Nanolipid-trehalose conjugates and nano-assemblies as putative autophagy inducers

Article abstract of DOI:10.3390/pharmaceutics11080422

The disaccharide trehalose is an autophagy inducer, but its pharmacological application is severely limited by its poor pharmacokinetics properties. Thus, trehalose was coupled via suitable spacers with squalene (in 1:2 and 1:1 stoichiometry) and with betulinic acid (1:2 stoichiometry), in order to yield the corresponding nanolipid-trehalose conjugates 1-Sq-mono, 2-Sq-bis and 3-Be-mono. The conjugates were assembled to produce the corresponding nano-assemblies (NAs) Sq-NA1, Sq-NA2 and Be-NA3. The synthetic and assembly protocols are described in detail. The resulting NAs were characterized in terms of loading and structure, and tested in vitro for their capability to induce autophagy. Our results are presented and thoroughly commented upon.

Full text of DOI:10.3390/pharmaceutics11080422

pharmaceutics
Article
Nanolipid-Trehalose Conjugates and
Nano-Assemblies as Putative Autophagy Inducers
1
1
2
3
Eleonora Colombo , Michele Biocotino , Giulia Frapporti , Pietro Randazzo ,
4
2
5
1,
Michael S. Christodoulou , Giovanni Piccoli , Laura Polito
and Daniele Passarella *
, Pierfausto Seneci *
1
,
1
Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy
CIBIO, Università di Trento, Via Sommarive 9, 38123 Povo (TN), Italy
Promidis Srl, San Raffaele Scientific Research Park, Torre San Michele 1, Via Olgettina 60, 20132 Milan, Italy
DISFARM, Sezione di Chimica Generale e Organica “A. Marchesini”, Universitdegli Studi di Milano,
via Venezian 21, 20133 Milano, Italy
2
3
4
5
ISTM-CNR, via Fantoli 16/15, 20138 Milan, Italy
*
Correspondence: pierfausto.seneci@unimi.it (P.S.); daniele.passarella@unimi.it (D.P.);
Tel.: +39-02-5031-4060 (P.S.); +39-02-5031-4081 (D.P.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 12 June 2019; Accepted: 8 August 2019; Published: 20 August 2019
Abstract: The disaccharide trehalose is an autophagy inducer, but its pharmacological application is
severely limited by its poor pharmacokinetics properties. Thus, trehalose was coupled via suitable
spacers with squalene (in 1:2 and 1:1 stoichiometry) and with betulinic acid (1:2 stoichiometry), in
order to yield the corresponding nanolipid-trehalose conjugates 1-Sq-mono
,
2-Sq-bis and 3-Be-mono
.
The conjugates were assembled to produce the corresponding nano-assemblies (NAs) Sq-NA1
,
Sq-NA2 and Be-NA3. The synthetic and assembly protocols are described in detail. The resulting
NAs were characterized in terms of loading and structure, and tested in vitro for their capability to
induce autophagy. Our results are presented and thoroughly commented upon.
Keywords: nano-assemblies; trehalose; squalene; betulinic acid; autophagy induction
1
. Introduction
Nano-vectors are used as therapeutics and diagnostics [
silica-based inorganic nano-vectors [ ] were tested as being a diagnostic (magnetic resonance imaging
reagents/MRIs [ ]), or as therapeutics (hyperthermia against tumors [ ], iron replacement therapies [ ]).
Liposome- [ ], micelle- [10] and polymer-based nano-assemblies (NAs) [11] are marketed, mostly as
1,2]. Iron-based [3], gold-based [4] and
5
6
7
8
9
anti-cancer agents [12]. Exploratory efforts [13] up to clinical trials [14] against diseases of the central
nervous system (CNS) were reported.
Trehalose [15,16] is a non-reducing disaccharide made by a 1,1 linkage between two d-glucose
molecules. It is bio-synthesized in lower organisms [16] to stabilize life processes and support survival
in extreme conditions (freezing [17], heat and desiccation [18]). Trehalose induces autophagy in vitro
and in vivo
chaperone and solvating them [20]. Reduction of aggregated huntingtin (Huntington Disease) [21],
synuclein (Parkinson Disease) [20] and amyloid species (Alzheimer Disease) [22] was observed in vitro
[19] and reduces protein misfolding and aggregation in vitro [20] by acting as a chemical
.
Trehalose was tested as a safe, cheap, neuroprotective agent in preclinical and clinical studies [16].
Unfortunately, high mM trehalose concentrations are needed in vivo for efficacy, due to its high
hydrophilicity and due to trehalase enzymes [23], that hydrolyze trehalose in the brush border cells of
the small intestine and in the proximal tubules of the kidneys, preventing its oral absorption.
Nano lipid-drug conjugates [24], obtained by the covalent coupling of a drug to bio-compatible
lipids, improve pharmacokinetics, decrease toxicity and increase the therapeutic index of the associated
Pharmaceutics 2019, 11, 422; doi:10.3390/pharmaceutics11080422
www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2019, 11, 422
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drugs. In particular, squalene-based amphiphilic conjugates have a proven track record for therapeutic
applications [25]. They spontaneously assemble in water into nano-assemblies (NAs), encasing the
bioactive payload, they do not show the drug on their surface, minimizing any side effect [26], they are
internalized by cells via endocytic pathways [27] and release the free drug at its site of action, when a
biologically labile linkage is used [28]. We also selected betulinic acid-based conjugates, a less studied
but promising class of self-assembling NAs [29,30].
In recent years we worked on anticancer drug-containing self-assembling drug conjugates that
spontaneously form NAs in aqueous media [31]. We reported NAs composed by conjugate releasable
compounds [32]; by single and dual drug fluorescent hetero-NAs [33
cyclopamine/taxol [34], cyclopamine/doxorubicin [35], ecdysteroid/doxorubicin [36]), and by
self-assembling conjugate dual drug NAs [37]. We prepared compounds containing a squalene [31 35
or a 4-(1,2-diphenylbut-1-en-1-yl)aniline tail [37 38] that leads to NAs ability to self-assemble in water.
,34], by dual drug hetero-NAs
(
–
]
,
We recently reported the assembly and characterization of squalene-thiocolchicine NAs that release
cytotoxic, free thiocolchicine in cancer cells through a disulfide bond or a p-hydroxybenzyl moiety [38].
Here we describe the synthesis of two squalene-trehalose conjugates 1-Sq-mono and 2-Sq-bis, and of a
betulinic acid-trehalose conjugate 3-Be-mono, their assembly and characterization as NAs (respectively
mono-Sq-NA1, bis-Sq-NA2 and mono-Be-NA3, Figure 1), and their effects in biological assays.
We measured cell viability to determine the safety of our NAs, while autophagy induction was selected
as a validated neuroprotective mechanism of action in multiple neurodegenerative diseases [39].
Trehalose-containing NAs may significantly increase the weak, high mM effects of trehalose on
autophagy induction [19], by facilitating its cellular internalization, and by releasing after NA
disassembly/ester hydrolysis.
Figure 1. Chemical structure of nanolipid-trehalose conjugates 1-Sq-mono, 2-Sq-bis, and 3-Be-mono.
2
. Materials and Methods
2
.1. Synthesis-General
Each reaction was carried out in oven-dried glassware, using dry solvents under a nitrogen
atmosphere. Unless otherwise stated, these solvents were purchased from Sigma Aldrich Italy (Milan,
Italy) and used without further purification. Chemical reagents were purchased from Sigma Aldrich,
and used as such. Thin layer chromatography (TLC) was performed on Merck-pre-coated 60F254 plates.
Reactions were monitored by TLC on silica gel, with detection by UV light (254 nm), or by charring
either with a 1% permanganate solution or a 50% H SO solution. Flash chromatography columns
2
4
were run using silica gel (240–400 mesh, Merck Italy, Milan, Italy).
1
H-NMR spectra were recorded on Bruker DRX-400 and Bruker DRX-300 instruments (Billerica,
13
MA, USA) in either CDCl , CD OD or DMSO-d6. C-NMR spectra were recorded on the same
3
3

