Liposomes Decorated with 2-(4′-Aminophenyl)benzothiazole Effectively Inhibit Aβ1-42Fibril Formation and Exhibit in Vitro Brain-Targeting Potential

Article abstract of DOI:10.1021/acs.biomac.0c00811

The potential of 2-benzothiazolyl-decorated liposomes as theragnostic systems for Alzheimer's disease was evaluated in vitro, using PEGylated liposomes that were decorated with two types of 2-benzothiazoles: (i) the unsubstituted 2-benzothiazole (BTH) and

Full text of DOI:10.1021/acs.biomac.0c00811

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Article
Liposomes Decorated with 2‑(4′-Aminophenyl)benzothiazole
Effectively Inhibit Aβ1−42 Fibril Formation and Exhibit in Vitro Brain-
Targeting Potential
Spyridon Mourtas,* Barbara Mavroidi, Antonia Marazioti, Maria Kannavou, Marina Sagnou,
Maria Pelecanou, and Sophia G. Antimisiaris
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ABSTRACT: The potential of 2-benzothiazolyl-decorated liposomes as
theragnostic systems for Alzheimer’s disease was evaluated in vitro, using
PEGylated liposomes that were decorated with two types of 2-
benzothiazoles: (i) the unsubstituted 2-benzothiazole (BTH) and (ii)
the 2-(4-aminophenyl)benzothiazole (AP-BTH). The lipid derivatives of
both BTH-lipid and AP-BTH-lipid were synthesized, for insertion in
liposome membranes. Liposomes (LIP) containing three different
concentrations of benzothiazoles (5, 10, and 20%) were formulated, and
their stability, integrity in the presence of serum proteins, and their ability
to inhibit β-amyloid (1−42) (Αβ42) peptide aggregation (by circular
dichroism (CD) and thioflavin T (ThT) assay), were evaluated.
Additionally, the interaction of some LIP with an in vitro model of the
blood−brain barrier (BBB) was studied. All liposome types ranged
between 92 and 105 nm, with the exception of the 20% AP-BTH-LIP that
were larger (180 nm). The 5 and 10% AP-BTH-LIP were stable when stored at 4 °C for 40 days and demonstrated high integrity in
the presence of serum proteins for 7 days at 37 °C. Interestingly, CD experiments revealed that the AP-BTH-LIP substantially
interacted with Αβ42 peptides and inhibited fibril formation, as verified by ThT assay, in contrast with the BTH-LIP, which had no
effect. The 5 and 10% AP-BTH-LIP were the most effective in inhibiting Αβ42 fibril formation. Surprisingly, the AP-BTH-LIP,
especially the 5% ones, demonstrated high interaction with brain endothelial cells and high capability to be transported across the
BBB model. Taken together, the current results reveal that the 5% AP-BTH-LIP are of high interest as novel targeted theragnostic
systems against AD, justifying further in vitro and in vivo exploitation.
efficiently target the blood−brain barrier (BBB) and also the
amyloid species.9,10 Indeed, the surface functionalization of NPs
has been extensively applied as a method to enhance the
accumulation of NP-encapsulated drugs at specific body sites
due to the increased binding affinity of the NPs to specific
receptors as a result of multivalent effects.11,12 Between the
various NP types, liposomes are usually preferred as drug
carriers because they are structurally versatile and also highly
biocompatible. Moreover, liposome PEGylation, which refers to
the coating of the vesicle surface with polyethylene glycol
molecules, can efficiently reduce the rapid clearance of
1. INTRODUCTION
Alzheimer’s disease (AD) is a disorder of the central nervous
system (CNS) that progressively leads to the degeneration of
neurons. Although the exact causes of AD are still not fully
understood, the amyloid and tau hypotheses currently dominate
AD research efforts. The amyloid hypothesis is related to the
aggregation of β-amyloid peptides (Aβ), which leads to their
accumulation in the brain and to the formation of extracellular
amyloid plaques, leading to inflammation.1−3 At the molecular
level, the unbalanced production and clearance of Aβ results in
increased levels of Aβ monomers in the brain, leading to the
formation of larger soluble oligomers that form aggregates, and
finally, insoluble amyloid fibrils, which are the main constituents
of the amyloid plaques. Several reports suggest that Aβ soluble
oligomers are responsible for synaptic dysfunction and cognitive
impairment (which are associated with AD).4−8
Received: May 26, 2020
Revised: October 13, 2020
In this context, strategies aiming at targeting the production,
aggregation, and clearance of Aβ species for theragnostic
purposes are currently being exploited. One such strategy is
the development of functionalized nanoparticles (NP) to
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liposomes by macrophages and prolong their circulation time in
the blood.13,14
that are immobilized on their surface due to multivalency, we
study for the first time, the activity of benzothiazolyl liposomes
toward Αβ species. In more detail, we compare two different
types of benzothiazolyl liposomes for their potential to interact
with Aβ42 and inhibit Aβ42 aggregation: (i) unsubstituted 2-
benzothiazole (BTH)-decorated liposomes (BTH-LIP), which
were formed as previously reported,51 and (ii) 2-arylsubstituted
benzothiazole (AP-BTH)-decorated liposomes (AP-BTH-LIP),
which were formed after establishing an appropriate method-
ology for the synthesis of stable vesicles.
The interactions of BTH-LIP and AP-BTH-LIP with Αβ42
species and their inhibitory effect on Aβ42 aggregation were
studied by circular dichroism (CD) spectropolarimetry52 and by
means of the thioflavin T (ThT) assay.53 Both LIP types were
additionally evaluated for their potential to be transported across
the blood−brain barrier (BBB) in an in vitro cellular
model,54−56to evaluate whether or not additional ligands
(homing devices) should be attached to consider them as
nanotherapeutic modalities for AD.
In several previous investigations, curcumin and various
curcumin derivatives were demonstrated to (i) effectively inhibit
the formation of Aβ oligomers, (ii) to rescue cells from Aβ-
induced toxicity (at micromolar concentrations in vitro)15−20
and (iii) to reduce the brain concentrations of amyloids in
vivo.21,22 Curcuminoid-decorated liposomes demonstrated high
affinity (KD: 1−5 nM) for Aβ42 fibrils,23 similar to the affinity
(KD: 0.5−2 nM) of liposomes that were functionalized with a
monoclonal antibody against Aβ.24 Phosphatidic acid (PA) and
cardiolipin (CL)25 are two other substances that were used for
the surface modification of liposomes. However, they exhibited
only moderate binding affinity for aggregated forms of Aβ42
(KD: 22−60 nM). Finally, liposomes decorated with tetracycline
derivatives have also been considered as systems for AD
diagnosis/therapy.26
The benzothiazole heterocycle is a versatile scaffold in
medicinal chemistry, possessing a wide spectrum of activ-
ities.27,28 In addition to their well-documented anticancer
properties, benzothiazole and its derivatives are well associated
with the search for therapeutics (or diagnostics) for Alzheimer’s
disease (AD). Unsubstituted 2-benzothiazole compounds as
well as substituted or more complicated compounds29−32 have
been found to effectively interact with Aβ42 species, in vitro and
in vivo, and to inhibit or delay the aggregation of Aβ42
monomers.33−36 In particular, the clinical testing of 2-(4-
aminophenyl)benzothiazole derivatives showed promising
results.37−39
In more detail, in the diagnostic area, many radiolabeled 2-
phenylbenzothiazole derivatives are continuously explored as
amyloid imaging agents, while an 18F-labeled derivative of
phenylbenzothiazole ([18F] flutemetamol, Vizamyl, GE Health-
care) gained approval in 2013 for clinical use as a positron
emission tomography (PET) diagnostic agent for AD.40 In the
therapeutic area of AD, the only available drugs are
cholinesterase inhibitors (donepezil, tacrine, rivastigmine, and
galantamine) that alleviate the symptoms of dementia but do not
treat the disease.41 Benzothiazoles represent a class of
heterocyclic compounds which serve as a scaffold for the design
of anti-amyloid agents against Alzheimer’s disease.42 More
specifically, benzothiazole derivatives and complexes27,43,44 have
been demonstrated to inhibit Aβ aggregation and reduce the
concentrations of soluble amyloid oligomers. Furthermore,
others have tried to develop substances that combine a
benzothiazole derivative with a cholinesterase inhibitor (tacrine,
in most cases) with the aim to achieve concomitant enhance-
ment of cholinergic neurotransmission and reduction of Aβ
aggregation.45−49
However, with the exception of the benzothiazole metal
complex,44 the issue of crossing the BBB has not been addressed,
despite the fact that it remains the major issue in the failure of
AD drug development.50 In fact, benzothiazole derivatives were
never considered up-to-date for the construction of potential
therapeutic nanosystems, via conjugation/immobilization on
the surface of NPs. The only benzothiazolyl-decorated NP that
is reported describes the development of unsubstituted 2-
benzothiazole liposomes,51 but their interaction with amyloid
species and their potential to be transported across the BBB were
not evaluated.
