Design and characterization of Squalene-Gusperimus nanoparticles for modulation of innate immunity

Article abstract of DOI:10.1016/j.ijpharm.2020.119893

Immunosuppressive drugs are widely used for the treatment of autoimmune diseases and to prevent rejection in organ transplantation. Gusperimus is a relatively safe immunosuppressive drug with low cytotoxicity and reversible side effects. It is highly hydrophilic and unstable. Therefore, it requires administration in high doses which increases its side effects. To overcome this, here we encapsulated gusperimus as squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles (NPs) were obtained from nanoassembly of the squalene gusperimus (Sq-Gus) bioconjugate in water, which was synthesized starting from squalene. The size, charge, and dispersity of the Sq-GusNPs were optimized using the response surface methodology (RSM). The colloidal stability of the Sq-GusNPs was tested using an experimental block design at different storage temperatures after preparing them at different pH conditions. Sq-GusNPs showed to be colloidally stable, non-cytotoxic, readily taken up by cells, and with an anti-inflammatory effect sustained over time. We demonstrate that gusperimus was stabilized through its conjugation with squalene and subsequent formation of NPs allowing its controlled release. Overall, the Sq-GusNPs have the potential to be used as an alternative in approaches for the treatment of different pathologies where a controlled release of gusperimus could be required.

Full text of DOI:10.1016/j.ijpharm.2020.119893

International Journal of Pharmaceutics 590 (2020) 119893
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Design and characterization of Squalene-Gusperimus nanoparticles for
modulation of innate immunity
,b,
Carlos E. Navarro Chicaa *, Bart J. de Haanb, M.M. Faasb, Alexandra M. Sminkb, Ligia Sierraa,
b
a
´
Paul de Vos , Betty L. Lopez
a
´
Grupo de Investigacion Ciencia de los Materiales, Instituto de Química, Facultad de Ciencias Exactas y Naturales, Universidad de Antioquia, Calle 70 No. 52-21,
Medellín, Antioquia, Colombia
b Department of Pathology and Medical Biology, Section of Immunoendocrinology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, EA11,
9713 GZ Groningen, The Netherlands
A R T I C L E I N F O
A B S T R A C T
Keywords:
Immunosuppressive drugs are widely used for the treatment of autoimmune diseases and to prevent rejection in
organ transplantation. Gusperimus is a relatively safe immunosuppressive drug with low cytotoxicity and
reversible side effects. It is highly hydrophilic and unstable. Therefore, it requires administration in high doses
which increases its side effects. To overcome this, here we encapsulated gusperimus as squalene-gusperimus
nanoparticles (Sq-GusNPs). These nanoparticles (NPs) were obtained from nanoassembly of the squalene gus-
perimus (Sq-Gus) bioconjugate in water, which was synthesized starting from squalene. The size, charge, and
dispersity of the Sq-GusNPs were optimized using the response surface methodology (RSM). The colloidal sta-
bility of the Sq-GusNPs was tested using an experimental block design at different storage temperatures after
preparing them at different pH conditions. Sq-GusNPs showed to be colloidally stable, non-cytotoxic, readily
taken up by cells, and with an anti-inflammatory effect sustained over time. We demonstrate that gusperimus
was stabilized through its conjugation with squalene and subsequent formation of NPs allowing its controlled
release. Overall, the Sq-GusNPs have the potential to be used as an alternative in approaches for the treatment of
different pathologies where a controlled release of gusperimus could be required.
Squalene-Gusperimus nanoparticles
In vitro evaluation
(RSM) response surface methodology
Block design
Immunosuppressant
1. Introduction
survival of grafts in both experimental animal and clinical studies in
allogenic and xenogeneic settings. In neovascularized graft transplants
Gusperimus is a drug derived from spergualin with anti-tumoral and
immunosuppressive properties (Takeuchi et al., 1981). Gusperimus ex-
erts its immunosuppressive effect through different mechanisms. Gus-
perimus binds to heat shock proteins and avoid translocation of the
nuclear factor-kB (NF-kB) to the nucleus (B.Kaufman et al., 1996) also it
inhibits key molecules such as Akt kinase (Kawada et al., 2002) and
deoxyhypusine synthase (Nishimura et al., 2002). During the immune
response, gusperimus attenuates different cytokines such as IFNγ, IL-6,
such as skin, thyroid, pancreatic islets, as well as in transplantation of
immediately vascularized organs such as kidneys, liver, pancreas, and
heart gusperimus has shown to prolong graft survival and prevent
rejection (Perenyei et al., 2014).
Gusperimus is a highly hydrophilic drug according to the United
States Pharmacopeia (USP) as it has a solubility higher than 100 mg/mL
in water (“Gusperimus | C17H37N7O3 - PubChem,” n.d.). Hydrophilic
drugs are often subject to low intracellular absorption, enzymatic
degradation, rapid elimination, distribution below optimal levels,
resistance development, poor pharmacokinetics, and low therapeutic
index (Arpicco et al., 2016). Gusperimus is in addition to this chemically
unstable (Fujii et al., 1989) and as a consequence is characterized as a
drug with low oral bioavailability. Therefore, it needs to be adminis-
tered intravenously or subcutaneously to avoid too much loss of
TNFα, and IL-10 (Perenyei et al., 2014). Its application has been stud-
ied for the treatment of different autoimmune diseases and it was shown
to be effective during prophylactic and treatment of vasculitis, glomer-
ulonephritis, and systemic lupus erythematosus (Perenyei et al., 2014).
Because of its efficacy in suppressing autoimmunity, it has also been
tested in organ transplantation. It has been shown to support the
* Corresponding author at: Pathology and Medical Biology, Section of Immunoendocrinology, University of Groningen, University Medical Center Groningen,
Hanzeplein 1, EA11, 9713 GZ Groningen, the Netherlands.
E-mail address: c.e.navarro.chica@umcg.nl (C.E. Navarro Chica).
https://doi.org/10.1016/j.ijpharm.2020.119893
Received 25 May 2020; Received in revised form 12 July 2020; Accepted 14 September 2020
Available online 19 September 2020
0378-5173/© 2020 Elsevier B.V. All rights reserved.