Pharmaceutics 2019, 11, 422
3 of 17
instrumentation (100 and 75 MHz) in either CDCl , CD OD or DMSO-d6. Chemical shifts (δ) for
3
3
proton and carbon signals are quoted in parts per million (ppm) relative to tetramethylsilane (TMS),
which was used as an internal standard. Electrospray ionization (ESI) MS spectra were recorded with a
Waters Micromass Q-Tof micro mass spectrometer (Milford, MA, USA); HR-ESI mass spectra were
recorded on a FT-ICR APEX (Bruker, Billerica, MA, USA), while EI mass spectra were recorded at
II
an ionizing voltage of 6 kEv on a VG 70-70 EQ. Specific rotations were measured with a P-1030-Jasco
polarimeter with 10 cm optical path cells and 1 mL capacity (Na lamp, λ = 589 nm).
2
.2. Synthesis-Squalene-Trehalose Conjugates 1-Sq-Mono and 2-Sq-Bis
-[(2R,3R,4S,5R,6R)-6-{[(2R,3R,4S,5R,6R)-6-(hydroxymethyl)-3,4,5-tris[(trimethylsilyl)oxy]oxan-2-
1
yl]oxy}-3,4,5-tris[(trimethylsilyl)oxy]oxan-2-yl]methyl10-(3E,7E,11E,15E)-3,7,12,16,20pentamethylheni-
cosa-3,7,11,15,19-pentaen-1-yl decanedioate/15-mono and 1-[(2R,3R,4S,5R,6R)-6-{[(2R,3R,4S,5R,6R)-6-
{[(10-oxo-10-{[(3E,7E,11E,15E)-3,7,12,16,20-penta-methylhenicosa-3,7,11,15,19-pentaen-1-yl]oxy}-
decanoyl)oxy]methyl}-3,4,5-tris[(trimethylsilyl)oxy]oxan-2-yl]oxy}-3,4,5-tris[(trimethylsilyl)oxy]-oxan-
2
-yl]methyl10-(3E,7E,11E,15E)-3,7,12,16,20-penta-methylhenicosa-3,7,11,15,19-pentaen-1-yl
decanedioate/16-bis EDC HCl (223 mg, 1.161 mmol) and DMAP (5 mg, 0.039 mmol) were
added under stirring at room temperature (RT) to a solution of hexaTMS-protected trehalose 40
300 mg, 0.387 mmol) in anhydrous toluene (8.3 mL). After 30 min, carboxylated squalene-linker
.
·
5
[ ]
(
◦
adduct
6 [32] (221 mg, 0.387 mmol) was added, and the reaction mixture was stirred at 50 C
overnight. Reaction monitoring (TLC, eluant: 9:1 n-hexane/AcOEt) confirmed the disappearance of
hexaTMS-protected trehalose . The solvent was then removed under reduced pressure, and the
crude oil was purified by flash chromatography (silicagel, eluant: 9:1 n-hexane/AcOEt) to obtain pure
5
1
5-mono (232.9 mg, 0.175 mmol, 45% yield) and pure 16-bis (101.2 mg, 0.054 mmol, 14% yield).
1
Analytical characterization. 15-mono: H-NMR (CDCl , 400 MHz):
δ
(ppm) = 5.21–5.05 (m, 5H),
3
4.94 (t, J = 2.8 Hz, 2H), 4.32 (dd, J = 11.8, 2.1 Hz, 1H), 4.12–3.98 (m, 4H), 3.98–3.82 (m, 3H), 3.71 (dd,
J = 6.4, 3.4 Hz, 2H), 3.54–3.41 (m, 4H), 2.41–2.26 (m, 4H), 2.14–1.96 (m, 16H), 1.79–1.67 (m, 5H), 1.62–1.58
m, 21H), 1.37–1.29 (m, 8H), 0.22–0.11 (m, 54H). 13C–NMR (CDCl , 400 MHz):
(ppm) = 173.33, 173.18,
34.88, 134.76, 134.07, 132.4, 132.00, 129.28, 129.17, 124.84, 124.57, 124.37, 93.83, 93.67, 73.77, 73.66,
(
δ
3
1
7
2
0
3.03, 72.05, 71.94, 70.59, 70.10, 68.28, 63.61, 63.24, 61.27, 39.73 (2C), 35.72, 33.98, 33.87, 29.45, 29.04 (3C),
8.99, 28.86, 28.18, 26.68, 26.43 (2C), 25.95, 25.01, 24.91, 22.38 (2C), 17.95, 16.28, 16.22, 16.05, (1.35, 1.18,
.36 = 18C). HR-ESI-MS: MW 1349.7973 calcd. for C H O Si Na, MW 1349.7982 found. Optical
67
130 14
6
20
◦
1
rotation, [α]D
:
−
61.9 . 16-bis: H-NMR ((CDCl , 400 MHz):
δ
(ppm) = 5.16–5.10 (m, 10H), 4.94 (d,
3
J = 3.0 Hz, 2H), 4.31–4.28 (m, 2H), 4.10–3.99 (d, J = 38.8 Hz, 8H), 3.95–3.90 (m, 2H), 3.52–3.44 (m,
4
0
H), 2.38–2.28 (m, 8H), 2.13–1.98 (m, 36H), 1.77–1.68 (m, 10H), 1.66–1.59 (m, 38H), 1.36–1.28 (m, 16H),
.21–0.11 (m, 54H). 13C-NMR (CDCl , 400 MHz):
δ(ppm) = 173.78 (2C), 173.62 (2C), 135.05 (2C), 134.90
3
(
2C), 134.82 (2C), 133.65 (2C), 131.15 (2C), 125.07 (2C), 124.40 (4C), 124.29 (4C), 94.40 (2C), 73.48 (2C),
7
2
2
2.67 (2C), 71.94 (2C), 70.74 (2C), 63.94 (2C), 63.30 (2C), 39.73 (4C), 35.80 (2C), 34.33 (2C), 34.09 (2C),
9.68 (2C), 29.11 (6C), 28.77 (2C), 28.25 (4C), 26.91 (2C), 26.76 (2C), 26.65 (4C), 25.67 (4C), 24.96 (2C),
4.74 (2C), 17.66 (2C), 16.02 (4C), 15.85 (2C), (1.05, 0.87, 0.44, 0.17 = 18C). HR-ESI-MS: MW 1902.2516
20
◦
calcd. for C104H190O Si Na, MW 1902.2524 found. Optical rotation, [α] : −43.9 .
17
6
D
1-(3E,7E,11E,15E)-3,7,12,16,20-pentamethylhenicosa-3,7,11,15,19-pentaen-1-yl
10-[(2R,3S,4S,
5
R,6R)-3,4,5-trihydroxy-6-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-
2
-yl]-methyl decanedioate/1-Sq-mono. Acetic acid (0.1 mL, 1.73 mmol) was added under stirring at
RT to a solution of 15-mono (230.0 mg, 0.173 mmol) in MeOH (3 mL), and the reaction mixture was
◦
stirred at 40 C overnight. Reaction monitoring (TLC, eluant: 9:1 n-hexane/AcOEt) confirmed the
disappearance of 15-mono. The solvent was then removed under reduced pressure, and the crude
solid was purified by flash chromatography (silicagel, eluant: 85:15 CH Cl /MeOH) to obtain pure
2
2
target 1-Sq-mono (153.2 mg, 0.171 mmol, quantitative yield).
1
Analytical characterization. H-NMR (DMSO-d6, 400 MHz):
δ(ppm) = 5.00–4.91 (m, 6H), 4.75
(d, J = 3.7 Hz, 2H), 4.72 (d, J = 3.6 Hz, 1H), 4.64 (t, J = 4.7 Hz, 2H), 4.55 (dd, J = 6.2, 1.8 Hz, 2H), 4.22