2. EXPERIMENTAL SECTION
All chemicals required for the synthesis of the lipid derivatives were
obtained from Sigma-Aldrich (Darmstadt, Germany). Silica-gel-60
F254 TLC plates were obtained from Merck (Darmstadt, Germany).
TLC visualization methods included UV light, charring with aqueous
solution of potassium permanganate (KMnO4) (freshly prepared),
ninhydrin, or molybdenum blue spray, all purchased from Sigma-
Aldrich (Darmstadt, Germany). Flash column chromatography for the
purification of the organic synthesis products was carried out on silica
gel (230−400 mesh) from Merck (Darmstadt, Germany). A Shimadzu
LC-2010 liquid chromatography HPLC system (Canby, OR, USA) was
used for sample analysis. Samples were eluted through an RP18 (5 μm
particle size) LiChroCART 250-4 column. A Brucker DPX 400 MHz
NMR instrument (Peoria, IL, USA) was used, and the sample spectra
were recorded at 25 °C. Chemical shifts were referenced to the
corresponding solvent peaks and are reported in ppm.
1,2-Distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC) and 1,2-
distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy-
(polyethyleneglycol)-2000] (PEG) were obtained from Avanti Polar
Lipids (Alabaster, AL, USA). Fluorescein-isothiocyanate-dextran-4000
(FITC), calcein, cholesterol (Chol), lucifer yellow-CH dilithium salt
(LY), Triton X-100, Sephadex G-50, and Sepharose CL-4B were
purchased from Sigma-Aldrich (Darmstadt, DE). Fetal calf serum
(FCS) was obtained from Invitrogen. For measurements of protein
concentrations, Bradford microassay was applied (Biorad, Hercules,
CA, USA). Any other chemicals used were purchased from Sigma-
Aldrich or Merck.
For the liposome preparation, a bath sonicator (Branson, Thermo
Fisher Scientific, Waltham, MA, USA) or a microtip-probe high-
intensity sonicator (Sonics and Materials, Leics, UK) was used. For
their purification, size exclusion chromatography was performed on
Sepharose CL-4B (Sigma-Aldrich, Darmstadt, Germany). A Shimatzu
RF-1501 spectrofluorometer (Shimatzu, Kyoto, JP) was used for the
measurement of the fluorescence intensity (FI) of calcein or FITC in
samples (EX/EM 490 nm/525 nm; 5 nm slits).
2.1. Synthesis of BTH and AP-BTH Lipid Derivatives.
2.1.1. Synthesis of BTH-Lipid (3). Palmitic acid (532 mg, 0.78 mmol)
dissolved in DCM (7.5 mL) was placed in a round-bottomed flask, and
the solution was cooled at 4 °C. Diisopropylcarbodiimide (DIC) (133
μL, 0.85 mmol, 1,1 equiv), which was used as an activation agent, was
added, and the palmitic acid was activated for 15 min at 4 °C. 2-
Aminophenyl disulfide dissolved in DCM (500 μL) was added to the
activated palmitic acid, and the reaction mixture was stirred overnight at
room temperature. It was then directly acidified using an aqueous
solution of citric acid (15% w/v, 5 mL), and the organic layer was
washed with H2O (3 × 5 mL) and further dried and evaporated. The
oily residue was further dissolved in a mixture of THF/MeOH (6 mL,
Herein, taking into consideration the capability of benzothia-
zoles to interact with Αβ42 and the known potential of
nanotechnologies to enhance the function of active compounds
B
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a
Table 1. Liposome Types, Lipid Membrane Composition (in Mole Ratios), and Physicochemical Properties
b
liposome type
Control
BTH-LIP (5%)
BTH-LIP (10%)
BTH-LIP (20%)
AP-BTH-LIP (5%)
AP-BTH-LIP (10%)
AP-BTH-LIP (20%)
DSPC/Chol/PEG/LIPID molar ratio
mean hydrodynamic diameter (nm)
polydispersity index (PDI)
ζ-potential (mV)
1:1:0.15
80.2 5.1
97.2 3.5
99.4 4.5
104.5 3.7
92.5 5.2
102.5 7.6
180.2 5.1
0.154
0.206
0.230
0.218
0.143
0.229
0.234
−1.32 0.17
−2.67 0.15
−3.21 0.71
−2.79 0.20
−2.51 0.35
−2.02 0.18
−1.54 0.02
1:1:0.15:0.05
1:1:0.15:0.10
1:1:0.15:0.20
1:1:0.15:0.05
1:1:0.15:0.10
1:1:0.15:0.20
a
The values reported are mean values from five measurements of three independent samples. The percent values used for the BTH-LIP and AP-
BTH-LIP naming is based on the content of BTH-lipid and AP-BTH-lipid (5%, 10 or 20%) in the vesicles expressed as percent of the DSPC
b
content. LIPID is AP-BTH-lipid or BTH-lipid.
5:1), and the solution was flushed with gaseous N2. To this, NaBH4 (34
mg, 0.90 mmol) was added, and the reaction mixture was stirred for 3 h
at room temperature and then acidified with AcOH (515 μL, 9.0 mmol)
followed by further stirring for 1 h (under an N2 atmosphere). Finally,
the solvents were evaporated under reduced pressure, and the oily
residue was crystallized using anhydrous EtOH and AcCN. Total yield:
55%. The final product showed a single spot in TLC analysis and was
prepared aqueous solution of potassium permanganate and molybde-
num blue reagent) and was further analyzed by HPLC and 1H NMR.
The HPLC chromatogram and the assignments of the 1H NMR
spectrum are shown in the Supporting Information (Figures S1B and
1
S2D). H NMR (MeOD) δ ppm: 0.90 (6Η, t), 1.28 (56H, m), 1.60
(4H, m), 2.30 & 2.33 (4H, 2 t), 2.60 (2H, t), 2.73 (2H, t), 3.34−3.62
(∼180H, m), 3.90 (2H, d), 3.99 (2H, t), 4.16−4.19 (2H, t & 1H, d),
4.42 (1H, 1d), 5.22 (1H, m), 7.42 (1H, t), 7.52 (1H, t), 7.78 (2H, d),
7.99 (d, 2H), 8.05 (d, 2H).
1
further analyzed by HPLC and H NMR. The HPLC chromatogram
and the assignments of 1H NMR spectrum are shown in the Supporting
Information (Figures S1A and S2A). 1H NMR (CDCl3) δ ppm: 0.88
(6H, t), 1.26 (58H, s), 1.59 (4H, m), 2.26 & 2.29 (4H, 2 t), 3.01 (2H,
t), 3.47 (2H, t), 4.13−4.36 (4H, 4dd), 5.26 (1H, m), 7.39 (1H, t), 7.48
(1H, t), 7.84 (1H, d), 8.00 (1H, d).