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
bioactivity (Perenyei et al., 2014). The entrapment of hydrophilic drugs
in colloidal delivery systems can in many cases overcome issues asso-
ciated with stability or off-target toxicity and facilitate their use (Arpicco
2.2. Synthesis of Sq-Gus bioconjugate
Sq-Gus bioconjugate synthesis started from squalene according to the
methodology proposed by Ceruti et al. (Ceruti et al., 1987) until
obtaining the aldehyde derived from squalene 1,1′,2-tris-nor-squalene
aldehyde. Briefly, a 0.4 M solution of squalene reagent in THF was
prepared and deionized water was added drop by drop under stirring
until the solution became opalescent, after which THF was added until
the solution turned clear again. To this solution, NBS in a molar ratio 1:1
respect to squalene was added and after stirring at room temperature for
1-hour, deionized water was added to form two phases. The organic
phase was extracted with PE, washed with brine, dried with Na2SO4, and
subjected to rotary evaporation. From this, squalene monobromohydrin
(Sq-MBH) as a pale-yellow oil was recovered by flash chromatography
using as eluent PE (100%) and DE (100%). The Sq-MBH was added to a
0.3 M methanolic solution of K2CO3 in a molar ratio 1:1.20 (Sq-MBH:
K2CO3), under stirring, and left for two hours at room temperature.
Afterward, the reaction mixture was diluted with water, and the organic
phase extracted with PE, washed with brine, dried with Na2SO4 and the
PE evaporated under reduced pressure to obtain squalene epoxide (Sq-
Ep). The Sq-Ep was added to a 0.5 M solution of H5IO6 in THF in a molar
ratio of 1:5 (Sq-Ep:H5IO6) under stirring and left reacting for 15 min
after which deionized water was added. The organic phase was extrac-
ted with PE, washed with brine, dried with Na2SO4 and the PE evapo-
rated under reduced pressure. The concentrated extract was subjected to
flash chromatography using PE/DE (95/5) as eluent, to obtain 1,1′,2-
tris-nor-squalene aldehyde (Sq-Ald).
¨
et al., 2016; Desmaele et al., 2012). Encapsulation in colloidal systems
can improve pharmacokinetics, protect the active substance against
degradation in vivo, maintain drug release over time, reduce side effects,
and, most importantly, increase patient comfort and applicability. There
are several systems available for entrapment of hydrophilic drugs
including liposomes, lipid NPs, polymer NPs, mesoporous silica NPs,
nanobubbles, and squalene-derived NPs (Arpicco et al., 2016). The
squalene-derived NPs are produced after binding squalene covalently to
the active principle obtaining a conjugate or prodrug that spontaneously
self-assemble as NPs in an aqueous medium (Couvreur et al., 2006). This
has several advantages over the other systems, such as the potential for
¨
high drug loading (Desmaele et al., 2012). Also, the covalent binding
enhances half-life time and therewith the drug stability, which avoids
“burst” release and allows fine-tuned controlled release. Finally, the
lipophilic properties of squalene can enhance the affinity of the bio-
conjugate to cell membranes resulting in a higher drug concentration
within cells, enhancing the pharmacological activity of the active prin-
ciple (Feng et al., 2017).
In this study, we designed and characterized Sq-GusNPs by binding
squalene covalently to gusperimus, to be used as a gusperimus
controlled release system that maintains its immunosuppressive prop-
erties over time. Sq-GusNPs were designed by applying a bottom-up
approach (Zhao et al., 2011) in which NPs are obtained starting from
the molecular units. From squalene and through different synthesis steps
the Sq-Gus bioconjugate was obtained. The characterization of the
different synthesis products was performed through the analysis of
Fourier transformed infrared spectroscopy (FTIR) and nuclear magnetic
resonance spectroscopy (NMR) data. The use of the nanoprecipitation
method and a response surface methodology (RSM) design allowed
obtaining the Sq-GusNPs. The Sq-GusNPs were characterized by dy-
namic light scattering (DLS), transmission electron microscopy (TEM),
and Z potential (ζ) measurements. The colloidal stability of the Sq-
GusNPs over time was tested using a block design at three different
pH values. Finally, the in vitro activity of Sq-GusNPs was studied using
the U-937 cell line through the determination of its uptake, cytotoxicity,
and anti-inflammatory capacity.
Sq-Ald was subsequently transformed to 1,1′,2-tris-nor-squalenic
acid (Sq-COOH) through an oxidation reaction following the method-
ology proposed by Sen and Prestwich (Sen and Prestwich, 1989). From
the carboxylic acid derivate, the squalenoyl N-hydroxysuccinimidyl
ester (Sq-COO-NHS) was obtained through reaction with NHS. The Sq-
Gus bioconjugate was obtained from a reaction of Sq-COO-NHS with
gusperimus (COUVREUR et al., 2009). To prepare the carboxylic acid
derivative, a 0.076 M solution of Sq-Ald at 0 ◦C reacted with silver oxide
(Ag2O), which was generated in situ by addition of an aqueous solution
0.3 M of AgNO3 in a molar ratio 1:1 (Sq-Ald:AgNO3) and the same
volume of 1.4 M NaOH solution. The reaction mixture was left under
stirring for 8 h at room temperature, after which it was filtered, the pH
adjusted to 1 with HCl and then the acid derivative was extracted with
DE. The DE concentrated extract (after DE evaporation under reduced
pressure), was subjected to flash chromatography using as eluent EA/
Hex (20/80) obtaining the Sq-COOH. To prepare the Sq-COO-NHS, a
0.06 M solution of Sq-COOH in DCM reacted with NHS and DCC, both in
a molar ratio of 2:1 respect to Sq-COOH. The reaction was left under
stirring for two hours. Afterward, the mixture was filtered, diluted with
EA, stirred for 30 min and the concentrated extract containing the Sq-
COO-NHS was obtained by evaporating under reduced pressure.
Finally, 2 mL of a gusperimus solution 0.01 M in dry DMF was added to
2 mL of Sq-COO-NHS solution in dry DMF in a molar ratio of 6:1 (Sq-
COO-NHS:Gus), followed by addition of 20 µL of TEA. The reaction was
left under stirring for 2 h. The reaction mixture was filtered and the Sq-
Gus bioconjugate extracted with 20 mL of DCM, after which the aqueous
phase was saturated with salt and the extraction step repeated. The
extract was concentrated by reduced pressure and after flash chroma-
tography, using as eluent DCM/EtOH 90/10 and EtOH/TEA 95/5, the
Sq-Gus bioconjugate, as a solid, was obtained.
2. Materials and methods
2.1. Materials
Squalene 98%, tetrahydrofuran (THF), N-bromosuccinimide (NBS),
diethyl ether (DE), sodium chloride (NaCl), sodium sulfate (Na2SO4),
periodic acid (H5IO6), silver nitrate (AgNO3), sodium hydroxide
(NaOH), hydrochloric acid 37% (HCl), ethyl acetate (EA), dime-
thylformamide (DMF), Silica gel 60 (0.040–0.063 mm) for column
chromatography (230–400 mesh ASTM), and absolute ethanol (EtOH)
were purchased from Merck (Lyon, France). Petroleum ether (PE),
methanol (MeOH), and hexane (Hex) were purchased from Avantor
(Radnor, PA, USA). Potassium carbonate (K2CO3), N-hydrox-
ysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), Nile Red,
3,3′-dihexyloxacarbocyanine iodide (DiOC6), propidium iodide (PI),
phorbol 12-myristate 13-acetate (PMA), and lipopolysaccharides (LPS)
from Escherichia coli O111:B4 purified by phenol extraction was pur-
chased from Sigma-Aldrich (St. Louis, MO, USA). Gusperimus was ob-
tained from Nordic Pharma SAS (Paris, France). Dichloromethane
(DCM), and triethylamine (TEA) were purchased from Panreac (Darm-
stadt, Germany). The alamarBlue™ reagent was purchased from Life
Technologies Europe BV (Bleiswijk, The Netherlands), and ELISA Duo-
2.2.1. Fourier-Transform Infrared spectroscopy (FTIR)
Characterization for all synthesis products was carried out through
FTIR using a Spectrum One FT-IR Spectrometer (PerkinElmer, Waltham,
MA, USA), dispersing the samples in KBr cells with 32 scans, a resolution
Set for human IL-10 and TNF
(Abingdon, United Kingdom).
α
were purchased from R&D systems
of 4 cmꢀ 1, and a wavelength range of 4000 cmꢀ 1 to 450 cmꢀ 1
.
2.2.2. Nuclear magnetic resonance (NMR)
NMR spectra of 1H and 13C were obtained using a Bruker Ascend III
2