Pharmaceutics 2019, 11, 422
4 of 17
(
(
t, J = 6.0 Hz, 1H), 4.13–4.10 (m, 1H), 3.92 (dd, J = 11.8, 5.4 Hz, 1H), 3.83 (t, J = 6.6 Hz, 2H), 3.80–3.76
m, 1H), 3.55–3.51 (m, 1H), 3.46–3.40 (m, 3H), 3.38–3.32 (m, 1H), 3.16–3.10 (m, 2H), 3.04–2.98 (m, 2H),
2
8
.1–2.12 (m, 4H), 1.95–1.79 (m, 18H), 1.56–1.49 (m, 5H), 1.44 (bs, 15H), 1.41–1.37 (m, 4H), 1.13 (bs,
H). C-NMR (DMSO-d6, 100 MHz): δ(ppm): δ 173.28, 173.22, 134.83, 134.77, 134.73, 133.97, 131.04,
13
1
24.82, 124.59, 124.52, 124.46, 124.35, 93.81, 93.71, 73.30, 73.26, 73.04, 72.05, 71.94, 70.59 (2C), 70.10, 63.63,
6
2
3.56, 61.27, 40.62, 39.64, 39.59, 39.37, 35.71, 33.98 (2C), 31.59, 30.28, 29.46, 29.06, 29.03, 28.91, 28.16,
6.82, 26.67, 26.42 (2C), 25.90, 24.96, 24.89, 17.94, 16.21, 16.06. HR-ESI-MS: MW 917.5602 calcd. for
2
0
◦
C H O Na, MW 917.5623 found. Optical rotation, [α] : −59.4 .
49
82 14
D
1-(3E,7E,11E,15E)-3,7,12,16,20-pentamethylhenicosa-3,7,11,15,19-pentaen-1-yl
10-[(2R,3S,4S,
5
2
R,6R)-3,4,5-trihydroxy-6-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-{[(10-oxo-10-{[(3E,7E,11E,15E)-3,7,12,16,
0-pentamethylhenicosa-3,7,11,15,19-pentaen-1-yl]oxy}decanoyl)oxy]methyl}oxan-2-yl]oxy}oxan-2-yl]-
methyl decanedioate/2-Sq-bis. Acetic acid (30 µL, 0.53 mmol) was added under stirring at RT to a
solution of 16-bis (100.0 mg, 0.053 mmol) in MeOH (1 mL), and the reaction mixture was stirred at
◦
4
0 C overnight. Reaction monitoring (TLC, eluant: 9:1 n-hexane/AcOEt) confirmed the disappearance
of 16-bis. The solvent was then removed under reduced pressure, and the crude solid was purified
by flash chromatography (silicagel, eluant: 85:15 CH Cl /MeOH) to obtain pure target 2-Sq-bis
2
2
1
(
69.1 mg, 0.047 mmol, 90% yield). Analytical characterization. H-NMR (DMSO-d6, 400 MHz):
δ
(ppm) = 5.12–5.04 (m, 12H), 4.88 (d, J = 4.9 Hz, 2H), 4.83 (d, J = 3.6 Hz, 2H), 4.75 (d, J = 6.1 Hz, 2H),
.25–4.21 (m, 2H), 4.03 (dd, J = 11.7, 5.6 Hz, 2H), 3.95 (t, J = 6.6 Hz, 4H), 3.92–3.87 (m, 2H), 3.58–3.52
4
(
1
m, 2H), 3.28–3.23 (m, 2H), 3.15–3.09 (m, 2H), 2.28–2.23 (m, 8H), 2.07–1.90 (m, 36H), 1.67–1.60 (m,
0H), 1.55–1.47 (m, 38H), 1.24 (s, 16H). 13C-NMR (DMSO-d6, 100 MHz):
δ(ppm): 173.90 (2C), 173.64
(
(
(
(
(
2C), 134.81 (2C), 134.72 (2C), 134.64 (2C), 133.63 (2C), 131.08 (2C), 124.94 (2C), 124.57 (2C), 124.52
2C), 124.47 (2C), 124.32 (2C), 93.75 (2C), 73.52 (2C), 72.56 (2C), 71.93 (2C), 70.71 (2C),63.99 (2C), 63.34
2C), 39.74 (4C), 39.65 (2C), 35.86 (2C), 34.38 (2C), 34.12 (2C), 29.74 (2C), 29.17 (2C), 29.10 (4C), 28.28
4C), 26.92 (2C), 26.81 (2C), 26.68 (2C), 26.65 (2C), 25.67 (4C), 24.98 (2C), 24.90 (2C), 17.94 (2C), 16.24
4C), 16.06 (2C). HR-ESI-MS: MW 1470.0147 calcd. for C H O Na, MW 1470.0146 found. Optical
86
142 17
20
◦
rotation, [α] : −33.7 .
D
2
.3. Synthesis—Betulinic Acid-trehalose Conjugate 3–Be-mono
Methyl(1R,3aS,5aR,5bR,9S,11aR)-9-hydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-icosa-
hydro-1H-cyclopenta[a]chrysene-3a-carboxylate/18. Trimethylsilyl diazomethane (2M in n-hexane,
.66 mL, 1.312 mmol) was added to a solution of betulinic acid 17 (500 mg, 1.093 mmol) in dry MeOH
0
(
(
10 mL) and dry toluene (15 mL). The reaction was stirred overnight at RT, and reaction monitoring
TLC, eluant 7:3 n-hexane/AcOEt with 1% HCOOH) confirmed the disappearance of starting material
1
6
. The reaction mixture was diluted with diethyl ether (13 mL) and 10% AcOH (10 mL). The aqueous
layer was extracted with diethyl ether (3 10 mL), and the collected organic phases were washed with
sat. Na CO (10 mL), dried with Na SO and then evaporated under reduced pressure to obtain pure
×
2
3
2
4
1
8 as a white solid (486.1 mg, 1.032 mmol, 95% yield).
1
Analytical characterization. H-NMR (CDCl 400 MHz):
δ(ppm) = 4.63 (bs, 1H), 4.49 (bs, 1H),
3,
3.56 (s, 3H), 3.07 (dd, J = 11.2, 5.1 Hz, 1H), 2.89 (td, J = 10.9, 4.4 Hz, 1H), 2.20–2.02 (m, 2H), 1.77 (dt,
J = 10.9, 5.9 Hz, 2H), 1.58 (s, 3H), 0.86 (s, 6H), 0.81 (s, 3H), 0.71 (s, 3H), 0.65 (s, 3H). HR-ESI-MS: MW
2
0
◦
93.3658 calcd. for C H O Na, MW 493.3661 found. Optical rotation, [α] : +5.1 .
31 50 3
D
4
1
0-oxo-10-[2-(trimethylsilyl)ethoxy]decanoic acid/19. Trimethylsilylethanol (313 mL, 2.181 mmol),
EDC.HCl (559 mg, 2.909 mmol) and DMAP (89 mg, 0.727 mmol) were added under stirring at RT to a
solution of sebacic acid 14 (1 g, 0.4942 mmol) in dry CH Cl (25 mL) and pyridine (2.5 mL). The reaction
2
2
mixture was stirred at RT overnight. The reaction mixture was then washed with 10% phosphoric acid
2 × 15 mL) and brine (20 mL).
The organic layer was dried with Na SO and evaporated under reduced pressure, and the crude
(
2
4
oil was purified by flash chromatography (silicagel, eluant: 8:2 n-hexane/AcOEt with 1% HCOOH) to
obtain pure 19 (408 mg, 1.342 mmol, 27% yield).

