storage at 4 C, for a period of 15 days, (liposome dispersions
with lipid concentration equal to 5 mg/mL were used), are presented in Figure 5. As seen, LIP1 and
especially LIP2 nanoliposomes tend to aggregate very fast after their preparation; thereby these specific
nanoliposome compositions could not be used for further in vitro/in vivo experiments. The aggregation
of LIP1 and LIP2 may be attributed to the BTH groups present on their surface, which may interact
either between one another, or with groups of the other lipids, leading to the formation of aggregates.
The negatively charged nanoliposomes (with DPPG in their lipid membrane) LIP3 and LIP4 also
demonstrate moderate stability during the 15 days storage period. Indeed, the PDI values of LIP 3
increased from 0.186 to 0.295, while for LIP4 the final PDI value was even higher (0.49), possibly due to
the higher amount of surface BTH groups on the surface of LIP4 (compared to LIP3). In comparison to
LIP1 and LIP2compositions, the DPPG containing nanoliposomes (LIP3 and LIP4) are more stable,
demonstrating reduced aggregation during storage, most probably as a result of their surface charge;
nevertheless, their PDI values were significantly increased after 9 days (especially in the case of
LIP4). Finally, pegylation seems to stabilize BTH-nanoliposomes, since the pegylated LIP5 and LIP6,
were found to be very stable (compared to the non-pegylated types), with sizes ranging between 113
and 121 nM (and PDI values around 0.200), after 15 days of storage at 4 ◦C. Due to their physical
stability, the former nanoliposome formulations are the best candidates for further in vitro
/in vivo
investigations. For all nanoliposome types tested the
15-day storage period.
ζ-potential values did not change during the
The integrity of the non-pegylated BTH-nanoliposomes LIP1 and LIP2, and of the pegylated
BTH-nanoliposomes LIP5 and LIP6 during incubation in presence of buffer (PBS) and serum proteins
(FCS) is presented in Figure 6A,B, respectively. Control nanoliposomes (without BTH) are also studied
in parallel. As seen, all nanoliposome types were stable during incubation in buffer. The fact that the
10% BTH non-pegylated nanoliposomes LIP1, and the 20% BTH non-pegylated nanoliposomes LIP2,
and also the corresponding control nanoliposomes are all equally stable in PBS, indicates that no phase
separation or fusion is taking place when lipid-BTH is inserted into the liposomal membrane, at 37 ◦C,
since if this was the case calcein would leak out of the vesicles.

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240
1,0
0,8
0,6
0,4
0,2
0,0
LIP1
LIP2
LIP3
LIP4
LIP5
LIP6
200
160
120
80
40
0
15
6
9
1
3
9
15
3
6
1
Storage Time (d)
Storage Time (d)
Figure 5. Size (d-hydrodynamic mean diameter, nM) and pdi values of the LIP1–LIP6 prepared
dispersions, in PBS buffer 7.4 at a lipid concentration of 5 mg/mL, during storage at 4 ◦C. Each value is
the mean of at least 5 different measurements and bars are the SD values of each mean.
100
100
80
80
60
60
40
40
control (no BTH, non-pegylted liposomes)
LIP1
LIP2
20
0
20
0
control (no BTH, pegylated liposomes)
LIP5
LIP6
A
B
0
24
48
72
96
120
144
0
24
48
72
96
120
144
time (h)
time (h)
Figure 6.
BTH-nanoliposomes (LIP5 and LIP6) and their corresponding control nanoliposomes (with no BTH on
their surface), during incubation in buffer ( ) or FCS (80% v/v) (
) at 37 ◦C. Each data point is the mean
Integrity of non-pegylated BTH-nanoliposomes (LIP1 and LIP2) and pegylated
A
B
of at least 3 different experiments and the bar is the SD of the mean.
In presence of plasma proteins (FCS), as anticipated due to the absence of PEG, the non-pegylated
nanoliposomes (LIP1, LIP2, and corresponding control vesicles) were unstable and their calcein
content was observed to rapidly leak out from the vesicles during incubation. On the contrary, both
BTH-pegylated nanoliposome types, LIP5 and LIP6, were highly stable in the presence of FCS during
the full incubation period of 144 h. Due to their increased integrity under conditions mimicking the
in vivo situation (compared to the other BTH-nanoliposome types), the pegylated-BTH-nanoliposomes
are the most appropriate to continue studies with, towards the development of potential theragnostic
systems for AD [44].