2.2. Preparation of Liposomes. Small unilamellar vesicle (SUV)
liposomes, having the lipid compositions reported in Table 1, were
prepared by the thin film hydration method and high-intensity
sonication, as reported in detail previously.51,55,56 Liposomes were
usually dispersed in phosphate-buffered saline (10 mM; pH 7.40; 300
mOsm). When FITC (36 mM) of calcein (100 mM) was encapsulated
in the liposomes, the osmotic pressure of the solutions was adjusted to
be isotonic. After formation, the liposome dispersions were annealed to
heal any structural defects at 60 °C for 1 h. Liposome purification from
nonentrapped materials was carried out, if needed, by size-exclusion
chromatography on a Sepharose 4B-CL or Sephadex G-50 (medium)
column (40 × 1 cm) that was eluted with PBS pH 7.40. The Stewart
assay was used for the measurement of the phospholipid concentration
of liposomes.57
2.3. Liposome Properties. 2.3.1. Physicochemical Properties
and Physical Stability. The mean hydrodynamic diameter and the
polydispersity index (PDI) of the vesicles (dispersed in PBS at 0.2 mg/
mL phospholipid concentration) was measured by dynamic light
scattering (DLS) at 25 °C (173o angle) on a Malvern Nano-ZS
(Malvern Instruments, Worcestershire, UK). Zeta potential (ζ-
potential) values were measured also at 25 °C in the same samples
by Doppler electrophoresis.
2.1.2. Synthesis of Lipid-2-(4-aminophenyl)benzothiazole (AP-
BTH-Lipid) (6). 2.1.2.1. Synthesis of 2-(4′-Aminophenyl)-
benzothiazole (AP-BTH) (4). 2-Aminobenzenethiol (1.25 g, 10
mmol) and 4-aminobenzoic acid (1.37 g, 10 mmol) were mixed in a
round-bottomed flask, and polyphosphoric acid (20 g) was added. The
reaction mixture was heated at 220 °C for 4 h, and the reaction mixture
was slowly poured into ice-cold aqueous Na2CO3 (10% w/v). The
precipitated product was filtered and washed with H2O, and finally, it
was recrystallized from a mixture of MeOH/H2O to afford 2-(4′-
aminophenyl)benzothiazole as a light brown powder (yield: 64%). The
isolated product was verified by 1H NMR analysis. The HPLC
1
chromatogram and the assignments of the H NMR spectrum are
shown in the Supporting Information (Figure S2B). 1H NMR (MeOD)
δ ppm: 6.75 (2H, d), 7.33 (1H, t), 7.45 (1H, t), 7.80 (2H, d), 7.89 (2H,
m).
2.1.2.2. Synthesis of N-[4-(Benzothiazol-2-yl)-phenyl]-succinamic
Acid [Also Named 3-(4-(Benzothiazol-2-yl)phenylcarbamoyl)-
propanoic Acid] (5). 2-(4′-Aminophenyl)benzothiazole (4) (99.3
mg, 0.44 mmol) and succinic anhydride (43.91 mg, 0.44 mmol) were
placed in a round-bottomed flask and dissolved in CHCl3 (approx. 2
mL), and the reaction mixture was stirred overnight at room
temperature. Next, the solvent was almost completely evaporated,
and Et2O was added. The solid that was formed was filtered, washed
with Et2O, and finally dried (yield: 90%). The isolated product was
verified by 1H NMR analysis. The HPLC chromatogram and the
The vesicle physical stability was studied by measuring the mean
diameter, PDI, and ζ-potential, periodically during storage (4 °C, 40
days).
2.3.2. Liposome Integrity in the Presence of Serum Proteins. To
monitor the integrity of AP-BTH-LIP and control liposomes (control-
LIP), calcein-encapsulating liposomes (1 mg/mL phospholipid) were
incubated at 37 °C in PBS as well as in FCS (80% v/v). As mentioned
above, calcein was encapsulated in the liposomes at 100 mM
concentration, where quenching of fluorescence is realized. Calcein
latency (%) and retention (%) values were calculated at various time
points, by taking samples from the incubated materials and measuring
calcein FI, as described in detail previously.51,55,56
2.4. Inhibition of Aβ1−42 (Aβ42) Peptide Fibril Formation by
Liposomes. 2.4.1. Preparation of Aβ and Liposome Samples. The
amyloid Aβ42 peptide (Anaspec, USA, >95% pure) was gradually
dissolved with gentle tapping (without vortexing) in Type 1 H2O
(Milli-Q) to 100 μM. The solutions of plain Aβ42 in phosphate buffer
(PB, 10 mM, pH 7.33) were then prepared by gradual dilutions until a
final concentration of 50 μM. In the preparation of the Aβ/liposome
mixtures, the ratios of 1:0.75, 1:0.50, 1:0.25, and 1:0.10 were calculated
based on the amount of Lipid-BTH or Lipid-AP-BTH incorporated to
liposomes (Section 2.2). Based on the lipid membrane composition (in
mole ratios), the proper volumes (μL) of liposome stock solutions were
added in Aβ solutions to achieve a final concentration of 37.5, 25, 12.5,
1
assignments of the H NMR spectrum are shown in the Supporting
Information (Figure S2C). 1H NMR (MeOD) δ ppm: 2.70 (4H, 2 t),
7.42 (1H, t), 7.52 (1H, t), 7.76 (2H, d), 7.99 (2H, d), 8.04 (2H, d).
2.1.2.3. Synthesis of AP-BTH-Lipid (6). N-[4-(Benzothiazol-2-yl)-
phenyl]-succinamic acid (21.4 mg, 0.0656 mmol) and N-hydrox-
ysuccinimide (NHS) (7.92 mg, 0.0688 mmol) were diluted in DMF/
DCM (300 μL, 1:1), and the mixture was added in a round-bottomed
flask. For carboxylic acid activation, diisopropylcarbodiimide (DIC)
(0.0688 mmol, 10.7 μL) was added, and the mixture was stirred for 5
min at room temperature. DSPE-PEG2000-NH2 (60 mg, 0.0219 mmol)
was added, and the reaction mixture was stirred overnight. Next, the
mixture was diluted with DCM (10 mL), and the organic phase was
washed with H2O (3 × 10 mL) and brine (3 × 5 mL) and then
separated and further dried and evaporated. The remaining oil was
purified using EtOAc/Hex at 65 °C and finally dried (yield: 55%). The
isolated DSPE-PEG2000-AP-BTH product (AP-BTH-lipid) showed a
single spot in TLC analysis (by UV light and by charring with a freshly
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Scheme 1. Synthesis of BTH-Lipid
and 5 μΜ Lipid-BTH or Lipid-AP-BTH. The solutions of plain Aβ42
and control liposomes (without BTH-lipid or AP-BTH-lipid) at the
same concentrations and incubation conditions were used as control
experiments for comparison.
2.4.2. Circular Dichroism (CD) Studies. Aβ structural changes were
monitored under quiescent conditions for 15 days at 33 °C. The CD
spectra were recorded in the range of 190−260 nm on a JASCO J-715
spectropolarimeter (Jasco Co., Japan). Each spectrum is the average
from three scans (rate 100 nm·min−1, resolution 0.5 nm). Three
independent experiments were performed for each condition and for
solutions of plain Aβ. Control liposomes (without BTH-lipid or AP-
BTH-lipid) were also studied. The CD data were analyzed using the
OriginPro 9 program.