Pharmaceutics 2019, 11, 422
5 of 17
1
Analytical characterization. H-NMR (CDCl , 400 MHz):
δ(ppm) = 4.21–4.12 (m, 2H), 2.38 (t,
3
J = 4.5 Hz, 2H), 2.32 (t, J = 4.3 Hz, 2H), 1.73–1.59 (m, 4H), 1.41–1.28 (m, 8H), 1.01–0.97 (m, 2H), 0.04 (s,
1
3
9
2
H). C-NMR: (CDCl , 100 MHz):
δ
(ppm) = 177.93, 173.60, 62.58, 34.32, 34.22, 29.46, 29.48, 29.30, 29.10,
3
5.12, 25.07, 17.05,
1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-
icosahydro-1H-cyclopenta[a]chrysen-9-yl 1-[2-(trimethylsilyl)ethyl] decanedioate/20
Dicyclohexylcarbodiimide (DCC, 201 mg, 0.973 mmol) and dimethylaminopyridine (DMAP,
−
1.53 (3C). HR-ESI-MS: MW 325.1811 calcd. for C H O SiNa, MW 325.1815 found.
15
30
4
(
.
3
0 mg, 0.243 mmol) were added under stirring to a solution of compound 18 (229 mg, 0.487 mmol) and
◦
compound 19 (221 mg, 0.731 mmol) in dry CH Cl (5 mL) at 0 C. The reaction was left stirring at RT
2
2
overnight. Reaction monitoring (TLC, eluant: 9:1 n-hexane/AcOEt) confirmed the disappearance of
starting material 18. The mixture was diluted with CH Cl (10 mL) and was filtered on a plug of
2
2
celite. The solvent was removed under reduced pressure, and the resulting crude oil was purified by
flash chromatography (silicagel, eluant: 96:4 n-hexane/AcOEt) to obtain pure 20 (339 mg, 0.449 mmol,
9
2% yield).
Analytical characterization. H-NMR (CDCl , 400 MHz):
1
δ
(ppm) = 4.74 (bs, 1H), 4.61 (bs, 1H),
.48 (dd, J = 10.1, 6.2 Hz, 1H), 4.21–4.12 (m, 2H), 3.67 (s, 3H), 3.06–2.95 (m, 1H), 2.33–2.13 (m, 6H),
.97–1.82 (m, 2H), 1.69 (s, 3H), 1.47–1.34 (m, 8H), 0.97 (s, 3H), 0.92 (s, 3H), 0.85 (s, 3H), 0.84 (s, 6H),
.05 (s, 9H). 13C-NMR: (CDCl , 100 MHz):
(ppm) = 176.67, 174.00, 173.64, 150.57, 109.63, 80.61, 62.37,
3
4
1
0
5
3
δ
3
6.57, 55.45 (2C), 51.25, 50.46, 49.48, 47.01, 42.40, 40.70, 38.40, 38.27, 37.85, 37.12, 36.98, 34.82, 34.52,
4.27, 32.18, 30.61, 29.68, 29.10 (3C), 27.97, 25.49, 25.12, 24.95, 23.75, 20.91, 19.36, 18.19, 17.33, 16.57,
1
6.18, 15.96, 14.69,
−
1.47 (3C). HR-ESI-MS: MW 777.5465 calcd. for C H O SiNa, MW 777.5469 found.
46 78 6
20
◦
Optical rotation, [α] : +10.2 .
D
1
0-{[(1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)
icosahydro-1H-cyclopenta[a]chrysen-9-yl]oxy}-10-oxodecanoic acid/21. Tetrabutylammonium fluoride
TBAF, 0.61 mL, 2.11 mmol) was added under stirring to a solution of compound 19 (318 mg,
.421 mmol) in dry THF (15 mL), and the reaction mixture was stirred at RT overnight. Reaction
monitoring (TLC, eluant: 9:1 n-hexane/AcOEt with 1% HCOOH) confirmed the disappearance of
starting material 20. The reaction was quenched by an addition of sat. NH Cl (10 mL). The aqueous
-
(
0
4
phase was extracted with AcOEt (2
×
10 mL), the collected organic phases were dried with Na SO
2 4
and evaporated under reduced pressure to obtain pure 21 (258 mg, 0.393 mmol, 93% yield).
1
Analytical characterization. H-NMR (CDCl , 400 MHz):
δ(ppm) = 4.72 (bs, 1H), 4.58 (bs, 1H),
3
4
.44 (dd, J = 10.1, 6.2 Hz, 1H), 3.65 (s, 3H), 3.03–2.94 (m, 1H), 2.23–2.16 (m, 6H), 1.92–1.82 (m, 2H), 1.67
13
(s, 3H), 1.47–1.34 (m, 8H), 0.95 (s, 3H), 0.90 (s, 3H), 0.84 (s, 3H), 0.82 (s, 6H). C-NMR (CDCl , 100
3
MHz):
δ
(ppm) 180.32, 177.30, 174.33, 151.11, 110.29, 81.31, 57.20, 56.08 (2C), 51.89, 51.10, 50.12, 47.63,
3.04, 41.34, 39.04, 38.90, 38.47, 37.76, 37.60, 35.43, 34.92, 34.70, 32.81, 31.25, 30.32, 29.68 (3C), 28.61,
6.13, 25.73, 25.29, 24.38, 21.56, 20.00, 18.84, 17.21, 16.81, 16.59, 15.33. HR-ESI-MS: MW 677.4757 calcd.
4
2
20
◦
for C H O Na, MW 677.4761 found. Optical rotation, [α] : +12.9 .
41
66
6
D
(1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-
icosahydro-1H-cyclopenta[a]chrysen-9-yl-1-[(2R,3R,4S,5R,6R)-6-{[(2R,3R,4S,5R,6R)-6-(hydroxy
met-hyl)-3,4,5-tris[(trimethylsilyl)oxy]oxan-2-yl]oxy}-3,4,5-tris[(trimethylsilyl)oxy]oxan-2-yl]methyl
decanedioate/22-monoand (1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-pentamethyl-
1
6
-(prop-1-en-2-yl)-icosahydro-1H-cyclopenta[a]chrysen-9-yl 1-[(2R,3R,4S,5R,6R)-6-{[(2R,3R,4S,5R,6R)-
-{[(10-{[(1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-penta-methyl-1-(prop-1-en-2-
yl)-icosahydro-1H-cyclopenta[a]chrysen-9-yl]oxy}-10-oxodecanoyl)oxy]-methyl}-3,4,5-tris
[
(trimethylsilyl)oxy]oxan-2-yl]oxy}-3,4,5-tris[(trimethylsilyl)oxy]oxan-2-yl]methyl decanedioate/23-bis
.
.
EDC HCl (15 mg, 0.0763 mmol) and DMAP (1 mg, 0.00763 mmol) were added under stirring at RT to a
solution of hexaTMS-protected trehalose 5 [40]. (59 mg, 0.0763 mmol) in anhydrous toluene (4 mL).
After 30 min, compound 21 (50 mg, 0.0763 mmol) was added, and the reaction mixture was stirred at
◦
5
0 C overnight. Reaction monitoring (TLC, eluant: 7:3 n-hexane/AcOEt) confirmed the disappearance
of starting material 21. The solvent was then removed under reduced pressure, and the crude oil was