LIP1 and LIP2 as well as control nanoliposomes (without BTH-lipid) were evaluated by Differential
Scanning Calorimetry (DSC) in order to understand the effect of BTH-lipid addition in the nanoliposome
membrane on their phase transition. As seen in Figure 7, in the case of the control nanoliposomes
no transition is observed, which is normal since the transition of DPPC should be abolished due to
◦
the presence of Chol [46]. On the contrary, a sharp peak at 54.5 C is observed in the case of the
nanoliposomes that contain lipid-BTH, and the peak recorded follows a lipid-BTH concentration
dependent manner. This result provides evidence that the insertion of lipid-BTH in the nanoliposome
membrane has an impact on the phase transition of the membrane. In any case no phase-separation
relevant results were observed in our other studies, since they were carried out at lower temperatures.

Molecules 2019, 24, 1540
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a
b
c
20
30
40
50
60
70
Temperature / oC
Figure 7. DSC thermographs of control nanoliposomes (no BTH) (a); LIP1 (10% BTH) (b); and LIP2
(20% BTH) (c).
3. Materials and Methods
3.1. Materials
3.1.1. Synthesis of BTH-Derivatives (Palm-BTH and Lipid-BTH)
All chemicals were purchased from Sigma-Aldrich OM, Athens, Greece, except
2-aminobenzenethiol which was a gift of CBL Patras S.A. (Industrial area of Patras, Building
block 1, GR-25018, Patras, Greece). All chemicals were used without further purification, while
all required anhydrous solvents were dried with molecular sieves, for at least 24 h prior to use.
Thin layer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck, Darmstadt,
Germany) and spot detection was carried out by UV light, or by charring with an aqueous solution of
K2CO3/KMnO4. Flash column chromatography was performed on silica gel 230–400 mesh (Merck,
Darmstadt, Germany). HPLC analysis was performed on a Shimadzu LC-2010 liquid chromatography
system (Canby, OR, USA) using a LiChrospher® 100 RP-18 (5 m) LiChroCART® 250-4; Mobile phase:
µ
THF/H2O; Gradient: 50% THF to 100% THF in 30 min; Flow rate: 1mL/min; Detection at 254 nM. nMR
spectra were recorded at 25 ◦C with a Brucker DPX 400 MHz instrument (Peoria, IL, USA). Chemical
shift assignments, reported in ppm, are referenced to the corresponding solvent peaks. MS were
recorded on a QTRAP system with ESI source (Applied Biosystems, Waltham, MA, USA).
3.1.2. Nanoliposome Preparation
1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-dipalmitoylsn-glycerol-3-
phosphatidyl glycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000-OMe) were purchased from Avanti
Polar Lipids (Alabaster, AL, USA). Cholesterol (Chol), SephadexG50 (course), calcein, Sepharose
CL-4B were purchased from Sigma-Aldrich OM, Athens, Greece. All other chemicals were reagent
grade. Ultrapure water was produced with a Millipore Direct-Q®3 with pump system (Millipore S.A.,
Molsheim, France). Fluorescence intensity (FI) of samples was measured with a Shimatzu RF-5301PC
◦
spectrofluorophotometer (Kyoto, Japan) at 37
±
0.1 C. In all cases, 5-nm slits were used. A bath
sonicator (Branson 2510E-DTH, Danbury, CT, USA)) and a probe sonicator equipped with a microtip
(Vibra-cell, Sonics and Materials, Inc., Newtown, CT, USA) were used for nanoliposome preparation.

Molecules 2019, 24, 1540
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3.2. Synthetic Procedures
3.2.1. Synthesis of Bis(2-Aminophenyl) Disulfide
20 mL of 2-aminobenzenethiol (187 mmol) were placed into a flask and hydrogen peroxide (H2O2)
35% was added drop-wise (7mL) at rt in 1h. Then, 150 mL diethylether (Et2O) was added and the
mixture was extracted, subsequently, with water, aq. NaOH and water. The organic layer was dried
with MgSO4 and the solvent was evaporated. The resulting yellow crystalline solid was delivered after
1
washing with Hexane (21.4 gr, yield: 92%). H-NMR (CDCl3): 7.21–7.08 (4H, m), 6.80–6.68 (2H, d),
6.65–6.55 (2H, t), and 4.5–4.0 (2H, ds, NH2)
3.2.2. Synthesis of Lipid-COOH
Synthesis of 3-Benzyl-sn-Glycerol
We placed 1.271 gr NaH (53 mmol) in a flask. Next, 90 mL THF and 5 gr (37.8 mmol) of
1,2-O-isopropylidene-sn-glycerol were added, and the mixture was stirred for 30 min at rt. Then 5.4 mL
(45.4 mmol) benzyl bromide were added in 3 portions (in 30 min) and the reaction was stirred overnight
at rt. Finally, 40 mL 10% aqueous Na2CO3 were added and the mixture was stirred for 30 min more
at rt. The mixture was then extracted with Et2O and the organic phase was washed with H2O ( 3) and
×
condensed until formation of an oily residue. This was directly hydrolyzed by 20 mL DCM/H2O/TFA
(10:1:2) for 30 min at rt and the mixture was extracted in Hex and 10% Na2CO3. The aqueous phase
was delivered and washed by Hex ( 2). EtOAc was added and the aqueous phase was saturated with
×
NaCl (or brine). The organic phase was delivered, dried with MgSO4 and condensed. The oily product
1
was delivered and dried over MgSO4 (6.49 gr). Yield: 94%. H-NMR (CDCl3): 7.37–7.28 (5H, m, Ar),
4.54 (2H, s, H4), 3.91–3.87 (1H, m, H2), 3.71–3.59 (2H, 2dd, H1, 10), 3.58–3.50 (2H, 2dd, H3,30).