2.4.3. Thioflavin T (ThT) Assay. For the ThT (Sigma-Aldrich, USA;
>97% pure) assay, the 15 day-aged CD solutions of Aβ42 (with or
without liposomes) were diluted with PBS (10 mM, pΗ 7.33) to obtain
theoretical Aβ42 concentration of 25 μM, and ThT in PBS (10 mM, pΗ
7.33) was added (5 μM). The mixture was well agitated, and the FIe was
monitored with a HITACHI F-2500 spectrofluorometer at an
excitation of 440 nm (emission slit = 2.5 nm, PMT voltage 700 V,
response 0.4 s). The analysis of the FIe data was performed using the
OriginPro 9 program.
2.5. Cell Studies. HEK cells (Human HEK-293 embryonic kidney
cells, American Type Culture Collection, VA) were cultured in RPMI
1640 medium (supplemented with 1% antibiotic−antimycotic solution
and 10% FBS (Invitrogen, Carlsbad, CA, USA)) at 37 °C, 5% CO2/
saturated humidity.
hCMEC/D3 cells (immortalized human cerebral microvascular
endothelial cells that were obtained under license from the Institut
National de la Sante et de la RechercheMedicale (INSERM, Paris, FR),
at passage 25−35) were cultured in EndoGro medium (Merck,
Darmstadt, DE), as described in detail previously.55,56
U-87 MG cells (human glioblastoma−astrocytoma cells, acquired
from the cell bank of the Laboratory of Radiobiology, Institute of
Nuclear & Radiological Sciences and Technology, Energy & Safety,
NCSR “Demokritos” (Athens, Greece)) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM), supplemented with 10% FBS,
penicillin, glutamine, and streptomycin. Cell cultures were maintained
in flasks and grown at 37 °C in a humidified atmosphere of 5% CO2 in
air.
2.5.1. Cytotoxicity Assay. The cytotoxicity of LIP toward HEK and
hCMEC/D3 was evaluated by incubating LIP (7 nmol) with cells (3 ×
104) for 4 h (37 °C, 5% CO2/saturated humidity). A 48 h incubation
was also performed, in some cases, to evaluate the potential cytotoxicity
of AP-BTH-LIP types. In all cases, the MTT assay was used as described
in detail previously.55,56
A cell cytotoxicity assay in U-87MG glioblastoma cells was also
conducted to investigate the effect of AP-BTH-LIP and BTH-LIP on
the Aβ-induced cell toxicity. In brief, U-87 MG cells (∼8000 cells/well)
were incubated with Aβ solutions (1 μM) in the presence of liposomes
(Aβ: liposome ratios of 1:0.5 and 1:1) for 24 h at 37 °C. MTT assay was
also used for this study.
2.5.2. Cell Uptake. The uptake of liposomes by cells was studied with
FITC-entrapping LIP after incubation with hCMEC/D3 cells (200
nmol liposomal lipid/106 cells) for 4 h at 37 °C. After washing the cells
(twice with ice-cold PBS), they were detached using scrappers and
suspended in PBS. After cell lysis with Triton X-100 (2% v/v, final
concentration), the FI was measured (EX-490 nm/EM-525 nm, slits 5
nm). Any autofluorescence of the empty cells was subtracted. The
protein content of the cell samples was measured by Bradford assay.
Finally, the calculated FITC uptake values (%) were normalized for
their protein concentration.
To exclude the possibility of the uptake of free dye (that may have
leaked out from LIP), free FITC was also incubated with the cells under
identical conditions, and it was proved that no free FITC was taken up
by the cells.
2.5.3. Cell-Monolayer Permeation Studies. For monolayer
preparation studies, the cells were seeded on Transwell filters
(polycarbonate six-well, pore size 0.4 μm; Millipore Merck, Darmstadt,
DE) precoated with type I collagen, at 5 × 104 cells/cm2. The detailed
procedure followed, and the tests carried out to verify that the
monolayer produced is intact (transendothelial electrical resistance
measurements (TEER) and Lucifer yellow (LY) permeability
calculation) are described in detail elsewhere.55,56
For liposome transport evaluation, FITC-containing liposomes were
placed on the monolayers (200 nmol per monolayer) in medium
containing 10% (v/v) FCS at 37 °C for 60 min. At selected time points,
the transport of FITC was calculated after measuring FITC-FI at 490−
520 nm, in the receptor compartment of the transwell systems. LY was
also used to verify that vesicles do not modify the properties of the
barrier. Control liposomes were also evaluated for comparison.
2.6. Transmission Electron Microscopy (TEM). The vesicular
morphology of the liposomal preparations was recorded by means of
TEM. Two types of AP-BTH-LIP (5 and 10%) were resuspended (1
mg/mL) in 10 mM HEPES and then stained with 1% neutral
phosphotungstic acid (PTA) (dissolved in dH2O), washed two times
with dH2O, drained with tissue paper, and observed with a JEOL (JEM-
2100) TEM (Jeol, Tokyo, JP) at 100.000 eV.58
TEM studies were also conducted with the Aβ42 peptide incubated
in the presence of 5% AP-BTH-LIP (ratio 1:1, 50 μΜ) for 15 days at 33
°C under quiescent conditions. As controls, Aβ42 plain peptides (a)
and 5% AP-BTH-LIP (b), incubated under identical conditions, were
also studied. The samples were stained with PTA, as mentioned above.
D
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Scheme 2. Synthesis of Lipid-2-(4-aminophenyl)benzothiazole (AP-BTH-lipid)
Figure 1. (A) Mean diameter values, (B) PDI values, and (C) and zeta potential values of 5% AP-BTH-LIP, 10% AP-BTH-LIP, and control-LIP during
storage at 4 °C, for up to 40 days. Liposomal dispersion in PBS buffer (lipid concentration 5 mg/mL). Each value is the mean of five measurements
(bars represent SD values of each mean). (D) TEM micrographs of the 5% AP-BTH-LIP and 10% AP-BTH-LIP.
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Figure 2. Calcein latency (%) in (A) control-LIP, (B) 5% AP-BTH-LIP, and (C) 10% AP-BTH-LIP during incubation in PBS buffer and FCS for 7
days at 37 °C. (D) Calcein retention (%) in the same liposome types during incubation in FCS. Liposomes were incubated at 1 mg/mL lipid
concentration.
2.7. Statistical Analysis. The results are reported as mean SD
(from three or four independent experiments). One-way ANOVA
followed by the Bonferroni post hoc test was applied for the calculation
of statistical significance (at p = 0.05). Two-way ANOVA was
performed when more factors were compared. The significance of all
comparisons made is presented in the figures.
formulations are reported in Table 1. In addition to AP-BTH-
LIP, with various concentrations of AP-BTH-lipid in their
membranes, BTH-LIP and control-LIP (without AP-BTH or
BTH group) were also studied for comparison. DSPC and Chol
were used to have rigid-membrane liposomes. PEG was used for
the prolongation of blood circulation of vesicles to improve their
potential to be distributed in tissues; indeed vesicles with
increased physical stability and strong interbilayer repulsion
provided by the PEGylated outer surface of the vesicles can
overcome attractive forces and be more stable (compared to
non-PEGylated vesicles) after in vivo administration.60,61 It
should be noted that the only difference between the control-
LIP and the BTH/AP-BTH-LIP is the presence of the BTH/AP-
BTH groups on the decorated ones, while all liposome
formulations (control and the decorated ones) have a DSPE-
PEG-OMe lipid, to introduce the PEG protecting group, at the
same percentage. The exact lipid and molar compositions of our
nanoliposomes are presented in Table 1.