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
HD 600 MHz spectrometer with 5 mm cryoprobe - TCl and a Bruker
Avance NEO 600 MHz spectrometer with 5 mm CryoProbe Prodigy BBO
(Bruker, USA). NMR analyses were only conducted on reaction products
that were obtained through a prior purification by flash chromatog-
raphy. NMR data were analyzed using MestReNova software version 12.
Conditions for NMR analysis are described in detail in the supplemen-
tary information.
2.4. Transmission electron microscopy (TEM)
The morphology of the NPs was examined by TEM using two
different methods for sample preparation. Cryo-TEM and staining were
done using a FEI Tecnai T20 cryo-electron microscope equipped with a
Gatan model 626 cryo-stage operating at 200 keV. For both methods, the
sample was prepared depositing 2 µL of Sq-GusNPs suspension at a
concentration of 0.76 mg/mL in a holey carbon-coated grid (Quantifoil
3.5/1, Quantifoil Micro Tools, Jena, Germany). For Cryo-TEM after
blotting the excess of liquid, the sample was vitrified in liquid ethane
(Vitrobot, FEI, Eindhoven, The Netherlands) and transferred to the TEM
microscope. For staining the excess of liquid was removed with a blot-
ting filter paper, after a drop of 2% uranyl acetate solution was added
and blotted again to remove the excess of the solution, finally, the
sample was transferred to the TEM microscope.
2.3. Nanoparticle preparation
2.3.1. Sq-GusNPs preparation
Sq-GusNPs were prepared through the nanoprecipitation method
(Barreras-Urbina et al., 2016). For this, the pure Sq-Gus bioconjugate,
obtained as previously described in section 2.2, was dissolved in abso-
lute EtOH at a concentration of 2 mg/mL. Later 380 µL of the previous
solution was added drop by drop to 1 mL of deionized water under
stirring (500 rpm) for 10 min after which EtOH was evaporated using a
rotary evaporator obtaining an aqueous suspension of pure NPs. No
additional components e.g. surfactants were used in nanoparticle prep-
aration and the aqueous nanoparticle suspension was used directly for
2.5. Colloidal stability of Sq-GusNPs
Colloidal stability of Sq-GusNPs was evaluated through a block
experimental design in which the time evaluated between 0 and 30 days
in 11 blocks was taken as a blocking factor. The size and ζ continually
were measured during the first five days as of day 0, followed by mea-
surements every 5 days until day 30. The experimental factor for this
design was the pH, which was evaluated at 3 different values 4.5, 7.4,
and 9.0. These pH values were established by adding NaOH or HCl so-
lutions to the water used for obtaining Sq-GusNPs. The experiment was
carried out at two storage temperatures i.e. 4 ◦C and 25 ◦C. It was ran-
domized, and for each temperature, this consisted of two replicas per
block with a total of 66 runs.
◦
further experiments. The prepared NPs suspension was stored at 4 C
and used until 1 month after its preparation.
2.3.2. Nile red labeled Sq-GusNPs preparation
To obtain labeled Sq-GusNPs an ethanolic solution of Nile Red at a
concentration of 0.1 mg/mL was prepared. From this 380 µL was taken
and EtOH was evaporated using a rotary evaporator to obtain dried
crystals of Nile Red. Later 380 µL of Sq-Gus bioconjugate dissolved in
EtOH at a concentration of 2 mg/mL was added to the dried Nile Red
crystals, vortexed, and NPs obtained as described in the previous section
(2.3.1). The aqueous suspension of labeled Sq-GusNPs was filtered
through a 0.22 µm filter to separate the Nile red precipitate after ethanol
evaporation obtaining a translucid suspension of labeled Sq-GusNPs.
The prepared NPs suspension was stored at 4 ◦C and used until 1
month after its preparation.
2.6. In vitro evaluation of the functionality of Sq-GusNPs
2.6.1. Flow cytometry
Flow cytometry was carried out using the Flow cytometer BD Facs-
Verse (BD Biosciences, Breda, The Netherlands) counting 10,000 events
for sample using a 488 nm probe for excitation and a 586/42 nm probe
for emission in the cellular uptake assay for Nile Red, and a 527/32 nm
and 586/42 nm probes for emission were used for DiOC6 and PI
respectively in the cytotoxicity assay. Data analysis was performed using
the software FlowJo version 10.6.1.
2.3.3. Response surface methodology (RSM)
Conditions for nanoparticle preparation were obtained through an
experimental design using the RSM in which three experimental factors
were evaluated: volume of bioconjugate solution dissolved in EtOH (Vol.
bioconjugate, µL), water volume (Vol. water, mL) which is added to the
bioconjugate solution, and stirring speed (stirring speed, rpm) at which
the NPs are obtained (Table 1). Response variables evaluated for the
RSM design were size (nm), ζ (mV), and dispersity (Ð). The size was
measured through DLS using a particle size analyzer HORIBA LB-550
(HORIBA, Kyoto, Japan), ζ was measured using a Zetasizer Nano Se-
ries – Nano Z (Malvern Instruments, Grovewood Road, UK), and Ð was
determined from size measurements according to its definition as the
standard deviation (SD) of the particle diameter distribution divided by
the mean particle diameter to the 2 power (Ð=(SD/Mean)2) (Clayton
et al., 2016). The experimental design created through the RSM was a
central composite design (CCD) in which the lower and upper levels
indicated in Table 1 were used. The experiment was completely ran-
domized, rotatable, and consisted of 17 treatments which included 3
central points.
2.6.2. Cellular uptake
To demonstrate cellular uptake of Sq-GusNPs we applied the human
monocyte U-937 cell line (ATCC® CRL1593.2™) (Cell culture is
described in detail in the supporting information). To this end, Sq-
GusNPs labeled with the fluorescent agent Nile Red (Sq-Gus-R) were
prepared as described in Section 2.3.2.
Sq-Gus-R at three different concentrations (Sq-Gus-R 1x/2x/4x) were
cultured with cells in a density of 2 × 105 cells/well in 24 well plates for
◦
three hours at 37 C and 5% CO2. Used concentrations of Sq-GusNPs
were equivalent to 2.8 μg/mL, 5.6 μg/mL (double concentration), and
11.2 μg/mL (fourfold concentration) of pure gusperimus for Sq-Gus-R
1x, Sq-Gus-R 2x, and Sq-Gus-R 4x respectively. After the three hours
culture period, cells were washed with plain medium and its fluores-
cence was measured by flow cytometry (the gating strategy is described
in Fig. S1 of the supplementary information).
To discard any interference in the obtained response, four controls
were used in these experiments, which were Sq-GusNPs without Nile
Red to the highest used concentration (Sq-Gus 4x), Sq-GusNPs labeled
with Nile Red to the highest concentration (Sq-Gus-R 4x), cells without
NPs (Cs), and cells with Sq-GusNPs at the highest concentration without
Nile Red (Cs + Sq-Gus 4x).
Table 1
Factors and response variables evaluated in the RSM design for Sq-GusNPs
obtention.
Factor
Unit
Level
Response variable
Unit
Lower
Upper
Cellular uptake was also confirmed using the fluorescence micro-
scope EVOS® FL Cell Imaging System (Thermo Fisher Scientific,
Landsmeer, The Netherlands). For this, cells were cultured with the
highest concentration of Sq-GusNPs (Cs + Sq-Gus 4x), Sq-GusNPs
Vol. Bioconjugate
Vol. Water
μ
L
300
2
500
5
Size
ζ
Ð
nm
mV
mL
Stirring speed
rpm
270
800
3