1
3
4
2
HO
O
OH
3-Benzyl-1,2-Dipalmitoyl-sn-Glycerol
We placed 6.230 gr (24.294 mmol) of palmitic acid and 3.26 mg (26.72 mmol) DMAP into a flask
and these were dissolved in 15 mL DCM and 4ml DMF. Then 4.18 mL DIC (26.72 mmol) were added
and the mixture was stirred for 5 min at rt. Following, 2.108 gr (11.568 mmol) 3-benzyl-sn-glycerol
were added, and the reaction mixture was stirred overnight. The formed 1,3-diisopropylurea was
filtered and the filtrate was extracted in 10% citric acid. The organic phase was washed with H2O ( 3).
×
Finally the organic phase was dried with MgSO4 and condensed. The oily residue was crystallized
and delivered by washings with anhydrous EtOH and the product was dried over MgSO4 (6.1 gr).
1
Yield: 80%. H-NMR (CDCl3): 7.37–7.23 (5H, m, Ar), 5.29–5.18 (1H, m, H18), 4.60–4.48 (2H, 2d, H20),
4.38–4.14 (2H, 2dd, H17,170), 3.65–3.53 (2H, d, H19), 2.38–2.22 (4H, 2t, H15,150), 1.68–1.55 (4H, m,
H14,140), 1.38–1.18 (53H, s, H2–13, H20–130), 0.94–0.84 (6H, t, H1,10).
O
2
12
11
4
8
10
6
14
O
16
1
3
5
7
9
13 15
17, 17'
18
11'
15'
9'
13'
3'
5'
7'
1'
16'
O
O CH2
20
2'
4'
6'
8' 10' 12' 14'
19
O
1,2-Palmitoyl-sn-Glycerol
We placed 2.3 gr (3.49 mmol) 3-benzyl-1,2-dipalmitoyl-sn-glycerol in a flask and these were
dissolved in 20 mL EtOAc (and some MeOH). Then 230 mg Palladium on Carbon catalyst (Pd-C)

Molecules 2019, 24, 1540
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(10% w/w) were added and H2 was passed and the reaction mixture was stirred overnight. The Pd-C
was the filtered and the mixture was condensed. The product was delivered in acetone and was finally
1
dried (1.79 gr). Yield: 90%. H-NMR (CDCl3): 5.14–5.05 (1H, m, H18), 4.37–4.20 (2H, 2dd, H17,170),
3.80–3.66 (2H, d, H19), 2.40–2.28 (4H, 2t, H15,150), 1.68–1.55 (4H, m, H14,140), 1.38–1.18 (55H, s, H2-13,
H20–130), 0.94–0.82 (6H, t, H1,10).
O
2
4
6
8
10 12 14
O
16
O
1
11
9' 11' 13'
3
5
7
9
13 15
17, 17'
18
15'
3'
5'
7'
1'
16'
OH
2'
4'
6'
8' 10' 12' 14'
19
O
1,2-Dipalmitoyl-sn-Glycerol-3-O-Succinic Acid Monoester
We placed 1.025 gr (10.245 mmol) succunic anhydride and 0.250 gr (2.05 mmol) DMAP
into a flask and these were dissolved with 16 mL DCM. In this mixture 1.166 gr (2.049 mmol)
1,2-palmitoyl-sn-glycerol was added and the reaction was stirred overnight. The reaction mixture was
then extracted with 10% citric acid and the organic layer was washed with H2O ( 3). The organic layer
×
was delivered and dried with MgSO4 and condensed until an oily residue. The final product was
1
delivered after crystallization in anhydrous EtOH (1.36 gr). Yield: 96%. H-NMR (CDCl3): 5.3–5.24
(1H, m, H18), 4.37–4.26 & 4.22–4.12 (4H, 4dd, H17,170,19,190), 2.72–2.64 (4H, m, H21,22), 2.35–2.28
(4H,2t, H15,150), 1.65–1.58 (4H, m, H14,140), 1.35–1.22 (53H, s, H2–13, H20–130), 0.94–0.82 (6H, t, H1,10).