3.4.1. Physicochemical Properties of AP-BTH-LIP. The mean
hydrodynamic diameter of the various AP-BTH-LIP was
approximately 100 nm or less (Table 1), and it was found to
be at a similar range for the AP-BTH-LIP as well, with the
exception of the 20% AP-BTH-LIP that had a significantly
increased mean diameter (180 nm). Polydispersity values were
always below 0.234, indicating that a highly uniform sample with
respect to the particle size was formed.62 The zeta potential
values were always close to zero, as anticipated due to the fact
that the vesicles were PEGylated, the lipids used were
zwitterionic, and the measurements were carried out in high
salt content.63
3. RESULTS
3.1. Synthesis of BTH-Lipid (3). BTH-lipid was synthe-
sized as already described by our group.51 In brief, lipid-COOH
(1) was activated with diisopropylcarbodiimide (DIC) and
reacted with 2-aminophenyl disulfide and then treated with
sodium borohydride (NaBH4) to achieve reduction and thiol
liberation. To this, acetic acid (AcOH) was added to favor the
ring closure51 and the formation of the BTH-lipid (3) (Scheme
1).
3.2. Synthesis of Lipid-2-(4-aminophenyl)-
benzothiazole (AP-BTH-Lipid) (6). For the synthesis of
lipid-2-(4-aminophenyl)benzothiazole (AP-BTH-lipid) (6), the
2-(4′-aminophenyl)benzothiazole derivative (AP-BTH) (4)
was first synthesized,59 and this was further reacted with
succinic anhydride to the corresponding N-[4-(benzothiazol-2-
yl)-phenyl]-succinamic acid (5). In the last step of the synthesis,
5 was conjugated with DSPE-PEG2000-NH2, in the presence of
N-hydroxysuccinimide (NHS)/DIC, to obtain the desired AP-
BTH-lipid (6) (Scheme 2).
3.3. Preparation of Liposomes (LIP). Small unilamellar
vesicles (SUVs) with the lipid compositions reported in Table 1
were prepared.51,55,56
3.4. Physicochemical Properties of AP-BTH-LIP. The
molecular composition and properties of the various liposomal
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The physical stability of 5 and 10% AP-BTH-LIP was studied
by monitoring their size distribution and ζ-potential, during
storage for 40 days at 5 °C. Control-LIP (without any lipid
conjugate) were also monitored. In Figure 1A−C, it is seen that
both the mean diameter and the PDI values remained
unchanged during the 40 days period. More specifically, mean
diameters ranged between 110 and 120 nm for 5% AP-BTH-LIP
and between 130 and 140 nm for the 10% AP-BTH-LIP, while
PDI values did not exceed 0.25 for all vesicles. The ζ-potential
values were also found to be stable during the 40 days storage
period, with values starting from −1.42 0.14 for the 5% AP-
BTH-LIP and − 1.47 0.16 for the 10% AP-BTH-LIP.
Moreover, these novel 5 and 10% AP-BTH-LIP exhibited a
round vesicular morphology as it was recorded by means of
TEM (Figure 1D), which also revealed the size of the vesicles
and confirmed those measured by DLS (Table 1).
3.4.2. Integrity of AP-BTH-LIP during Incubation in Serum
Proteins (Calcein Studies). Both 5 and 10% AP-BTH-LIP (as
well as control liposomes) were studied for their integrity during
their incubation in buffer and FCS for a 7 day time period at 37
°C. The results are reported in Figure 2. As seen from the calcein
latency results, both types of liposomes were very stable when
incubated in PBS even after 7 days (Figure 2B,C), as it was also
shown for the control-LIP (Figure 2A). Additionally, all LIP
types exhibited high integrity when incubated in serum proteins,
having calcein latency values higher than 80% after 48 h of
incubation. Their high integrity in serum proteins was also
retained during continued incubation, since calcein latency
values higher than 70% were measured even after 7 days of
incubation (in FCS) for all the LIP types. This was anticipated
for the control-LIP, due to their lipid composition (see above, in
Section 2.4). Nevertheless, the calcein latency results of the AP-
BTH-LIP types indicate that the incorporation of AP-BTH-lipid
at the specific concentrations tested does not decrease the
integrity of the control-LIP in the presence of serum proteins. In
fact, the addition of AP-BTH-lipid in the liposome membrane
seems to increase the calcein latency values of the liposomes
during their incubation in serum proteins. This is better depicted
in Figure 2D, where the retention of calcein in the liposomes is
reported; calcein retention in liposomes is slightly but
significantly (p = 0.026) increased as increasing amounts of
AP-BTH-lipid are incorporated in their membranes.
3.5. Inhibition of Aβ42 Aggregation Efficiency.
3.5.1. CD Studies. To assess the effect of the decorated
liposomes (AP-BTH-LIP and BTH-LIP) on the aggregation
course of Aβ42, we used circular dichroism (CD) during a 15
day study. The aggregation of Aβ produces characteristic CD
spectra that reflect the conformational changes of the peptide
from a β-sheet assembly (red line, negative maximum at 222
nm) to larger aggregates and finally to insoluble amyloid fibrils
that precipitate from solution with the loss of the CD signal
(Figure 3A).52 The presence of inhibitors of aggregation causes
changes in the typical CD spectral pattern over time, providing
evidence of interaction.
Figure 3. CD spectra of 50 μM plain solution of (A) Aβ42 as well as in
the presence of (B) AP-BTH-LIP (5%), (C) AP-BTH-LIP (10%), and
(D) AP-BTH-LIP (20%) at ratio 1:0.10 and in the presence of (E) AP-
BTH-LIP (5%), (F) AP-BTH-LIP (10%), and (G) AP-BTH-LIP
(20%) at ratio 1:0.25. Spectra were recorded for a period of 15 days at
33 °C. Representative spectra from n = 3 independent experiments are
presented.
unaffected up to day 15, indicating an intervention of the
liposome itself, in the aggregation course of Aβ42. At the lower
ratios of 1:0.25 and 1:0.10, it is obvious that the interaction of
the LIP with Aβ42 monomers or oligomers takes place, as
observed by the presence of composite peaks (especially clear in
Figure S3D, 1:0.25 ratio, negative max at 214 nm); however, the
equilibrium is eventually shifted toward aggregation, resulting in
the complete loss of the signal at day 15, as observed for plain
Aβ42 (Figure 3A). Based on the above, the Aβ/LIP ratios of
1:0.10 and 1:0.25 were judged appropriate for the assessment of
the effect of the decorated liposomes AP-BTH-LIP and BTH-
LIP on Aβ42 aggregation. In addition, the CD spectra of the
dispersions of plain LIP, BTH-LIP (5, 10, and 20%), and the
corresponding AP-BTH-LIP dispersions in the absence of Aβ42
were obtained under the same conditions, as the ones used in the
Aβ42−liposome interaction studies, to serve as controls. They
all presented a weak absorption with a negative maximum at 205
nm, relatively stronger at the 20% load, which remained
unchanged for the duration of the study (Figure S4, Supporting
Information).
Before the actual studies, the solutions of plain undecorated
liposomes (LIP) with Aβ42 at different Aβ/LIP ratios, 1:0.10,
1:0.25, 1:0.50 and 1:0.75 ratios, were studied with CD to assess
the effect of the presence of liposomes on the aggregation of
Aβ42 and to define the optimum Aβ/liposome ratio for the CD
studies. As shown in Figure S3 (Supporting Information) at the
higher ratios of Aβ/LIP 1:0.75 and 1:0.50, the presence of the
liposomes stabilized a β-sheet-like structure (maximum at 216
nm) of Aβ42 that did not aggregate further but remained
The CD spectra of Aβ42 solutions in the presence of AP-
BTH-LIP (5, 10, and 20% load) at the Aβ42/LIP ratios of 1:0.10
and 1:0.25 are shown in Figure 3. At the 1:0.10 ratio, relatively
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Figure 4. CD spectra of 50 μM Aβ42 solution in the presence of (A) BTH-LIP (5%), (B) BTH-LIP (10%), and (C) BTH-LIP (20%) at ratio 1:0.25.
Spectra were recorded for a period of 15 days at 33 °C. Representative spectra from n = 3 independent experiments are presented.