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
labeled with Nile Red (Cs + Sq-Gus-R 4x) and cells without NPs (Cs) for
3 h. Finally, cells were washed with plain medium and observed using
bright light and fluorescence light with the filter EVOS™ LED Cube,
CY®5.
Technologies Inc., USA). The experiments with cells were repeated at
least 3 times and statistical analysis was carried out in GraphPad Prism,
Version 8.2.0 (GraphPad Software Inc., USA). The normal distribution of
data was confirmed using the D’Agostino-Pearson omnibus (K2) test.
Comparisons were made using one-way ANOVA with Dunnet’s post hoc
test or two-way ANOVA with Tukey’s post hoc test. A p-value < 0.05 was
considered statistically significant. Results are expressed as mean ±
standard deviation (SD).
2.6.3. Cytotoxicity
Cytotoxicity of Sq-GusNPs was measured through flow cytometry in
U-937 monocytes using two viability fluorophores: DiOC6 as an indi-
cator of mitochondrial damage and apoptosis (Shapiro, 2000; Wang
et al., 2006) and PI as an indicator of cytoplasmatic membrane damage
(Rosenberg et al., 2019). The use of DiOC6 allows the detection of cells
with mitochondrial damage in the early stage of apoptosis which cannot
be detected with PI (Odaka et al., 1998). Briefly, 2 × 105 cells/well in 24
well plates were treated with three different concentrations of Sq-
GusNPs, free gusperimus (at the same equivalent concentrations used
for gusperimus previously), and free squalene as NPs (SqNPs, see sup-
plementary information for SqNPs preparation). After treatment for
24 and 48 h, the fluorescence was measured through flow cytometry
culturing the cells for 20 min with both fluorophores at a concentration
of 0.06 µg/mL for DiOC6 and 1 µg/mL for PI (the gating strategy is
described in Fig. S2 of the supplementary information).
3. Results
3.1. Preparation and characterization of Sq-Gus bioconjugate
The synthesis of the Sq-Gus bioconjugate involved several reaction
steps with different intermediate products, which were identified by
FTIR and NMR after their purification by flash chromatography. Fig. 1
shows the reaction steps with the respective yields. The reaction starts
with the electrophilic addition of Br in one of the external olefinic bonds
of squalene (1), followed by subsequent incorporation of an OH group
through a nucleophilic water attack (Clayden et al., 2012) forming the
Sq-MBH (2). After obtaining Sq-MBH, bromine is displaced following a
SN2 mechanism in basic medium to produce Sq-Ep (3) (Clayden et al.,
2012). This compound is transformed to Sq-Ald (4) through a reaction in
which a cyclic intermediary with the periodic acid affords the epoxide
cleavage (Nagarkatti and Ashley, 1973). Sq-COOH (5) is obtained as a
result of the Sq-Ald oxidation by in situ formation of Ag2O (Sen and
Prestwich, 1989). From the reaction of Sq-COOH with NHS, using the
coupling reagent DCC, the intermediary product Sq-COO-NHS (6) is
obtained, which provides an active leaving group and reacts with gus-
perimus through its amine primary group for the obtaining of Sq-Gus
bioconjugate (7), (“Conversion of Carboxylic acids to amides using
DCC as an activating agent - Chemistry LibreTexts,” n.d.).
2.6.4. ELISA
A sandwich ELISA (DuoSet ELISA R&D systems) for human IL-10 and
TNF
α were performed according to the manufacturer’s instructions
using the microplate spectrophotometer Benchmark Plus BIO-RAD at
450 nm with correction at 540 nm.
2.6.5. Anti-inflammatory capacity determination assay
The anti-inflammatory capacity of Sq-GusNPs was tested as follows:
Human U-937 monocytes at a density of 5 × 105 cells/well in 24-well
plates were differentiated into macrophages by adding PMA at a con-
centration of 200 nM (Haque et al., 2019). After 24 h, cells were washed
with plain medium and treated with the highest concentration used in
the previous experiments which was equivalent to 11.2 µg/mL of free
gusperimus and 11.1 µg/mL of free squalene supplied as NPs (SqNPs)
(Sq-Gus 4x, Sq 4x, and Gus 4x). This concentration was chosen since it
showed an enhanced effect on cytokine downregulation at 6 h after LPS
stimulation (see Fig. S3 of the supplementary information). After two
3.1.1. FTIR characterization
The presence of characteristic functional groups of the Sq-Gus bio-
conjugate and the intermediary products was confirmed by FTIR and
their chemical structure by NMR. Fig. 2 shows the FTIR spectra of the
different compounds involved in the synthesis of the Sq-Gus bio-
conjugate. For Sq-MBH, it is observed the presence of bands at 3470
cmꢀ 1 and 633 cmꢀ 1, associated with the stretching of the OH and C-Br
bonds respectively (Silverstein et al., 2005). Two bands are observed for
Sq-EP that correspond to the epoxide ring breathing at 1249 and 1124
cmꢀ 1, as well as bands at 899 and 873 cmꢀ 1 corresponding respectively
to asymmetric and symmetric deformations (Ceruti et al., 1992). For Sq-
Ald and Sq-COOH, stretches for the carbonyl groups bands appear at
1729 cmꢀ 1 and 1711 cmꢀ 1 respectively. In the case of Sq-COO-NHS,
three bands are observed at 1744 cmꢀ 1, 1788 cmꢀ 1, and 1818 cmꢀ 1
which are associated with the stretching of three different carbonyl
groups in this molecule. The FTIR spectrum of Sq-Gus bioconjugate
(Fig. 2B) clearly presents the bands associated with its functional groups:
at 3447 cmꢀ 1 the O-H stretching band; between 3050 cmꢀ 1 and 2830
cmꢀ 1 the C-H alkene and alkane stretching bands, respectively; and
between 1600 cmꢀ 1 and 1750 cmꢀ 1 the bands associated with the
presence of the carbonyl bonds of the secondary amide groups of this
molecule (Silverstein et al., 2005). The bands between 2400 cmꢀ 1 and
2800 cmꢀ 1 are associated with the presence of the guanidine group of
gusperimus moiety, which exhibits in this regions stretching vibrations
corresponding to C-N, C=N, and N-H bonds, as it happens in molecules
that contain the guanidine group such as arginine (Ebrahiminezhad
et al., 2012) and the guanidine-derived salt GULAS (Thangaraj et al.,
2017).
hours incubation, LPS (1
μ
g/mL) was added to stimulate inflammatory
responses and IL-10 (an anti-inflammatory cytokine) and TNF
α
(a pro-
inflammatory cytokine) production were quantified in the supernatant
6, 24, 48, 72, and 96 hours later through ELISA. Cells without any
treatment served as negative control while cells treated only with LPS
served as the positive control. To exclude that the observed effect in
cytokine production was due to cell death, viability was measured with
alamarBlue™ for exposition times between 24 and 96 h (see Fig. S4 of
the supplementary information).
2.6.6. Cell viability with alamarBlue™
To determine cell viability with alamarBlue™ the reagent was
diluted in culture medium (10% v/v). After treatment of the cells, at 24,
48, 72, and 96 hours supernatants were collected for cytokine deter-
mination. Cells were washed with plain medium and incubated for four
hours with the diluted reagent. The absorbance was measured at 570 nm
with correction at 600 nm with the microplate spectrophotometer
Benchmark Plus BIO-RAD (Bio-Rad Laboratories B.V, Veenendaal, The
Netherlands). Cell viability was determined respect to the controls
without any treatment (Cs) for each evaluated timepoint taking as 100%
of viability the absorbance value for these controls.
2.7. Statistics
3.1.2. NMR characterization
For the RSM and block designs the experimental design, analysis of
data, and determination of optimal conditions were performed using
STATGRAPHICS Centurion XVI software, Version 16.0.07 (StatPoint
Fig. 3 shows the 1H and 13C NMR spectra of the compounds involved
in the synthesis of the Sq-Gus bioconjugate, for which a previous puri-
fication process by flash chromatography was done. The 1H NMR spectra
4

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
Fig. 1. Schematic representation of the synthesis of Sq-Gus bioconjugate (1) Squalene, (2) Sq-MBH, (3) Sq-Ep, (4) Sq-Ald, (5) Sq-COOH, (6) Sq-COO-NHS, (7) Sq-Gus
bioconjugate. The quantities stated as a percentage were calculated based on the obtained yield.
of Sq-COOH (Fig. 3G) and Sq-Gus bioconjugate (Fig. 3I) present, in
deuterated EtOH, low-intensity signals for their hydrophilic hydrogens
(OH of the carboxylic acid group of Sq-COOH and amide, amine, and
hydroxyl groups of Sq-Gus). The amphiphilic character of these com-
pounds makes them adopt micelle like conformations in a moderately
polar solvent like EtOH. This conformation results from interactions of
the molecule lyophobic part with the solvent forming the shell. At the
same time, the lyophilic parts of the molecules interact with themselves
forming the nuclei with restricted mobility and short proton T1 relaxa-
tion times producing low-intensity signals (Heald et al., 2002; Podo
et al., 1973). In the case of the 13C NMR spectrum of the Sq-Gus bio-
conjugate, it was not possible to observe all carbons of the molecule due
to the low solubility of the conjugate in different deuterated solvents or
combination of them (data not shown). This together with especially
short T1 values for certain carbons decrease even more the already low
sensibility of 13C to the NMR experiment.
3.2. Use of RSM to transform Sq-Gus bioconjugate into NPs
Sq-Gus bioconjugate was transformed into NPs through the nano-
precipitation method using an ethanolic solution of the Sq-Gus bicon-
jugate (2 mg/mL), which was added to distilled water to form
nanoassemblies. The optimal conditions for NPs preparation were
established through an experimental design applying the RSM, which
allowed the determination of factors influenced size, charge, and dis-
persity of the NPs. Treatments and responses obtained from the RSM
5

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
Fig. 2. Infrared spectra of the compounds involved in the Sq-Gus bioconjugate synthesis. (A) Stacked spectra of all synthesis products in the different reaction steps.
(B) Infrared spectra of Sq-Gus bioconjugate.
design are shown in Table 2.
them. Neither the volume of the bioconjugate, nor the volume of water,
or interactions between factors, had significant effects on NPs charge.
3.2.1. Effect of the volume of bioconjugate, the volume of water, and the
stirring speed on NPs size
3.2.3. Effect of the volume of bioconjugate, the volume of water, and the
stirring speed on NPs dispersity
The Fig. 4A and Table S1 of the supplementary information show
that any of the studied factors had a statistically significant effect on NPs
size. Of these factors, stirring speed and the volume of the bioconjugate
in EtOH had the most pronounced influence on the size compared to
water volume and the interactions between factors. The stirring speed
showed a negative effect on the size indicating that when it is increased,
the NPs size decreases. The volume of the bioconjugate in ethanol,
added to the water phase, had a positive effect on the NPs size, so an
increase in the volume of the bioconjugate will enhance the NPs size.
This indicates that an increase in the concentration of the bioconjugate
implies an increase in the size of the NPs. The interaction between fac-
tors had a minor effect on NPs size.
The Fig. 4C and Table S3 of the supplementary information show
that the Ð of the NPs was affected at a higher degree by the volume of the
bioconjugate with a negative effect on its variation. According to this, an
increase in the concentration (Vol. bioconjugate) of the bioconjugate
will produce NPs with a more uniform size or lower dispersity. Stirring
speed and its interaction with the volume of the bioconjugate had
contrary effects on Ð but similar in magnitude. Therefore, an increase in
the stirring speed and volume of the bioconjugate at the same time will
produce smaller nanoparticles. It is to note that only the volume of the
bioconjugate had a significant effect while the volume of water, the
stirring speed and the interactions between factors showed a non-
statistically significant effect on Ð.
3.2.2. Effect of the volume of bioconjugate, the volume of water, and the
stirring speed on NPs charge
3.2.4. Optimization using the RSM
The surface charge of the NPs was most affected by the stirring speed
(Fig. 4B and Table S2 of the supplementary information) with a negative
effect on the variation of the z potential on NPs. The surface charge of
the NPs became more negative when the stirring speed was enhanced.
Therefore, increasing the stirring speed will enhance the stability of the
NPs due to the increase of repulsive electrostatic interactions between
The previously studied response variables were normally distributed,
with a constant variance, and independent of each other complying with
´
the assumptions of the experimental design (Gutierrez Pulido and de la
Vara Salazar, 2008). According to the aforementioned, the obtained
response surface (Fig. 4D) can precisely describe the behavior of the
studied variables and could be used to predict the optimal values
6