O
2
4
6
8
10 12 14
11
11' 13' 15'
O
16
O
1
3
5
7
9
13 15
O
3'
5'
7'
9'
22
16'
O
18
23
O
1'
OH
O
2'
4'
6'
8' 10' 12' 14'
20
21
3.2.3. Lipid-BTH
We placed 532.07 mgr (0.78 mmol) of palmitic acid in a flask and this was dissolved in 7.5 mL
DCM and the solution was stirred at 4 ◦C. Then 132.8
L (0.857 mmol) DIC were added and the mixture
was stirred for 15 min. Bis(2-aminophenyl) disulfide dissolved in 500 L DCM was added and the
reaction mixture was stirred overnight. The reaction mixture was sequentially extracted with 15%
citric acid ( 1) and H2O ( 3). The organic phase was further dried with Na2SO4 and condensed until
an oily residue was formed. This was dissolved in 6 mL THF/MeOH 5:1 and 34 mg (0.90 mmol) NaBH4
were added and the reaction mixture was stirred under N2 atmosphere for 3 h at rt. Then 515
µ
µ
×
×
µL
AcOH (9.0 mmol) were added and the mixture was stirred for 1 h under N2. Finally the mixture was
condensed until an oily residue was formed and the solid product was delivered and recrystallized
from anhydrous EtOH (or/and AcCN). The product was finally dried. Total yield: 55%; ESI-MS (M+H)+
found: 758.56, calculated: 758.54; 1H-NMR (CDCl3): 8.02–7.99 (1H, d, H-Ar), 7.86–7.83 (1H, d, H-Ar),
7.50–7.46 (1H, t, H-Ar), 7.41–7.36 (1H, t, H-Ar), 5.28–5.23 (1H, m, H18), 4.38–4.26 & 4.23–4.10 (4H, 4dd,
H17,170,19,19’), 3.5–3.43 (2H, t, H22), 3.04–2.98 (2H, t, H21), 2.32–2.25 (4H, 2t, H15,150), 1.62–1.55 (4H,
m, H14,140), 1.35–1.20 (58H, s, H2–13, H20–130), 0.90–0.86 (6H, t, H1,10); 13C-NMR (CDCl3): 173.5, 173
(C16,20), 171.5 (C23), 126, 125, 122.5, 121.5, 115 (C-Ar), 69 (C18), 63, 62 (C17,19), 34 (C15,150), 32.5 (C21),
32 (C22), 29–30 (C3-13, C30-130), 25 (C14,140), 22.5 (C2,20), 14 (C1,10).

Molecules 2019, 24, 1540
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O
2
4
6
8
10 12 14
11
9' 11' 13' 15'
O
16
O
1
3
5
7
9
13 15
O
3'
5'
7'
22
16'
O
18
23
N
1'
S
O
2'
4'
6'
8' 10' 12' 14'
20
21
3.3. Nanoliposome Preparation
BTH Decorated Nanoliposomes
For the preparation of SUV nanoliposomes incorporating lipid-BTH conjugate, the appropriate
amounts of lipids (DPPC, DPPG, DSPE-PEG2000-OMe, Chol, and lipid-BTH) were dissolved in a
chloroform/methanol (2:1 v/v) mixture, placed in a round bottom flask and evaporated under vacuum
until the formation of a thin lipid film. The lipid film was treated with gas N2 and was subsequently
connected to a vacuum pump overnight, in order to remove any traces of organic solvent. The lipid film
was hydrated with PBS buffer (pH 7.4) at 60 ◦C [or a 100 mM solution of calcein, prepared in the same
buffer, in case of integrity experiments]. After complete lipid hydration and formation of multilamellar
liposomes (LMV), the vesicle dispersion was placed under the microtip of a probe sonicator (Sonics &
Materials, Inc., Newtown, CT, USA) for 10min, or until the liposome dispersion became completely
clear. The nanoliposome dispersion was left in peace for annealing of potential structural defects,
at a temperature above the lipid transition temperature for 1–2 h. In case of calcein-encapsulating
nanoliposomes, non-encapsulated calcein was removed by size exclusion chromatography.