Figure 5. Fluorescence emission spectra of ThT upon binding to fibrils of Αβ42 (25 μΜ, 15 days) (A) in the presence of the AP-BTH-LIP at an Aβ/
liposome ratio of 1:0.10, (B) in the presence of the AP-BTH-LIP at an Aβ/liposome ratio of 1:0.25, and (C) in the presence of the BTH-LIP at an Aβ/
liposome ratio of 1:0.25. Fluorescence was monitored after excitation at λ = 440 nm. Representative spectra from n = 3 independent experiments are
presented.
weak composite negative peaks with maxima at 205 and 222 nm
are present, stronger in the case of the 20% load, attributed to
AP-BTH-LIP and β-sheet assemblies, respectively, which
present small changes in the duration of the study. The same
holds true for the 1:0.25 ratio with the exception of the 20% load
in which quick advancement of the aggregation is noted and
already at day 9, the CD signal is reduced to its final value,
indicating precipitation from solution. Hence, the overall, strong
interaction of the AP-BTH-LIP with Aβ42 is evident, which
interferes with the aggregation process. The quick precipitation
noted in the presence of the reduced interaction of 20% AP-
BTH-LIP with Αβ42 could be linked to the substantially larger
size of these particular vesicles (compared to the other two AP-
BTH-liposome types, Table 1). Another potential explanation
may be that the reduced interaction is due to higher intra or
intermolecular interactions of AP-BTH groups between the
different liposome particles due to the hydrophobic character of
AP-BTH on their surface, which may result in liposome
aggregation.
The interaction of the BTH-LIP-bearing benzothiazole-lipid
at 5, 10, and 20% load51 was also studied through CD at the
ratios Aβ42/BTH-LIP of 1:0.10 and 1:0.25. The CD spectra of
the 1:0.10 ratio are shown in Figure 4. At both ratios,
interactions are present, as evidenced by the weak spectra and
the shift of the β-sheet-like negative peak to lower wavelengths
(maxima at 218 nm); however, aggregation appears to precede
faster compared to plain Aβ42 solutions, and the CD signal is
almost at the baseline at day 15.
Figure 5A,B (black lines), the fluorescence intensity of the plain
solutions of Aβ42 was considerably enhanced compared to the
solutions of the plain ThT of the same concentration (orange
lines), indicating the presence of amyloid fibrils. In the 1:0.10
ratio (Figure 5A), the presence of both 5 and 10% AP-BTH-LIP
resulted in the complete inhibition of fibril formation, as
observed by the lack of fluorescence intensity, while in the 20%
load, a very weak fluorescence signal is present, indicating the
presence of small amount of fibrils. The noted shift of the
maximum of the signal to higher wavelengths may also be
indicating the formation of alternative aggregated structures. At
the higher ratio of 1:0.25 (Figure 5B), a great reduction in the
fluorescence signal was also recorded in the presence of AP-
BTH-LIP but less pronounced compared to the 1:0.10 ratio.
The noted shift of the maximum of the signal to higher
wavelengths in the case of the 5 and 10% AP-BTH-LIP, as well as
the reduced amount of fibrils in the case of the 20% load while
the CD absorbance has reached the baseline, may be indicating
the formation of the aggregates of differentiated structure
compared to typical amyloid fibrils, or even the presence of
amorphous aggregates that precipitate from solution, but do not
generate signal in the ThT assay.
The ThT assay results are in complete agreement with the CD
study that shows the interaction of the AP-BTH liposomes with
Aβ42. The interaction can only be ascribed to the presence of 2-
(4′-aminophenyl)benzothiazole as both the plain non-deco-
rated liposomes (LIP, red lines in Figure 5) and the liposomes
bearing the unsubstituted benzothiazole (BTH-LIP, Figure 5C)
fail to prevent fibril formation, as evidenced by the strong,
comparable to that of plain Aβ42 solutions, fluorescence signal
of their solutions in the ThT assay.
3.5.2. ThT Assay. The ThT dye, which is almost non-
fluorescent in solution, is known to display enhanced
fluorescence upon binding to typical amyloid fibrils.64 The
results of the ThT assay for both the 1:0.10 and 1:0.25 Aβ42/
ΑP-BTH-LIP ratios are shown in Figure 5. As it can be seen in
3.5.3. TEM Studies. To clarify the structure of Aβ42 peptides
in the presence of 5% AP-BTH-LIP, TEM studies were
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Figure 6. TEM micrographs of (A) plain Aβ42 peptides (50μΜ, 15 days), (B) Aβ42 peptides in the presence of 5% AP-BTH-LIP at an Aβ/liposome
ratio of 1:1 (50 μΜ, 15 days), and (C) 5% AP-BTH-LIP (50 μΜ, 15 days).
Figure 7. (A) Uptake of various liposome types by hCMEC/D3 cells. (B) TEER values at various time periods during the monolayer-barrier
development. (C) LY permeability values in presence of the various types of liposomes tested for their permeation across the monolayers. (D) Time-
frame of liposome-associated-FITC transport (%) across the in vitro BBB. (E) Permeability values of the various types of liposomes, calculated from
the FITC-transport results. Each result is the mean of four independent samples. The SDs of mean values are added as bars. Asterisks show the
significance of comparisons (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). In the column graphs, the significance of difference from control-
LIP is denoted as white asterisks in each column; other significant differences are shown individually.
conducted. Opposed to the typical fibril structures that are
formed when plain Aβ42 peptides are used (Figure 6A), in the
presence of the liposomes (Aβ42 + 5% AP-BTH-LIP), non-
typical threadlike amyloid aggregate formations are present
(Figure 6B). Thus, the TEM experiments are in good correlation
with the CD and ThT findings.
their interaction with the brain−blood barrier (BBB), vesicle
uptake and transport (across cell monolayers) experiments were
carried out on hCMEC/D3 cells (Figure 7), which are known to
have a brain endothelial phenotype and are widely used as a
cellular model of the human BBB.54
Herein, the uptake of 5 and 10% AP-BTH-LIP and BTH-LIP,
as well as control-LIP by hCMEC/D3 cells, was evaluated. It is
important to mention that none of the liposomal types studied
3.6. Cell Interaction Studies. To investigate whether the
insertion of AP-BTH-lipid in the liposome membrane affects
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for their interaction with hCMEC/D3 cells exhibited any
cytotoxicity against the endothelial cells under the particular
experimental conditions used (Figure S5, Supporting informa-
tion).
tively), were found to have great physicochemical stability and
integrity upon incubation with serum proteins.
The results obtained through the CD studies and the ThT
binding assays clearly demonstrate the strong interaction of the
decorated AP-BTH-LIP with Aβ42. The results can only be
ascribed to the AP-BTH moiety, as both the non-decorated
liposomes (LIP) and the unsubstituted 2-benzothiazole (BTH)-
decorated liposomes (BTH-LIP) did not prevent the formation
of Aβ42 fibrils. The almost complete inhibition of the formation
of the amyloid fibrils of Aβ42, which is a peptide with high
aggregation propensity, is an important result. In addition to the
reduction of amyloid fibrils, the AP-BTH-LIP may well interact
with lower-order entities (oligomers, protofibrils) that are also
implicated in the pathogenesis of Alzheimer’s disease and, in
fact, are considered to be even more important pathologic agents
than amyloid fibrils.65
As seen in Figure 7A, the level of liposomal uptake was
significantly affected by the type of liposomes used (p < 0.0001),
and more specifically by the addition of BTH or AP-BTH (and
the amount [5 or 10%] added) on their surface. All the
liposomes demonstrated higher uptake compared to the control-
LIP, with the exception of the 10% BTH-LIP. The highest cell
uptake was exhibited by the 5% AP-BTH-LIP, for which uptake
values were between 1.6 and 2.8 times higher than those of the
other liposome-types evaluated. Interestingly, 5% AP-BTH-LIP
exhibited significantly higher uptake than the 10% AP-BTH-LIP,
suggesting that perhaps the 5% amount is closer to the optimal
amount of AP-BTH-lipid that should be incorporated in vesicles
for optimal biological properties.