Pharmaceutics 2019, 11, 422
6 of 17
purified by flash chromatography (silicagel, eluant: 85:15 n-hexane/AcOEt) to obtain pure 22-mono
(
23 mg, 0.0163 mmol, 21% yield) and pure 23-bis (24 mg, 0.0117 mmol, 15% yield).
1
Analytical characterization. 22-mono: H-NMR (CD OD, 400 MHz):
δ
(ppm) = 4.96 (dd, J = 3.0,
3
1
1
.9 Hz, 2H), 4.74 (d, J = 2.1 Hz, 1H), 4.67–4.58 (m, 1H), 4.47 (dd, J = 10.6, 5.7 Hz, 1H), 4.43–4.34 (m,
H), 4.12–4.02 (m, 2H), 3.99 (td, J = 9.0, 3.1 Hz, 2H), 3.87 (dt, J = 9.5, 3.0 Hz, 1H), 3.70–3.68 (m, 5H),
3
5
(
.61–3.45 (m, 4H), 3.34–3.32 (m, 2H), 3.02 (td, J = 10.8, 4.7 Hz, 1H), 2.44–2.23 (m, 6H), 1.90 (tt, J = 11.7,
.8 Hz, 2H), 1.78–1.70 (m, 6H), 1.36 (s, 3H), 1.04 (s, 3H), 0.97 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.88
s, 3H), 0.25–0.14 (m, 54H). 13C-NMR (CD OD, 400 MHz):
δ
(ppm) = 176.69, 173.89, 173.68, 150.33,
3
108.96, 94.45, 94.19, 80.81, 73.58, 73.54, 73.31, 72.66 (2C), 72.01, 71.21, 70.72, 62.91, 60.41, 56.48, 55.45
(
2
1
1
δ
4
2C), 50.44, 49.24, 47.09, 42.17, 40.53, 38.24, 38.19, 37.52, 36.91, 36.47, 34.16, 34.06, 33.62, 31.74, 30.24,
9.42, 28.78 (2C), 28.73, 28.62, 27.22, 25.38, 24.80, 24.58, 23.38, 20.72, 18.21, 17.90, 15.75, 15.42, 15.20,
3.83, (0.19, 0.25, 0.99, 1.07 = 18C). HR-ESI-MS: MW 1433.8185 calcd. for C H O Si Na, MW
−
−
−
71
134 16
6
20
◦
1
433.8191 found. Optical rotation, [α] : +60.3 . 23-bis: H-NMR (CDCl , 400 MHz, detected signals):
D
3
(ppm) = 4.94 (d, J = 3.0 Hz, 2H), 4.76 (d, J = 2.0 Hz, 2H), 4.62 (d, J = 3.2 Hz, 2H), 4.53–4.44 (m, 2H),
.29 (dd, J = 11.8, 2.0 Hz, 2H), 4.07 (dd, J = 11.8, 4.3 Hz, 2H), 4.02 (ddd, J = 9.4, 4.2, 2.1 Hz, 2H), 3.92
(
t, J = 9.0 Hz, 2H), 3.69 (s, 6H), 3.50 (t, J = 9.0 Hz, 2H), 3.46 (dd, J = 9.3, 3.1 Hz, 2H), 3.01 (td, J = 10.8,
4
6
.2 Hz, 2H), 2.40–2.16 (m, 12H), 1.98–1.83 (m, 4H), 1.71 (s, 6H), 1.68–1.56 (m, 16H), 1.28 (s, 6H), 0.98 (s,
H), 0.93 (s, 6H), 0.86 (s, 6H), 0.85 (s, 12H), 0.16 (m, 54H).
13
C-NMR (CDCl , 100 MHz):
δ
(ppm) = 176.66 (2C), 173.68 (2C), 173.60 (2C), 150.55 (2C), 109.62
3
(
2C), 94.44 (2C), 80.61 (2C), 73.49 (2C), 72.67 (2C), 71.94 (2C), 70.75 (2C), 63.31 (2C), 56.57 (2C), 55.46 (2C),
5
3
2
1.23 (2C), 50.47 (2C), 49.49 (2C), 47.01 (2C), 42.40 (2C), 40.71 (2C), 38.40 (2C), 38.27 (2C), 37.84 (2C),
7.13 (2C), 36.97 (2C), 34.81 (2C), 34.28 (2C), 34.11 (2C), 32.18 (2C), 30.61 (2C), 29.68 (2C), 29.14 (2C),
9.11 (2C), 29.09 (2C), 27.97 (2C), 25.49 (2C), 25.12 (2C), 24.75 (2C), 23.75 (2C), 20.91 (2C), 19.35 (2C),
1
8.19 (2C), 16.56 (2C), 16.16 (2C), 15.96 (2C), 14.69 (2C), 14.10 (2C), (1.06, 0.88, 0.18 =18C). HR-ESI-MS:
20
◦
MW 2070.2939 calcd. for C112H198O Si Na, MW 2070.2949 found. Optical rotation, [α] : +41.2 .
21
6
D
(
1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-
icosahydro-1H-cyclopenta[a]chrysen-9-yl 1-[(2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-{[(2R,3R,4S,5S,6R)-3,4,
-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl]methyl decanedioate/3-Be-mono. Acetic acid
5
(
18
µ
L, 0.324 mmol) was added under stirring at RT to a solution of 22-mono (23 mg, 0.0162 mmol) in
◦
MeOH (1 mL), and the reaction mixture was stirred at 40 C for two days. Reaction monitoring (TLC,
eluant: 98:2 CH Cl /MeOH) confirmed the disappearance of starting 22-mono. The solvent was then
2
2
removed under reduced pressure to obtain pure target 3-Be-mono (13 mg, 0.0133 mmol, 82% yield).
1
Analytical characterization. H-NMR (CDCl , 400 MHz):
δ
(ppm) = 4.98 (dd, J = 3.0, 1.9 Hz, 2H),
3
4
.76 (d, J = 2.0 Hz, 1H), 4.65–4.59 (m, 1H), 4.52–4.42 (m, 1H), 4.45–4.37 (m, 1H), 4.11–4.02 (m, 2H), 3.97
(td, J = 8.9, 3.1 Hz, 2H), 3.84 (dt, J = 9.4, 3.1 Hz, 1H), 3.72–3.67 (m, 5H), 3.60–3.43 (m, 4H), 3.35–3.33 (m,
2
0
H), 3.01 (td, J = 10.8, 4.7 Hz, 1H), 2.43–2.17 (m, 6H), 1.98–1.85 (m, 2H), 1.75–1.68 (m, 6H), 1.28 (s, 3H),
.98 (s, 3H), 0.94 (s, 3H), 0.87 (s, 3H), 0.85 (s, 6H). 13C-NMR (CDCl , 100 MHz):
(ppm) = 176.64, 173.80,
δ
3
173.72, 150.50, 109.65, 94.42, 94.15, 80.74, 73.59, 73.51, 73.30, 72.66, 72.62, 72.04, 71.25, 70.68, 62.90, 60.37,
5
3
6.56, 55.44, 51.24, 50.46, 49.49, 47.00, 42.40, 40.71, 38.40, 38.26, 37.85, 37.13, 36.96, 34.82, 34.28, 34.18,
4.12, 34.09, 32.18, 30.62, 29.69, 29.35, 29.17, 28.01, 25.48, 25.14, 24.81, 23.75, 20.92, 19.38, 18.21, 16.61,
1
6.18, 15.96, 14.71. HR-ESI-MS: MW 1001.5813 calcd. for C H O Na, MW 1001.5819 found. Optical
53 86 16
20
◦
rotation, [α] : +70.6 .
D
Attempted synthesis of (1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-pentamethyl-
-(prop-1-en-2-yl)-icosahydro-1H-cyclopenta[a]chrysen-9-yl 1-[(2R,3S,4S,5R,6R)-6-{[(2R,3R,4S,5S,6R)-
1
6
-{[(10-{[(1R,3aS,5aR,5bR,9S,11aR)-3a-(methoxycarbonyl)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-
icosahydro-1H-cyclopenta[a]chrysen-9-yl]oxy}-10-oxodecanoyl)oxy]methyl}-3,4,5-trihydroxyoxan-
-yl]oxy}-3,4,5-trihydroxyoxan-2-yl]methyl decanedioate/4-Be-bis. Acetic acid (13 L, 0.234 mmol)
2
µ
was added under stirring at RT to a solution of 23-bis (24 mg, 0.0117 mmol) in MeOH (1 mL), and
◦
the reaction mixture was stirred at 40 C for four days. Reaction monitoring (TLC, eluant: 98:2
CH Cl /MeOH) showed the formation of a series of uncharacterizable degradation products.
2
2