3.4. Characterization of Nanoliposomes
3.4.1. Size Distribution, ζ-Potential, and Stability Studies
Particle size of vesicle dispersions (0.2 mg/mL lipid, in 10 mM PBS pH 7.40) was measured by
dynamic light scattering (DLS) technique (Malvern Nano-ZS; Malvern Instruments, Worcestershire,
UK) at 25 ◦C at a 173-degree angle. Zeta potential was measured for the same samples (dispersed
◦
in 10 mM PBS, pH 7.40) at 25 C, by the same instrument (utilizing the Doppler electrophoresis
technique). For some of the nanoliposome types, the physical stability (size, polydispersity index and
zeta-potential) of the vesicle dispersions (in buffer) was monitored during storage at 4 ◦C, for a period
of 15 days.
3.4.2. Nanoliposome Integrity Studies
The integrity of decorated vesicles was evaluated during incubation of calcein-encapsulating
◦
nanoliposomes at 37 C, in presence and absence of serum proteins (80% v/v, FCS). Calcein was
encapsulated in the vesicles at a concentration (100 mM) at which its fluorescence is quenched.
Nanoliposome dispersions (1 mg/mL) were incubated with buffer or FCS, and at various time points,
20 µL samples were drawn for calculation of calcein latency (%). For this, the samples (20 µL) were
diluted with 4 mL buffer, pH 7.40 and the fluorescence intensity (FI) was measured (EX 470 nM,
EM 520 nM) before and after addition of Triton X-100 at a final concentration of 1% v/v (that ensures
nanoliposome disruption and release of all encapsulated (and latent) dye). Latency (%) was calculated
from the equation:
[1.1 × Fat] − [1.1 × Fbt]
Latency (%) = 100 × (
)
(1)
[1.1 × Fat]
where Fbt and Fat are calcein fluorescence intensities before and after the addition of Triton
X-100, respectively.

Molecules 2019, 24, 1540
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Integrity (%) was estimated by setting the latency (%) at time zero as integrity 100 (%). At each
time point, the integrity (%) during nanoliposome incubation in PBS and FCS was calculated from
the equation:
Latency (%)measured
Integrity (%) = 100 ×
(2)
Latency (%)at time 0
3.4.3. Nanoliposome Differential Scanning Calorimetry (DSC) Study
DSC measurements were carried out with a high-sensitivity differential scanning calorimeter
DSC Q100 TA Instruments (TA Instruments, New Castle, DE, USA). An aliquot sample of 4 mg of
dehydrated nanoliposomes was put into the DSC specimen container. The specimen was scanned at a
heating rate of 10 ◦C/min and the temperature range was from 10 to 80 ◦C.
4. Conclusions
In order to take advantage of the affinity of BTH-groups towards A
potential of surface functionalized nanoliposomes for enhanced targeting due to multivalency [14
β
species [27
–
36] and the
16],
–
we prepared nanoliposomes decorated with BTH. For this, we initially synthesized a lipid-BTH
derivative, with high yield, after identifying an optimal methodology. The newly synthesized
derivative (lipid-BTH) was incorporated at different amounts (10 and 20 mol%) into nanoliposomes
with different lipid compositions, which were characterized for their physicochemical properties,
integrity and stability. From all the nanoliposome types evaluated, the pegylated-nanoliposomes
(DPPC/Chol/DSPE-PEG2000-OMe with 10% lipid-BTH or 20% lipid-BTH nanoliposomes) were the
most stable in terms of size stability and membrane integrity. Thus, we conclude that pegylated
BTH-nanoliposomes decorated with 10 mol% or 20 mol% BTH, are potential candidate nanoliposome
types that deserve further exploitation for the development of improved diagnostic/therapeutic systems
for AD.
Author Contributions: S.M., K.B. and D.G. designed the synthetic methodologies; S.M. and S.G.A. designed the
nanoliposome formulation studies; S.M. and P.C. performed experiments (syntheses, nanoparticle preparations,
analyses); S.M., S.G.A., P.K. designed and P.K. performed the DSC study/analysis; S.G.A. and S.M. wrote the
manuscript; All authors approved the final version of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
The authors acknowledge C.B.L. Patras S.A., Patras, Greece, for kindly
providing 2-aminobenzenethiol.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2, 6, 7 and 8 are available from the authors.
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