In all previous studies of benzothiazole pharmacophore
derivatives and complexes presented in the Introduction section,
the % of inhibition of the Aβ fibril formation, as determined by
the ThT test, ranges between 16 and 72%, compared to the
respective controls.45−49 In our case, as shown in the ThT test,
the novel AP-BTH-LIP prevented almost 100% the formation of
fibrils at the 5 and 10% load and the 1:0.10 Aβ/AP-BTH-LIP
ratio, while the inhibition results were great also at the 1:0.25
ratio. The results obtained by the biophysical studies on the Aβ
fibrillation inhibitory activity of the liposomes in this study are
considerably better than those mentioned above for benzothia-
zole derivatives, as in the case of the AP-BTH-LIP, almost 100%
inhibition of fibril formation was effected. The results are also
considerably better than those of other liposomic preparations
incorporating bioactive compounds, such as curcumin66,67 or a
modified ApoE-derived peptide,68 as in all these cases, a smaller
% of the inhibition of Aβ42 fibril formation is reported
compared to the controls. To the best of our knowledge, this
is the first time that the direct interaction of the decorated
liposomes with Aβ42 and the disruption of their typical
aggregation course is demonstrated through CD studies, and
fully confirmed by the ThT fibril binding assay. A literature
search for relevant CD and ThT studies employing other types
of Aβ targeting nanoformulations reveals that a relatively small
number of reports exist that provide evidence of the modulation
of the aggregation course of Aβ, including gold nanoparticles,69
self-assembled curcumin-poly(carboxybetaine methacrylate)
nanogels,70 chitosan-based nanoparticles,71 and functionalized
gadolinium (Gd) nanoparticles;72 none of the studies, however,
results in the complete absence of Aβ typical fibrils, as seen in the
case of the AP-BTH-LIP.
Moreover, it is highly important that the two best performing
(in CD and ThT studies) types of AP-BTH-LIP (5 and 10%),
and especially the 5% AP-BTH-LIP, exhibited very high
capability to target and penetrate hCMEC/D3 cell membranes,
although they do not have any known BBB-specific ligand on
their surface. A similar BBB-targeting effect was observed
previously for two other types of amyloid-targeting lip-
osomes,56,73 and this unexpected result was proven to implicate
the RAGE transporter, which is responsible for the transport of
amyloid peptides from the blood to the brain.73 In more detail,
immunoliposomes decorated with a monoclonal antibody
against Aβ42 peptides (Aβ-Mab) were found to be transported
across the hCMEC/D3 cellular model of the BBB by a
mechanism that implicates the RAGE transporter, since the
transport was substantially reduced when the monolayer was
pretreated with an anti-RAGE monoclonal antibody, while the
As monolayer transport experiments were also carried out, it is
important to report the TEER value of the monolayer barrier as
well as to confirm that the barrier was not affected by the applied
vesicles. Indeed, as seen in Figure 7B, the monolayers achieved a
TEER value of 110.6 6.5 Ω cm2, which is very close to the
previously reported values.55,56 Furthermore, the permeability of
LY was not modulated by the presence of any of the liposomes
types evaluated, as seen in Figure 7C, proving that the liposome
samples do not affect the barrier function of the monolayers.
The results of the transport experiments confirmed the
increased interaction of AP-BTH-LIP with the human brain
cells, compared to the BTH-LIP types. In fact, similarly to the
uptake study, the most effectively transported liposomal
formulation across the monolayer was the 5% AP-BTH-LIP
(Figure 7D), which also showed the highest permeability value
(Figure 7E), compared to all the other liposome types. The
permeability value calculated for 5% AP-BTH-LIP was
significantly higher than that of 10% AP-BTH-LIP (p < 0.05)
(Figure 7E). This is in good agreement with the higher uptake of
the former-type liposomes by hCMEC/D3 cells, which was
described earlier (Figure 7A). Both the AP-BTH-LIP evaluated
had significantly higher permeability values compared with the
corresponding BTH-LIP, which did not manage to exceed
transport across the cellular model of the BBB, compared to the
control liposomes.
Moreover, preliminary MTT cytotoxicity studies in U-87MG
glioblastoma cells for 24 h provide evidence that the presence of
5% AP-BTH-LIP alleviates the Aβ-induced cell toxicity (approx.
25% at 1 μΜ of 5% AP-BTH-LIP, Figure S6, Supporting
Information), while the 5% BTH-LIP or the control-LIP have no
significant effect.
4. DISCUSSION
In continuation of our efforts to identify active nanosystems that
can efficiently interfere and inhibit Αβ42 aggregation, we herein
exploited for the first time the known anti-amyloid properties of
2-benzothiazole (BTH) and its 2-(4′-aminophenyl)-
benzothiazole derivative (AP-BTH) in the construction of
novel liposomal nanoformulations against AD. Their con-
jugation to lipids and incorporation into liposome lipid-
membranes is considered to be an advanced method for
increasing their effectiveness due to the fact that liposomes have
proven potential for crossing the BBB and several liposome-
based drugs are in clinical use for brain-targeted delivery. The
two types of liposomes constructed bearing either BTH or AP-
BTH on their surface (BTH-LIP and AP-BTH-LIP, respec-
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hCMEC/D3 cells were proven to express RAGE (by western
blotting). Presumably, the liposomes can bind residual Aβ
peptides (which are present in the serum in which the
experiments were carried out) or to Aβ peptides that are
added externally, and these Aβ peptides, which are strongly
bound on the surface of the liposomes, are transported by RAGE
across the BBB model together with the liposome that is linked
to the specific Aβ-binding compounds.73
Surprisingly, the permeability value calculated herein for the
transport of the 5% AP-BTH-LIP across the cellular model of the
BBB monolayer (Figure 7) was equal to 1.87 × 10−4 1.79 ×
10−5, much higher compared to the corresponding permeability
value calculated previously for liposomes with a curcumin
derivative on their surface (5.78 × 10−5 0.58 × 10−5 cm/min
for TREG-LIP).56 In fact, the permeability of the AP-BTH-LIP
was similar to that reported before for dually brain-targeted
liposomes and multifunctional liposomes (1.90 × 10−4 2.8 ×
10−5 cm/min and 2.51 × 10−4 3.2 × 10−5/min, respectively).