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
A
B
D
C
E
F
G
H
I
Fig. 3. 1H and 13C NMR for squalene derivatives obtained during the synthesis of Sq-Gus bioconjugate. (A) 1H NMR of squalene in CD3Cl. (B) 13C NMR of squalene in
CD3Cl. (C) 1H NMR of Sq-MBH in CD3Cl. (D) 13C NMR of Sq-MBH in CD3Cl. (E) 1H NMR of Sq-Ald in CD3Cl. (F) 13C NMR of Sq-Ald in CD3Cl. (G) 1H NMR of Sq-COOH
in EtOD. (H) 13C NMR of Sq-COOH in CD3Cl. (I) 1H NMR of Sq-Gus in EtOD.
7

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
labeled Sq-GusNPs uptake by the cells through flow cytometry, which
was observed as a displacement towards the NR+ side (Fig. 6A). An
increase of 6.1, 14.3, and 17.5 times the peak area was observed after
exposing the cells to Nile-Red labeled NPs with respectively 1x, 2x, and
4x concentrations (Fig. 6B and C). As shown in Fig. 6C uptake was
effective and concentration dependent. We were also able to visualize
fluorescence in the cells (Fig. 6D) confirming NPs uptake.
Table 2
Treatments and responses obtained for the RSM design.
Treatment
No.
Vol.
Vol.
Stirring
speed
Size
ζ
Ð
Bioconjugate
(µL)
water
(mL)
(nm)
(mV)
(rpm)
1
400
232
300
400
400
400
500
400
500
300
500
568
300
400
500
400
300
3.5
3.5
2.0
3.5
3.5
1.0
2.0
6.0
2.0
5.0
5.0
3.5
2.0
3.5
5.0
3.5
5.0
535
535
800
980
535
535
800
535
270
800
800
535
270
90
134.5
108.1
114.6
111.2
114.8
140.2
118.1
131.7
145.2
97.1
–33.5
–33.5
ꢀ 34.0
–32.3
ꢀ 35.2
ꢀ 30.0
ꢀ 30.1
–32.6
ꢀ 27.3
ꢀ 36.5
ꢀ 35.5
ꢀ 28.0
ꢀ 28.5
ꢀ 26.4
ꢀ 27.5
ꢀ 31.8
ꢀ 34.1
0.39
0.57
0.61
0.45
0.34
0.08
0.13
0.33
0.11
0.75
0.19
0.16
0.10
0.30
0.20
0.24
0.38
2
3
4
3.4.2. Cytotoxicity of Sq-GusNPs
5
Cytotoxicity of Sq-GusNPs was tested through the determination of
cell viability in U-937 monocytes with the two fluorophores PI and
DiOC6 through flow cytometry after exposure of the cells for 24 and 48 h
to either free gusperimus, SqNPs, or Sq-GusNPs in equivalent concen-
trations (Fig. 7A and B). None of the treatments were toxic for the cells
(Fig. 7C and D) in the times and ranges of concentrations evaluated
which were 2.8 to 11.2 µg/mL for gusperimus and squalene, and 5.6 to
22.3 µg/mL for Sq-GusNPs.
6
7
8
9
10
11
12
13
14
15
16
17
124.5
128.3
120.7
152.7
155.0
124.4
112.7
270
535
270
3.4.3. Anti-inflammatory effect of Sq-GusNPs
To investigate the efficacy of Sq-GusNPs in attenuating inflammatory
responses, U-937 monocytes were differentiated into macrophages by
the addition of PMA (Haque et al., 2019). Subsequently, the cells were
activated with LPS and 6 to 96 h later the anti-inflammatory capacity
considering the interaction between the different responses. The ob-
tained response surface is in terms of the desirability function (Table S4
of the supplementary information), which can take values between
0 and 1 and is defined according to the values set as the goal to obtain an
optimal response. Based on the foregoing, we selected 140 nm for the
particle size, ꢀ 30 mV for ζ, and a minimum value for Ð. The values
established as a goal for each one of the response variables were chosen
considering the observed behavior after the evaluation of the 17 treat-
ments, where for treatment 6 the lowest dispersity was obtained
(Table 2), and therefore the size and ζ for this treatment were chosen.
The conditions for nanoparticle preparation (Table 3) were obtained
by optimization using the predicted values and the mathematical model
of the RSM. As was expected, a variation in the responses was caused by
slight changes in conditions used for nanoparticle preparation because
of instrumental limitations as indicated in Table 3. A change in the
obtained responses compared to the predicted values of 15.4%, 13.3%,
and 11.1% for size, charge, and dispersity was obtained, respectively.
Through TEM a spherical shape for the NPs was observed without in-
ternal organization (Fig. 4E and F), whose size agreed with DLS mea-
surements (Table 3).
was studied by quantifying secretion of TNF
α and IL-10. Cells were
incubated with either Sq-GusNPs, free gusperimus, or SqNPs. Untreated
cells and cells stimulated with LPS served as controls. After 6 h, Sq-
GusNPs showed a clear anti-inflammatory effect. It significantly
reduced LPS-induced TNFα secretion by the macrophages 2.7 times and
IL-10 secretion 3.9 times compared with the control LPS (p < 0.0001)
(Fig. 8A and B). There was no statistically significant difference between
Sq-GusNPs and the other two treatments free gusperimus and SqNPs in
the reduction of the inflammatory response. After 24, 48, and 72 h of
exposition to LPS (Figure S5 of the supplementary information) there
were less pronounced effects of gusperimus either as free or in encap-
sulated form, although only the treatment with Sq-GusNPs showed a
tendency to the downregulation of both cytokines. After 96 h LPS
exposition, Sq-GusNPs significantly reduced LPS-induced TNF
α pro-
duction 1.4 times compared to the control (p < 0.005) meanwhile free
gusperimus, enhanced TNFα production 1.3 times higher than the LPS
stimulated cells (p < 0.005) (Fig. 8A). Sq-GusNPs also reduced LPS-
induced IL-10 secretion after 96 h (p < 0.05) (Fig. 8B).
3.3. Colloidal stability of Sq-GusNPs
4. Discussion
The colloidal stability for Sq-GusNPs was determined using a block
experimental design in which the change in size and surface charge of
the NPs was evaluated for 30 days at pH values of 4.5, 7.4, and 9.0 at
Here we present the design and characterization of a nano-
encapsulation system for the immunosuppressive drug gusperimus using
the platform originally proposed by Couvreur et al. (Couvreur et al.,
2006). Gusperimus was bound covalently to squalene to obtain a bio-
conjugate or prodrug, which has the unique characteristic to self-
assemble forming NPs in aqueous medium. The ability of the Sq-Gus
bioconjugate to form nanoassemblies is due to its amphiphilic char-
acter where the squalene part provides the lyophobic moiety and the
gusperimus the lyophilic one (Arpicco et al., 2016). The Sq-Gus bio-
conjugate chemical composition was confirmed by NMR analysis. The
presence of the hydrophobic squalene moiety and the consequent in-
crease in surface activity on the aqueous phase when the ethanolic so-
lution of the biconjugate was added are responsible of the formation of
nanoassemblies (Couvreur et al., 2006). In the self-aggregation of the
molecules occurring by lowering the surface tension, the interacting
4
◦C and 25 ◦C. The colloidal stability of Sq-GusNPs showed to be
temperature and pH-dependent (Table S5 of the supplementary infor-
mation). Higher stability could be obtained at 4 ◦C (Fig. 5A) since at this
temperature at both pH 4.5 and 7.4, the NPs were stable for 30 days and
at pH 9.0 for 20 days. When the NPs were stored at 25 ◦C at pH 7.4, the
NPs were stable for 25 days while at pH 9.0 and 4.5 they were only stable
for 15 and 10 days respectively (Fig. 5B). The highest stability was ob-
tained when NPs were prepared at pH 7.4 at both temperatures. An
increase in the magnitude of the NPs surface charge was observed when
the pH was increased at both temperatures (Fig. 5C and D).
3.4. Uptake, toxicity, and efficacy in attenuating inflammatory responses
of Sq-GusNPs in the U-937 cell line
¨
squalene moieties form the nuclei of the NPs (Desmaele et al., 2012).
The homogeneous morphology and granulometry of the obtained NPs
are the result of the more rigid conformation of the squalene derivatives
as previously reported by Pogliani et al. (Pogliani et al., 1994). This
nanoencapsulation procedure improved drug stability and availability
as we demonstrated with stability and in vitro studies, respectively. Sq-
GusNPs might also allow enhanced in vivo biodistribution of the active
3.4.1. Cell uptake of Sq-GusNPs
As gusperimus is efficacious in suppressing cells of the innate im-
munity (Perenyei et al., 2014) we evaluated cellular uptake, toxicity,
and the anti-inflammatory capacity of Sq-GusNPs in the human U-937
cell line. Pinocytosis of Sq-GusNPs was followed by monitoring Nile-Red
8