Pharmaceutics 2019, 11, 422
7 of 17
2
.4. NA Assembly and Characterization
Mono-Sq-NA1. In accordance with standard solvent evaporation protocols [41] the squalene-trehalose
conjugate 1-mono (4.0 mg) was first dissolved in THF (1 mL) in a vial while stirring at RT. The resulting
solution was added dropwise to a round bottom flask containing MilliQ grade distilled water (2 mL)
under magnetic stirring (500 rpm). The resulting suspension was stirred for 5 min, then THF was
thoroughly evaporated under reduced pressure, obtaining pure mono-Sq-NA1 as an opalescent
suspension (2 mL, 2 mg/mL).
Bis-Sq-NA2. In accordance with standard solvent evaporation protocols [41] the squalene-trehalose
conjugate 2-bis (4.0 mg) was first dissolved in THF (1 mL) in a vial while stirring at RT. The resulting
solution was added dropwise to a round bottom flask containing MilliQ grade distilled water (2 mL)
under magnetic stirring (500 rpm). The resulting suspension was stirred for 5 min, then THF was
thoroughly evaporated under reduced pressure, obtaining pure bis-Sq-NA2 as opalescent suspension
(2 mL, 2 mg/mL).
Mono-Be-NA3. In accordance with standard solvent evaporation protocols [41] the betulinic
acid-trehalose conjugate 3-mono (4.0 mg) was first dissolved in THF (1 mL) in a vial while stirring
at RT. The resulting solution was added dropwise to a round bottom flask containing MilliQ grade
distilled water (2 mL) under magnetic stirring (500 rpm). The resulting suspension was stirred for
5
min, then THF was thoroughly evaporated under reduced pressure, obtaining pure mono-Be-NA3
as opalescent suspension (2 mL, 2 mg/mL).
NA Characterization. NAs were characterized by dynamic light scattering (DLS), using a 90 Plus
Particle Size Analyzer from Brookhaven Instrument Corporation (Holtsville, NY, USA) operating at
1
5 mW of a solid-state laser (
λ
= 661 nm), using a 90-degree scattering angle. The
ζ
-potential was
◦
determined at 25 C using a 90 Plus Particle Size Analyzer from Brookhaven Instrument Corporation
(Holtsville, NY, USA) equipped with an AQ-809 electrode, operating at an applied voltage of 120 V.
Each sample was diluted to a concentration of 0.2 mg/mL and sonicated for 3 min before each
experiment. Ten independent measurements of 60 s duration were performed for each sample.
Hydrodynamic diameters were calculated using Mie theory, considering the absolute viscosity and
refractive index values of the medium to be 0.890 cP and 1.33, respectively. The same aqueous samples
at a concentration of 0.2 mg/mL were used for
ionic strength (no addition of KCl). The -potential was calculated from the electrophoretic mobility of
nanoparticles, by using the Smoluchowski theory [42].
ζ-potential measurement, without any change for the
ζ
2
.5. Biology
Cell cultures. HeLa cells (ATCC: CCL-2) were cultured in DMEM with 10% FBS, 1% penicillin/
◦
streptomicin and 1% glutamine in a humidified atmosphere of 5% CO at 37 C (all reagents from
Euroclone). Cultures were treated with lipid-trehalose conjugates or NAs for 2 or 48 h at 37 C at the
2
◦
concentration indicated in the text.
Cytotoxicity assay. We performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay to measure culture vitality. HeLa cells were cultured in a 96-well plate at
3
2
◦
a concentration of 5
×
10 cell/cm and incubated at 37 C for 24 h. MTT was added in cell medium
◦
at a final concentration of 0.25 mg/mL. Incubation lasted 30 min at 37 C. Then, the medium was
removed and formazan precipitates were collected in 200 µL of DMSO. The absorbance measured
at 570 nm using a spectrophotometer reflects cell viability. Cell viability was expressed as fold over
control condition set at 100%.
Autophagy assay. We assessed autophagy by monitoring LC3 conversion by western-blotting as
previously described [43]. Briefly, upon a wash in PBS, cells were solubilized in RIPA buffer (150 mM
NaCl, 50 mM HEPES, 0.5% NP40, 1% sodium-deoxycholate). After 1 h under mild agitation, the lysate
was clarified by centrifugation for 20 min at 16,000 g. All experimental procedures were performed at
◦
4
C. Protein concentrations were evaluated via Bradford assay (Bio-Rad, Segrate, Italy). For Western
blotting experiments, an equal amount of proteins was diluted with 0.25% 5X Laemmli buffer, separated

Pharmaceutics 2019, 11, 422
8 of 17
onto 10% SDS-PAGE gels and transferred onto nitrocellulose membrane (Sigma-Aldrich Italy, Milan,
◦
Italy) at 80 V for 120 min at 4 C.
Primary antibodies (source in parentheses) included: Mouse anti-LC3, 1:500 (Enzo Life Sciences
AG, Lausen, Switzerland), and mouse anti β-actin 1:1000 (Sigma Aldrich Italy, Milan, Italy) which were
applied overnight in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat
dry milk). Proteins were detected using the ECL prime detection system (GE Healthcare). Images were
acquired with the imaging ChemiDoc Touch system (Bio Rad Laboratory Italy, Segrate, Italy), and the
optical density of the specific bands was measured with ImageLab software (Bio Rad).
2
.6. Statistical Analysis
All data are reported as mean
±
standard error of the mean (SEM). The entire data-set was logged
into GraphPad Prism and analyzed via unpaired Student’s T-test (two classes) or ANOVA followed by
Tukey’s posthoc test (more than two classes). Number of experiments (n) and level of significance (p)
are indicated throughout the text.
3
. Results
3
.1. Synthesis of Target 1-mono and 2-bis Squalene-Trehalose Conjugates
In order to obtain either a mono-(target compound 1-Sq-mono, 1:1 squalene-trehalose conjugate)
and a bis-squalenylated trehalose construct (target compound 2-Sq-bis, 2:1 squalene-trehalose
conjugate), we focused our attention onto the hexaTMS-protected trehalose derivative 5 [38] and the
carboxylated squalene-linker adduct 6 [31] (Figure 2).
Figure 2. Chemical structure of target squalene-trehalose conjugates 1-Sq-mono and 2-Sq-bis, and of
key synthetic intermediate hexaTMS-protected trehalose and carboxylated squalene-linker adduct
5
6.
The synthesis of key intermediates 5 [38] and 6 [31] is reported respectively in Schemes 1 and 2.
Scheme 1. Synthesis of hexaTMS-protected trehalose 5.