Nevertheless in addition to the high affinity for amyloid peptides
the high permeability of the 5% AP-BTH-LIP is probably also
attributed (at least partially) to the small size of these particular
vesicles (<100 nm), compared to the sizes of some of the
previously studied liposome types (which ranged between 111
and 155 nm for the curcumin-derivative liposomes and the
brain-targeted and multifunctional liposomes, respectively55,56),
in accordance with previous reports about the effect of the size of
vesicles on their interaction with the hCMEC/D3 BBB model.74
Patras, Rio Patras 26510, Greece; Institute of Chemical
Engineering Sciences, Foundation for Research and Technology
Hellas (FORTH/ICES), Rio Patras 26504, Greece;
orcid.org/0000-0003-1204-1574; Phone: +30 2610-
962332; Email: mourtas@upatras.gr
Authors
Barbara Mavroidi − Institute of Biosciences & Applications,
National Center for Scientific Research “Demokritos”, Athens
15310, Greece
Antonia Marazioti − Laboratory of Pharmaceutical Technology,
Dept. of Pharmacy, School of Health Sciences, University of
Patras, Rio Patras 26510, Greece; Institute of Chemical
Engineering Sciences, Foundation for Research and Technology
Hellas (FORTH/ICES), Rio Patras 26504, Greece
Maria Kannavou − Laboratory of Pharmaceutical Technology,
Dept. of Pharmacy, School of Health Sciences, University of
Patras, Rio Patras 26510, Greece; Institute of Chemical
Engineering Sciences, Foundation for Research and Technology
Hellas (FORTH/ICES), Rio Patras 26504, Greece
Marina Sagnou − Institute of Biosciences & Applications,
National Center for Scientific Research “Demokritos”, Athens
15310, Greece; orcid.org/0000-0002-4231-6658
Maria Pelecanou − Institute of Biosciences & Applications,
National Center for Scientific Research “Demokritos”, Athens
15310, Greece; orcid.org/0000-0002-7669-2424
Sophia G. Antimisiaris − Laboratory of Pharmaceutical
Technology, Dept. of Pharmacy, School of Health Sciences,
University of Patras, Rio Patras 26510, Greece; Institute of
Chemical Engineering Sciences, Foundation for Research and
Technology Hellas (FORTH/ICES), Rio Patras 26504, Greece
5. CONCLUSIONS
The combined effectiveness of the novel, physicochemically
stable liposomes AP-BTH-LIP (5 and 10% AP-BTH-load) in
inhibiting almost completely the fibrillation of Aβ42 and
demonstrating a strong BBB penetration potential is unique in
the field of the development of agents against AD. The
remarkable multifaceted 2-(4’-aminophenyl)benzothiazole
pharmacophore becomes a big asset in the formulation of the
AP-BTH-LIP as it simultaneously bestows them with the two
most desired properties in AD research, the anti-amyloid action
and the BBB crossing capacity. Our results strongly indicate the
highly advantageous nature of the 5% AP-BTH-liposomes as an
ideal candidate for the Aβ-targeting therapeutic and/or
diagnostic system against AD, which deserves further in vivo
exploitation.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biomac.0c00811
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
M.K., A.M., and S.G.A. acknowledge support of this work by the
project ″Development of Innovative Neuroprotective Neuro-
generative Synthetic Micro-Neurotrophins″ (MIS-5032840),
which is implemented under the Special Service of the
Operational Program Competitiveness Entrepreneurship and
Innovation. The project is funded by the Operational
Programme ″Competitiveness, Entrepreneurship and Innova-
tion″ (NSRF 2014−2020) and co-financed by Greece and the
European Union (European Regional Development Fund).
B.M., M.S., and M.P. acknowledge support of this work by the
projects ″Target Identification and Development of Novel
Approaches for Health and Environmental Applications″ (MIS
5002514), which is implemented under the Action for the
Strategic Development on the Research and Technological
Sectors. The project is funded by the Operational Programme
″Competitiveness, Entrepreneurship and Innovation″ (NSRF
2014−2020) and co-financed by Greece and the European
Union (European Regional Development Fund). B.M. gratefully
acknowledges the financial support by the Stavros Niarchos
Foundation (SNF) and the State Scholarships Foundation
(ΙΚΥ) through the implementation of the program of Industrial
Fellowships at NCSR ″Demokritos″ and ″Reinforcement of
Postdoctoral Researchers − 2nd Cycle” (MIS-5033021)
(European Social Fund (ESF) − Operational Programme
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.0c00811.
■
sı
HPLC chromatograms of the synthesized lipid deriva-
tives; 1H NMR spectra of the synthesized products; CD
spectra of the plain solution of Aβ42 as well as in the
presence of LIP (control); CD spectra of plain solution of
LIP, AP-BTH-LIP, and BTH-LIP; cell viability of
hCMEC/D3 and HEK cells in the presence of BTH/
AP-BTH-LIP; effect of the BTH/AP-BTH-LIP on the
cytotoxicity of Aβ42 in U-87MG glioblastoma cells
(PDF)
AUTHOR INFORMATION
Corresponding Author
Spyridon Mourtas − Laboratory of Pharmaceutical Technology,
Dept. of Pharmacy, School of Health Sciences, University of
■
K
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(7) Reiss, A. B.; Arain, H. A.; Stecker, M. M.; Siegart, N. M.;
Kasselman, L. J. Amyloid toxicity in Alzheimer’s disease. Rev. Neurosci.
2018, 29, 613−627.
″Human Resources Development, Education and Lifelong
Learning″), respectively.
Notes
(8) Takahashi, R. H.; Nagao, T.; Gouras, G. K. Plaque formation and
the intraneuronal accumulation of β-amyloid in Alzheimer’s disease.
Pathol. Int. 2017, 67, 185−193.
The authors declare no competing financial interest.
(9) Saraiva, C.; Praca̧ , C.; Ferreira, R.; Santos, T.; Ferreira, L.;
ACKNOWLEDGMENTS
■
Bernardino, L. Nanoparticle-mediated brain drug delivery: Over-
coming blood−brain barrier to treat neurodegenerative diseases. J
Controlled Release 2016, 235, 34−47.
The authors are thankful to Dr. Mary Kollia and the Laboratory
of Electron Microscopy and Microanalysis (L.E.M.M.), Faculty
of Natural Sciences, University of Patras, for the TEM studies.
(10) Agrawal, M.; Ajazuddina; Tripathi, D. K.; Saraf, S.; Saraf, S.;
Antimisiaris, S. G.; Mourtas, S.; Hammarlund-Udenaes, M.; Alexander,
A. Recent advancements in liposomes targeting strategies to cross
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ABBREVIATIONS
■
MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; AcCN,acetonitrile; AcOH,acetic acid; AD,Alzheimer’s
disease; AP-BTH,2-(4-aminophenyl)benzothiazole;
BBB,blood−brain barrier; BTH,2-benzothiazole; CD,circular
dichroism; CDCl3,chloroform-d; CHCl3,chloroform; Chol,cho-
lesterol; CL,cardiolipin; CNS,central nervous system;
CO2,carbon dioxide; DCM,dichloromethane; dH2O,distilled
water; DIC,diisopropylcarbodiimide; DLS,dynamic light scat-
tering; DMF,dimethylformamide; DMSO,dimethyl sulfoxide;
DSPC,1,2-distearoyl-sn-glycerol-3-phosphatidylcholine; DSPE-
PEG2000-amine,1,2-distearoyl-sn-glycero-3-phosphoethanol-
amine-N-[amino(polyethylene glycol)-2000] (ammonium
salt); Et2O,diethyl ether; EtOAc,ethyl acetate; EtOH,ethanol;
FCS,fetal calf serum; FI,fluorescence intensity; FITC,fluores-
cein-isothiocyanate-dextran-4000; H2O,water; hCMEC/D3,im-
mortalized human cerebral microvascular endothelial cells;
HEK,human HEK-293 embryonic kidney cells; HEPES,(4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid); Hex,hexane;
HPLC,high-performance liquid chromatography; IgG,immuno-
globulin G; KMnO4,potassium permanganate; LIP,liposomes;
LY,lucifer yellow-CH dilithium salt; MAb,monoclonal antibody;
MeOD,methanol-d; MeOH,methanol; N2,nitrogen;
NaBH4,sodium borohydride; NHS,N-Hydroxysuccinimide;
NMR,nuclear magnetic resonance; NP,nanoparticles; PA,phos-
phatidic acid; PBS,phosphate-buffered saline; PDI,polydisper-
sity index; PEG,1,2-distearoyl-sn-glycerol-3-phosphoethanol-
amine-N-[methoxy(polyethyleneglycol)-2000]; ppm,parts per
million; PTA,phosphotungstic acid; RES,reticuloendothelial
system; SD,standard deviation; TEER,transendothelial electri-
cal resistance; TEM,transmission electron microscopy; THF,te-
trahydrofuran; ThT,thioflavin T; TLC,thin-layer chromatog-
raphy; UV,ultraviolet; (Aβ, Aβ1−42),β-amyloid peptide
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https://dx.doi.org/10.1021/acs.biomac.0c00811
Biomacromolecules XXXX, XXX, XXX−XXX