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
Fig. 4. Pareto diagrams for each one of the studied variables, response surface for Sq-GusNPs preparation though a CCD, and TEM images of the obtained NPs (A)
Pareto diagram for Size. (B) Pareto diagram for ζ. (C) Pareto diagram for Ð. (D) Response surface obtained for the CCD experimental design. (E) Cryo-TEM images for
Sq-GusNPs. (F) TEM images of Sq-GusNPs obtained after staining with a 2% of uranyl acetate solution. Arrowheads show the NPs; scale bars represent 200 nm.
Table 3
Predicted, used, and obtained values according to the RSM. NPs were prepared by triplicate and the responses are expressed as mean ± standard deviation.
Factor
Predicted optimal point
Experimental optimal point
Response
Predicted value
Obtained response
Vol. Bioconjugate (µL)
Vol. Water (mL)
380.24
1.04
380
1
Size (nm)
ζ (mV)
Ð
135.75
ꢀ 30.00
0.09
158.0 ± 48.8
ꢀ 34.0 ± 0.7
0.1 ± 0.01
Stirring speed (rpm)
510.83
500
principle as it has been previously demonstrated for other squalene
nanoparticulate delivery systems (Rouquette et al., 2019). The NPs were
obtained through a bottom-up approach starting from the molecular
components squalene and gusperimus until the formation of the NPs.
The bioconjugate was synthesized and characterized using
spectrometric techniques. NPs characteristics such as size, charge, dis-
persity, and stability were tuned and evaluated using the experimental
design approaches of RSM and block design. For evaluation of the in vitro
efficacy of Sq-GusNPs, we used the U-937 cell line and demonstrated its
uptake, absence of toxicity, and anti-inflammatory capacity.
9

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
1100
pH
400
300
200
100
0
pH
4.5
7.4
9.0
4.5
7.4
9.0
A
B
900
700
500
300
100
-100
0
1
2
3
4
5
10 15 20 25 30
0
1
2
3
4
5
10 15 20 25 30
-33
-37
-41
-45
-49
-32
-35
-38
-41
-44
-47
C
D
4.5
7.4
pH
9.0
4.5
7.4
pH
9.0
Fig. 5. Colloidal stability of Sq-GusNPs over time at different pH values evaluated using an experimental block design. (A) Size for Sq-GusNPs stored at 4 ◦C for 30
days. (B) Size for Sq-GusNPs stored at 25 ◦C for 30 days. (C) Z potential for Sq-GusNPs stored at 4 ◦C for 30 days. (D) Z potential for Sq-GusNPs stored at 25 ◦C for 30
days. The experiment included 11 blocks, was randomized, and for each temperature, this consisted of two replicas per block with a total of 66 runs.
By using FTIR and NMR we identified and characterized the different
intermediate products from Sq-MBH until the formation of the Sq-Gus
bioconjugate. In the reaction required to produce Sq-MBH, we
observed selectivity of binding to one of the terminal olefinic bonds. This
selectivity can be caused by the folded conformation of squalene in polar
solvents induced by the water added to the squalene solution in THF
during the first reaction step. In this folding phase, the internal olefinic
bonds are protected from Br electrophilic addition due to intramolecular
colloidal system as particles in the nanosized range tend to approach
each other and form large aggregates due to interactions such as van der
Waals forces (Selvamani, 2019). Colloidal stability can be reached
through electrostatic or steric stabilization mechanisms (Myers, 1999).
Our system showed to be very stable for at least 30 days (Fig. 5A) due to
the high surface charge of the NPs, which was between –33 and ꢀ 49 mV
(Fig. 5C and D) at a wide range of pH values (Dosio et al., 2010). This
finding provides insight into the stabilization mechanism of Sq-GusNPs,
which is assumed to be due to electrostatic repulsion interactions
(Gumustas et al., 2017).
¨
interactions of squalene (Desmaele et al., 2012). The squalene folding
conformation in polar solvents has been previously demonstrated by
Pogliani et al. (Pogliani et al., 1994), where, through NMR studies a
diminution of relaxation times (T1) in carbons located at the center of
the squalene molecule was observed. The folding of the molecule is
accompanied by an increased stiffness that goes from the end to the
center of the squalene chain. This is an explanation for the availability of
the olefinic extremes of the squalene chain for reacting with NBS to form
the Sq-MBH due to its enhanced mobility. This is needed to obtain the
subsequent derivatives and finally the Sq-Gus bioconjugate.
Monocytes are innate immune cells and responsible for the first line
of defense against pathogens with the potential to differentiate into
macrophages or dendritic cells (Abbas et al., 2007). Monocytes are key
players in modulating inflammation by producing pro-inflammatory
cytokines such as IL-1β, IL-6, and TNF-
α as well as iNOS and anti-
inflammatory cytokines as IL-10 and TGF-β1. In this way, monocytes
steer immune responses in both autoimmunity and graft rejection
(Karlmark et al., 2012). Macrophages are more phagocytic cells and
have some other, more specialized functions than monocytes and serve
as cells responsible for antigen presentation, chemotaxis, and release of
pro-inflammatory and anti-inflammatory cytokines (Bradlet, 2009).
This was the reason to choose the U-937 cell line for this study as it is
from human origin and because of its versatile properties to be used as
monocytes or macrophages depending on the applied stimuli.
RSM is an experimental and analytical strategy that allows identifi-
cation of the optimal operating conditions in a process allowing to set
optimal values for one or several characteristics of the experimental unit
´
(Gutierrez Pulido and de la Vara Salazar, 2008) in this case the NPs.
With the use of the parameters found with RSM (Table 2), Sq-GusNPs
with an average size of 158 nm could be obtained. The NPs size distri-
bution was uniform as was corroborated by a dispersity value of 0.1
(Danaei et al., 2018). The surface charge of the NPs was relatively high
with an average value of ꢀ 34.0 mV facilitating electrostatic repulsion
forces that conferring them stability (Selvamani, 2019). This high sur-
face charge of Sq-GusNPs influence features as uptake and intracellular
trafficking (Gumustas et al., 2017). The gusperimus loading capacity of
the NPs is 50.19%, as determined by the weight-contribution of the
gusperimus to the Sq-Gus bioconjugate (Gaudin et al., 2016). However,
the encapsulation efficiency, or conjugation efficiency, is 66% as
determined by the yield obtained from the final reaction step in the
synthesis process.
Sq-GusNPs was shown to be readily taken up by monocytes without
any cytotoxic side-effect in the range between 5.6 and 22.3 µg/mL. The
Sq-GusNPs had a robust anti-inflammatory effect since both TNFα and
IL-10 production were attenuated after LPS-stimulation at 6 and 96 h. A
¨
surprising observation was that not only gusperimus (Kalsch et al.,
2006) but also squalene showed an anti-inflammatory effect at 6 h. This
´
corroborates the findings of Ana Cardeno et al. who studied the anti-
inflammatory properties of squalene in neutrophils, monocytes, and
´
macrophages in vitro (Cardeno et al., 2015). The effects of squalene and
gusperimus, however, were not as profound as that of Sq-GusNPs, which
had a stronger tendency to attenuate both TNFα and IL-10 between 24
Colloidal stability is a measure for the long-term integrity of a
and 96 h (Fig. S5 of the supplementary information). In contrast,
10