Pharmaceutics 2019, 11, 422
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Scheme 2. Synthesis of carboxylated squalene-linker adduct 6.
HexaTMS-protected trehalose
5
was obtained through per-silylation of commercially-available
trehalose with TMS-Cl (per-silylated 8, step a), followed by a selective deprotection of primary, more
7
easily accessible, hydroxyls (step b, Scheme 1).
Commercial squalene 9 was sequentially submitted to halohydration (bromohydrine 10, step a),
base-promoted elimination (epoxide 11, step b), oxidative cleavage (aldehyde 12), reduction (alcohol 13)
and mono-esterification with diacid 14, to provide target carboxylated squalene adduct 6 (step e,
Scheme 2).
Finally, key intermediates
5 and 6 were coupled in equimolar amounts in an esterification protocol,
obtaining a ≈ 3:1 mixture of 15-mono and 16-bis hexaTMS-protected compounds (step a, Scheme 3).
Scheme 3. Synthesis of target 1-Sq-mono and 2-Sq-bis squalene-trehalose conjugates.
After chromatographic separation, both hexaTMS protected compounds 15-mono and 16-bis were
submitted to acidic deprotection, yielding respectively 1-Sq-mono and 2-Sq-bis targets, respectively
in 45% and 13% overall yields from 5 and 6 (step b, Scheme 3).
3
.2. Synthesis of Target 3-Be-mono Betulinic Acid-Trehalose Conjugate, Attempted Synthesis of Target 4-Be
bis Betulinic Acid-Trehalose Conjugate
In order to obtain the target 3-Be-mono betulinic acid-trehalose conjugate we adopted a similar
strategy, focusing our attention onto the same hexaTMS-protected trehalose derivative 5 [38] and the
carboxylated betulinic-linker adduct 21 (Figure 3).

Pharmaceutics 2019, 11, 422
10 of 17
Figure 3. Chemical structure of target betulinic-trehalose conjugates 3-Be-mono and 4-Be-bis, of key
synthetic intermediate hexaTMS-protected trehalose 5 and carboxylated betulinic-linker adduct 21.
The synthesis of key intermediate 21 is reported in Scheme 4.
Scheme 4. Synthesis of carboxylated betulinic-linker adduct 21.
Betulinic acid 17 was first esterified (methyl ester 18, step a), then coupled with mono-protected
diacid 19 (prepared by a controlled esterification of sebacic acid 14, step b) to provide silyl-protected
construct 20 (step c). Carboxylic acid deprotection finally provided target carboxylated betulinic-linker
adduct 21 (step d, Scheme 4).
Finally, key intermediates
5
and 21 were coupled in equimolar amounts in an esterification
protocol, obtaining a
Scheme 5).
≈
1.5:1 mixture of 22-mono and 23-bis hexaTMS-protected compounds (step a,
After chromatographic separation, hexaTMS protected compound 22-mono was submitted to
acidic deprotection, yielding target 3-Be-mono betulinic acid–trehalose conjugate, in 21% overall
yield from
5 and 21 (step b, Scheme 5). The same reaction, targeting 4-Be-bis from 23-bis, leads to
uncharacterizable degradation products.

Pharmaceutics 2019, 11, 422
11 of 17
Scheme 5. Synthesis of target 3-Be-mono betulinic-trehalose conjugate.
.3. NA Assembly and Structural Characterization
Both 1-Sq-mono and 2-Sq-bis squalene-trehalose conjugates, and 3-Be-mono betulinic acid-trehalose
3
conjugate were assembled into their corresponding NAs (mono-Sq-NA1, left, bis-Sq-NA2, middle,
and mono-Be-NA3, right, Scheme 6) following a standard experimental protocol [38].
Scheme 6. Assembly and graphic representation of mono-Sq-NA1, bis-Sq-NA2 and mono-Be-NA3.
Self-assembled mono-Sq-NA1, bis-Sq-NA2 and mono-Be-NA3 were characterized in terms of
hydrodynamic diameter and ζ-potential, as shown in Table 1.
Table 1. Mono-Sq-NA1, mono-Be-NA3 and bis-Sq-NA2: characterization.
Nanovector/Test
mono-Sq-NA1
bis-Sq-NA2
Hydrodynamic Diameter (HD, nm)
ζ-Potential (mV)
−25.12 ± 0.79
−25.43 ± 0.69
−23.53± 0.29
Polydispersity Index
0.121 ± 0.019
90.4 ± 0.7
132.8 ± 0.9
463 ±29
0.072 ± 0.010
mono-Be-NA3
0.126 ± 0.010
The size of the hydrodynamic diameters shows an increase from mono-Sq-NA1 to bis-Sq-NA2
,
while the self-assembly of mono-Be-NA3 results in much larger NAs with a mean HD centered at

Pharmaceutics 2019, 11, 422
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about 460 nm. However, the polydispersity index confirms the mono-dispersion of the colloidal
solution of each NA. Moreover, the self-assembled NAs show good colloidal stability as confirmed by
their
ζ
-potential value (< 20.0 mV), and by the stability of their hydrodynamic diameter (HD) which
−
is not affected even after 10 days’ storage in aqueous solution.
Furthermore, the TEM images and UV spectra of mono-Sq-NA1 (respectively Figures S1 and S2),
of bis-Sq-NA2 (Figures S3 and S4) and of mono-Be-NA3 (Figures S5 and S6) are provided in the
Supplementary Materials.
3
.4. Biological Profiling
Finally, the set of three NAs was submitted to biological profiling in HeLa cells for cytotoxicity
(
safety determination) and autophagy induction (activity determination). Namely, we treated HeLa
◦
cultures for 2 and 48 h at 37 C with either the three NAs (either estimated, adjusted 20
µ
M concentrations
M concentrations for 48 h), their non-assembled
squalene-trehalose precursors 1-Sq-mono and 2-Sq-bis (either 20 M in DMSO for 2 h, or 40 M for
M in EtOH for 2 h, or 40 M for
8 h), or each individual component (100 mM trehalose in water, either 20 M squalene in DMSO and
M betulinic acid in EtOH for 2 h, or 40 M with both for 48 h), and relative vehicle (DMSO or
of trehalose in water for 2 h, or estimated, adjusted 40
µ
µ
µ
4
4
2
8 h) and betulinic acid-trehalose precursor 3-Be-mono (either 20
µ
µ
µ
0
µ
µ
EtOH). Assays were carried out at 48 h to ensure the release of free trehalose from NAs.
At first, we determined the in vitro safety profile of each sample via the MTT cytotoxicity assay
(Figure 4).
Figure 4. Cytotoxicity assay on betulinic acid, squalene, 1-Sq-mono, 2-Sq-bis
mono-Sq-NA1 bis-Sq-NA2 and mono-Be-NA3. MTT assay, 2 h/20 M (left) and 48 h/40
trehalose = 100 mM.
,
3-Be-mono
,
,
µ
µM (right),
The tested samples do not elicit an overt toxicity upon 2 hours of treatment. Instead, while the set
of three NAs confirmed lack of cytotoxicity at 48 h, both the 2-Sq-bis construct ( 65% viable cells at
8 h) and free betulinic acid ( 25% viable cells at 48 h) show significant cytotoxicity (p < 0.001 versus
not treated, n = 4).
≈
4
≈
Next, we assessed if NAs, non-assembled precursors and individual components, could induce
autophagy by western-blotting. In accordance with cytotox results, we tested NAs at both timelines
(
2 h/Figure 5, and 48 h/Figure 6), while non-assembled precursors and individual components were
tested only at 2 h. Autophagy can be monitored by tracking the mobility shift from LC3I to LC3II
Figures 5 and 6, right), that is a bona fide reporter of the induction of autophagy; and by the amount
(
of LC3II, that correlates with the formation of autophagosomes (Figures 5 and 6, left).
used as an internal control in the assays.
α-Tubulin was