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
Fig. 6. Cellular uptake of Sq-GusNPs by monocytes after 3 h of incubation with Nile Red labeled NPs. (A) Dot plots obtained through flow cytometry for the applied
treatments (NR+: Nile Red positive cells, NR-: Nile Red negative cells). (B) Stacked histograms showing the increment of fluorescent cells with the concentration of
labeled Sq-GusNPs. (C) Cellular uptake of Sq-GusNPs expressed as Nile Red positive events for the applied treatments (ns: no significant difference). All treatments
were statistically different from the control group (Cs) except the treatment Cs + Sq-GusNPs. (D) Micrographs obtained with bright light and fluorescent light for the
cellular uptake experiment after 3 h of incubation at the highest evaluated concentration of Sq-GusNPs, scale bar represent 100 µm. Sq-Gus 4x (no labeled NPs); Sq-
Gus-R 4x (labeled Sq-GusNPs); Cs (cells without NPs); Cs + Sq-Gus 4x (cells treated with no labeled NPs); Cs + Sq-Gus-R (cells treated with labeled NPs). x is
equivalent to 2.8
μg/mL of free gusperimus. Comparisons were made using one-way ANOVA with Dunnett’s post hoc test. Data represent mean values ± SD of 5
independent experiments.
11

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
Fig. 7. Cytotoxicity of Sq-GusNPs in monocytes for 24 and 48 h. (A) Representative dot plots obtained through flow cytometry at 24 h for the treatments. (B)
Representaive dot plots obtained through flow cytometry at 48 h for the treatments. (PI+ superior box PI-positive cells, DiOC6 Low inferior left box, DiOC6 low
absorption, DiOC6 + inferior right box DiOC6 positive cells). (C) Cell viability at 24 h for each treatment. (D) Cell viability at 48 h for each treatment. No statistical
differences between the control (Cs) and the treatments for the evaluated time points were founded. Cs (Cells without any treatment); Cs+Sq-Gus (Cells treated with
Sq-GusNPs); Cs+Gus (Cells treated with free gusperimus); Cs+Sq (Cells treated with SqNPs). x is equivalent to 2.8 μg/mL of free gusperimus or free squalene.
Comparisons were made using one-way ANOVA with Dunnett’s post hoc test. Data represent mean values ± SD of 3 independent experiments.
12

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
gusperimus has been shown to have high efficacy (Slobbe, 2012).
Finally, further studies are necessary to determine important pharma-
cological characteristics and potential clinical applications involving the
use of Sq-GusNPs as a therapeutic agent. These studies could include but
are not limited to the determination of half-maximal inhibitory con-
centration (IC50), internalization mechanisms involved in the uptake of
the Sq-GusNPs, in vivo studies for determination of NPs biodistribution,
residence time, and effect in different animal models of autoimmune
diseases or organ transplantation.
CRediT authorship contribution statement
Carlos E. Navarro Chica: Conceptualization, Methodology, Inves-
tigation, Writing - original draft. Bart J. de Haan: Formal analysis,
Resources. M.M. Faas: Writing - review & editing. Alexandra M.
Smink: Formal analysis, Resources. Ligia Sierra: Writing - review &
editing. Paul de Vos: Writing - review & editing, Supervision, Project
´
administration, Funding acquisition. Betty L. Lopez: Writing - review &
editing, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Fig. 8. Anti-inflammatory capacity of Sq-GusNPs. (A) TNFα production in LPS
Acknowledgments
stimulated U-937 human macrophages at 6 and 96 h, only the treatment with
Sq-GusNPs produced downregulation of the cytokine over time. (B) IL-10 pro-
duction in LPS stimulated U-937 human macrophages after exposure to the
different treatments, for the treatment with Sq-GusNPs was obtained the lowest
concentration of the cytokine. Cs (Cells without any treatment); LPS (Cells
treated with LPS); Sq-Gus (Cells treated with Sq-GusNPs); Gus (Cells treated
with free gusperimus); Sq (Cells treated with SqNPs). Comparisons were made
using two-way ANOVA with Tukey’s post hoc test. Data represent mean values
± SD of 3 independent experiments. p < 0.0001 (****); p < 0.005 (**); p <
0.05 (*).
This work was supported by the Abel Tasman Talent Program of the
University of Groningen and the COLCIENCIAS project “Preparation and
characterization of Gusperimus nanocarriers with potential application
in the process of implantation of cellular islets for the treatment of type 1
diabetes mellitus“ contract 747-2018, N◦ 111580763077.
Data availability
Datasets generated during and/or analyzed during the current study
are in the supplementary data of this paper. Additional datasets are
available from the corresponding author upon reasonable request.
squalene and gusperimus treatments showed no attenuation of the LPS-
induced TNFα and IL-10 production over time demonstrating the spec-
ificity of Sq-GusNPs in attenuating inflammatory responses. We did
observe, however, that treatment with free gusperimus caused a higher
production of TNF
α
at 96 h. This can be explained by the high hydro-
Appendix A. Supplementary data
philic character of gusperimus which in the culture medium is unstable
and hydrolyzed causing loss of its activity (Fujii et al., 1989). Further, it
is oxidized to by-products that have shown cytotoxic effects on lym-
phocytes and leukemia cells (Fujii et al., 1989; Kerr et al., 1994). We
showed that nanoencapsulation of gusperimus as Sq-GusNPs allows the
induction of anti-inflammatory effects in the absence of toxicity and
instability issues.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2020.119893.
References
Abbas, A.K., Lichtman, A.H., Pillai, S., 2007. Cellular and molecular immunology, 6th ed.
Saunders Elsevier.
Arpicco, S., Battaglia, L., Brusa, P., Cavalli, R., Chirio, D., Dosio, F., Gallarate, M.,
Milla, P., Peira, E., Rocco, F., Sapino, S., Stella, B., Ugazio, E., Ceruti, M., 2016.
Recent studies on the delivery of hydrophilic drugs in nanoparticulate systems.
J. Drug Deliv. Sci. Technol. 32, 298–312. https://doi.org/10.1016/j.
jddst.2015.09.004.
5. Conclusion
This study demonstrates that by using the squalenoylation platform
(Couvreur et al., 2006) the immunosuppressive drug gusperimus can be
nanoencapsulated as Sq-GusNPs, which has a profound influence on the
bioactivity of gusperimus. These NPs are shown to be stable, have a high
drug loading capacity, and in vitro a high uptake, lack of toxicity, and
anti-inflammatory activity on both human monocytes and macrophages.
This encapsulation system allows stabilization and controlled release of
gusperimus, which was corroborated by the maintenance of gusperimus
B.Kaufman, D., F.Gores, P., Kelley, S., M.Grasela, D., G. Nadler, S., EleanorRamos, 1996.
15-deoxyspergualin: Immunotherapy in solid organ and cellular transplantation.
Transplant. Rev. 10, 160–174. https://doi.org/https://doi.org/10.1016/S0955-
470X(96)80025-5.
´
Barreras-Urbina, C.G., Ramírez-Wong, B., Lopez-Ahumada, G.A., Burruel-Ibarra, S.E.,
´
´
Martínez-Cruz, O., Tapia-Hernandez, J.A., Rodríguez Felix, F., 2016. Nano- and
Micro-Particles by Nanoprecipitation: Possible Application in the Food and
Agricultural Industries. Int. J. Food Prop. 19, 1912–1923. https://doi.org/10.1080/
10942912.2015.1089279.
downregulatory effects over time for TNFα and IL-10 (Perenyei et al.,
Bradlet, S., 2009. Conversion of the U937 Monocyte into “Macrophage-Like” Populations
Exhibiting M1 or M2 Characteristics. Wright State University, Brows. all Theses Diss.
2014). These results suggest that the Sq-GusNPs are a promising ther-
apeutic for the treatment of autoimmune diseases such as vasculitis,
glomerulonephritis, systemic lupus erythematosus, rheumatoid
arthritis, Crohn’s disease, and management of graft rejection where
´
Cardeno, A., Aparicio-Soto, M., Montserrat-de la Paz, S., Bermudez, B., Muriana, F.J.G.,
´
Alarcon-de-la-Lastra, C., 2015. Squalene targets pro- and anti-inflammatory
mediators and pathways to modulate over-activation of neutrophils, monocytes and
macrophages. J. Funct. Foods 14, 779–790. https://doi.org/10.1016/j.
jff.2015.03.009.
13