Pharmaceutics 2019, 11, 422
13 of 17
2h – 20 M
LC3B I
LC3B II
-tubulin
-
19 kDa
-17 kDa

-52 kDa
Figure 5. Autophagy induction on betulinic acid, squalene, 1-Sq-mono, 2-Sq-bis
mono-Sq-NA1 bis-Sq-NA2 and mono-Be-NA3. LC3BII amount (left), LC3BII/LC3BI ratio (right),
h/20 µM, trehalose = 100 mM).
, 3-Be-mono,
,
2
48h – 40 M
48h
LC3B I
-19 kDa
17 kDa
-
LC3B II

-tubulin
-52 kDa
Figure 6. Autophagy induction on mono-Sq-NA1, bis-Sq-NA2 and mono-Be-NA3. LC3BII amount
(
left), LC3BII/LC3BI ratio (right), 48 h/40 µM, trehalose = 100 mM).
At 2 h, we only observed a moderate effect by the 2-Sq-bis construct that did not reach statistical
significance (Figure 5, left and right). At 48 h, surprisingly, the three NAs did not show any effect on
autophagy induction/progress (Figure 6).

Pharmaceutics 2019, 11, 422
14 of 17
We investigated the fate of our NAs in the biological medium, to rationalize their lack of biological
effects. Thus, HeLa cell lysates were treated following a published procedure [44], obtaining two
protein-free aqueous (
analysis could neither detect trehalose as such at the expected
lipid-trehalose intermediates (see Figures S7–S10, mono-Sq-NA1; and Figures S11–S14 mono-Be-NA3
≈
4:3 MeOH:water) and organic (
≈
3:1 MeOH:chloroform) layers. Their LC-MS
µM concentration, nor the most likely
,
Supplementary Materials). We could not rule out the presence of trehalose at lower, nM concentrations
that would not elicit an autophagy-inducing effect in cells, due to its detection LC-MS limits; and
we suggest that highly lipophilic lipid-trehalose intermediates 1-Sq-mono and 3-Be-mono remain
trapped within the protein pellet.
4
. Discussion
Accumulating evidence indicates that induction of autophagy can be clinically relevant in the
context of neurodegenerative disorders characterized by protein aggregation [39]. Pre-clinically,
trehalose alleviates protein aggregation and cellular toxicity in pathological deposition of
amyloid/Alzheimer [22] and alpha synuclein/Parkinson [20]. It may act by binding to extra-cellular
GLUT transporters and by inducing AMPK-dependent autophagy [19], and/or by cytosolic activation
of the TFEP pathway [45]. Trehalose is highly hydrophilic, preventing its passive cell permeation [46].
Moreover, trehalases hydrolyze it to glucose at the GI barrier [23]. Thus, trehalose PK in humans is
challenging. By conjugating trehalose with squalene and betulinic acid, and self-assembling three
constructs into NAs, we generated entities with a putatively higher permeability profile, hopefully
leading to higher effects on autophagy at lower dosages.
The 2:1 2-Sq-bis construct showed per se an indication of higher potency than trehalose on
autophagy induction at much lower,
≈
40 µM, concentrations. We hypothesize that its higher
lipophilicity, compared to its 1:1 1-Sq-mono and 3-Be-mono counterparts, may yield better cell
permeability after 2 hours of incubation, with a significant effect on autophagy induction.
The three NAs did not show any cytotoxicity, supporting their testing in the autophagy induction
assay. The three mono-Sq-NA1
autophagy induction; their inactivity could be justified by the absence of
,
bis-Sq-NA2 and mono-Be-NA3 NAs did not show any effect upon
M-free trehalose in cell
µ
lysates (LC-MS determination), possibly due to limited degradation of NAs in 48 h.
Our next efforts will include the design and execution of modified assays to measure autophagy
induction at longer times and/or more NA degradation-prone conditions, and the synthesis of modified
self-assembly trehalose-nanolipid conjugates to fine-tune their properties.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/11/8/422/s1,
Figure S1: TEM images of mono-Sq-NA1, Figure S2: UV-vis spectrum of mono-Sq-NA1, Figure S3: TEM images
of bis-Sq-NA2, Figure S4: UV-vis spectrum of bis-Sq-NA2, Figure S5: TEM images of mono-Be-NA3, Figure S6:
UV-vis spectrum of mono-Be-NA3, Figure S7: HPLC (220 nm, top) and TIC spectrum (ESI+, bottom) of the
aqueous methanolic phase from the treatment of HeLa cells with mono-Sq-NA1
compound searching for 2-Sq-bis (MW = 1448, first lane), 1-Sq-mono (MW=895, second lane), squalene alcohol
MW = 386, third lane) and trehalose (MW = 342 Da, fourth lane) in the aqueous methanolic phase from the
treatment of HeLa cells with mono-Sq-NA1 EC-37, Figure S9: HPLC (220 nm, top) and TIC spectrum (ESI+,
bottom) of the lipophilic methanol-chloroform phase from the treatment of HeLa cells with mono-Sq-NA1 EC-37
/EC-37, Figure S8: MS analysis /
(
/
/
,
Figure S10: MS analysis / compound searching for 2-Sq-bis (MW = 1448, first lane), 1-Sq-mono (MW = 895,
second lane), squalene alcohol (MW = 386, third lane) and trehalose (MW = 342 Da, fourth lane) in the lipophilic
methanol-chloroform phase from the treatment of HeLa cells with mono-Sq-NA1
/
EC-37, Figure S11: HPLC
(
220 nm, top) and TIC spectrum (ESI+, bottom) of the aqueous methanolic phase from the treatment of HeLa cells
with mono–Be-NA3
/
MIC-17, Figure S12: MS analysis / compound searching for 3-Be-mono (MW = 979, first lane),
betulinic acid (MW = 456, second lane), betulinic acid methyl ester (MW = 470, third lane) and trehalose (MW = 342
Da, fourth lane) in the aqueous methanolic phase from the treatment of HeLa cells with mono-Be-NA3
Figure S13: HPLC (220 nm, top) and TIC spectrum (ESI+, bottom) of the lipophilic methanol-chloroform phase
from the treatment of HeLa cells with mono-Be-NA3 MIC-17, Figure S14: MS analysis / compound searching for
-Be-mono (MW = 979, first lane), betulinic acid (MW = 456, second lane), betulinic acid methyl ester (MW = 470,
/MIC-17,
/
3
third lane) and trehalose (MW = 342 Da, fourth lane) in the lipophilic methanol-chloroform phase from the
treatment of HeLa cells with mono-Be-NA3/MIC-17.

Pharmaceutics 2019, 11, 422
15 of 17
Author Contributions: Conceptualization, P.S., G.P. and D.P.; chemistry, M.B. and E.C.; structural characterization,
L.P.; biology, G.F.; bioanalytical studies, P.R.; writing—original draft preparation, E.C. and P.S.; HPLC-MS analysis,
P.R.; structure elucidation, M.S.C.; writing—review and editing, P.S., G.P. and D.P.
Funding: This research was funded by Fondazione Telethon (grant number TDPG00514TA) and MIUR
(PRIN-2017ENN4FY) for G.P.
Acknowledgments: D.P. expresses his gratitude to MAECI Italia-India Strategic Projects 2017–2019.
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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