C.E. Navarro Chica et al.
International Journal of Pharmaceutics 590 (2020) 119893
¨
Ceruti, M., Balliano, G., Viola, F., Cattel, L., Gerst, N., Schuber, F., 1987. Synthesis and
biological activity of azasqualenes, bis-azasqualenes and derivatives. Eur. J. Med.
Chem. 22, 199–208. https://doi.org/10.1016/0223-5234(87)90050-X.
Ceruti, M., Balliano, G., Viola, F., Grosa, G., Rocco, F., Cattel, L., 1992. 2,3-Epoxy-10-aza-
10,11-dihydrosqualenae, a High-Energy Intermediate Analogue Inhibitor of 2,3-
Oxidosqualene Cyclase. J. Med. Chem. 35, 3050–3058. https://doi.org/10.1021/
jm00094a020.
Kalsch, A.I., Schmitt, W.H., Breedijk, A., Marinaki, S., Weigerding, S., Nebe, T.C.,
Nemoto, K., Van Der Woude, F.J., Yard, B.A., Birck, R., 2006. In vivo effects of cyclic
administration of 15-deoxyspergualin on leucocyte function in patients with
Wegener’s granulomatosis. Clin. Exp. Immunol. 146, 455–462. https://doi.org/
10.1111/j.1365-2249.2006.03231.x.
Karlmark, K., Tacke, F., Dunay, I., 2012. Monocytes in health and disease — Minireview.
Eur. J. Microbiol. Immunol. 2, 97–102. https://doi.org/10.1556/eujmi.2.2012.2.1.
Kawada, M., Masuda, T., Ishizuka, M., Takeuchi, T., 2002. 15-Deoxyspergualin inhibits
Akt kinase activation and phosphatidylcholine synthesis. J. Biol. Chem. 277,
27765–27771. https://doi.org/10.1074/jbc.M200318200.
Clayden, J., Greeves, N., Warren, S., 2012. Organic Chemistry, Second. ed. Oxford
University Press Inc.
Clayton, K.N., Salameh, J.W., Wereley, S.T., Kinzer-Ursem, T.L., 2016. Physical
characterization of nanoparticle size and surface modification using particle
scattering diffusometry. Biomicrofluidics 10, 1–14. https://doi.org/10.1063/
1.4962992.
Kerr, P.G., Ntkolic-Paterson, D.J., Lan, H.Y., Tesch, G., Rainone, S., Atkins, R.C., 1994.
Deoxyspergualin suppresses local macrophage proliferation in rat renal allograft
rejection. Transplantation 58, 596–601. https://doi.org/10.1097/00007890-
199409150-00012.
Conversion of Carboxylic acids to amides using DCC as an activating agent - Chemistry
LibreTexts [WWW Document], n.d. URL https://chem.libretexts.org/Bookshelves/
Organic_Chemistry/Supplemental_Modules_(Organic_Chemistry)/Carboxylic_Acids/
Reactivity_of_Carboxylic_Acids/Conversion_of_Carboxylic_acids_to_amides_using_
DCC_as_an_activating_agent (accessed 10.30.19).
Myers, D., 1999. Surfaces, interfaces, and colloids: Principles and Applications, Second.
ed. John Wiley & Sons, Inc. https://doi.org/10.1002/0471234990.
Nagarkatti, J.P., Ashley, K.R., 1973. Periodic acid cleavage of epoxides in aqueous
medium. Tetrahedron Lett. 14, 4599–4600. https://doi.org/10.1016/S0040-4039
(01)87286-5.
Couvreur, P., Lakkireddy, Harivardhan, R., Dosio, F., Stella, B., Cattel, L., 2009.
Nanoparticles of therapeutic agents having low water solubility, Patent
WO2009071850A2.
Nishimura, K., Ohki, Y., Fukuchi-Shimogori, T., Sakata, K., Saiga, K., Beppu, T.,
Shirahata, A., Kashiwagi, K., Igarashi, K., 2002. Inhibition of cell growth through
inactivation of eukaryotic translation initiation factor 5A (eIF5A) by
deoxyspergualin. Biochem. J. 363, 761–768. https://doi.org/10.1042/0264-6021:
3630761.
Couvreur, P., Stella, B., Harivardhan Reddy, L., Hillaireau, H., Dubernet, C.,
ˇ
ˆ
Desmaeie, D., Lepetre-Mouelhi, S., Rocco, F., Dereuddre-Bosquet, N., Clayette, P.,
Rosilio, V., Marsaud, V., Renoir, J.M., Cattel, L., 2006. Squalenoyl nanomedicines as
potential therapeutics. Nano Lett. 6, 2544–2548. https://doi.org/10.1021/
nl061942q.
Odaka, C., Toyoda, E., Nemoto, K., 1998. Immunosuppressant deoxyspergualin induces
apoptotic cell death in dividing cells. Immunology 95, 370–376. https://doi.org/
10.1046/j.1365-2567.1998.00606.x.
Danaei, M., Dehghankhold, M., Ataei, S., Hasanzadeh Davarani, F., Javanmard, R.,
Dokhani, A., Khorasani, S., Mozafari, M.R., 2018. Impact of particle size and
polydispersity index on the clinical applications of lipidic nanocarrier systems.
Pharmaceutics 10, 1–17. https://doi.org/10.3390/pharmaceutics10020057.
Perenyei, M., Jayne, D.R.W., Floßmann, O., 2014. Gusperimus: Immunological
mechanism and clinical applications. Rheumatol. (United Kingdom) 53, 1732–1741.
https://doi.org/10.1093/rheumatology/ket451.
¨
Desmaele, D., Gref, R., Couvreur, P., 2012. Squalenoylation: A generic platform for
Podo, F., Ray, A., Nemethy, G., 1973. Structure and hydration of nonionic detergent
micelles. High resolution nuclear magnetic resonance study. J. Am. Chem. Soc. 95,
6164–6171. https://doi.org/10.1021/ja00800a003.
nanoparticular drug delivery. J. Control. Release 161, 609–618. https://doi.org/
10.1016/j.jconrel.2011.07.038.
Dosio, F., Reddy, L.H., Ferrero, A., Stella, B., Cattel, L., Couvreur, P., 2010. Novel
nanoassemblies composed of squalenoyl-paclitaxel derivatives: Synthesis,
characterization, and biological evaluation. Bioconjug. Chem. 21, 1349–1361.
https://doi.org/10.1021/bc100154g.
Pogliani, L., Ceruti, M., Ricchiardi, G., Viterbo, D., 1994. An NMR and molecular
mechanics study of squalene and squalene derivatives. Chem. Phys. Lipids 70,
21–34. https://doi.org/10.1016/0009-3084(94)90044-2.
Rosenberg, M., Azevedo, N.F., Ivask, A., 2019. Propidium iodide staining underestimates
viability of adherent bacterial cells. Sci. Rep. 9, 1–12. https://doi.org/10.1038/
s41598-019-42906-3.
Ebrahiminezhad, A., Ghasemi, Y., Rasoul-Amini, S., Barar, J., Davaran, S., 2012. Impact
of amino-acid coating on the synthesis and characteristics of iron-oxide
nanoparticles (IONs). Bull. Korean Chem. Soc. 33, 3957–3962. https://doi.org/
10.5012/bkcs.2012.33.12.3957.
Rouquette, M., Lepetre-Mouelhi, S., Dufrançais, O., Yang, X., Mougin, J., Pieters, G.,
Garcia-Argote, S., IJzerman, A.P., Couvreur, P., 2019. Squalene-adenosine
nanoparticles: Ligands of adenosine receptors or adenosine prodrug? J. Pharmacol.
Exp. Ther. 369, 144–151. https://doi.org/10.1124/jpet.118.254961.
Selvamani, V., 2019. Stability Studies on Nanomaterials Used in Drugs, Characterization
and Biology of Nanomaterials for Drug Delivery. Elsevier Inc. https://doi.org/
10.1016/b978-0-12-814031-4.00015-5.
Feng, J., Lepetre-Mouelhi, S., Couvreur, P., 2017. Design, preparation and
characterization of modular squalene-based nanosystems for controlled drug release.
Curr. Top. Med. Chem. 17, 2849–2865. https://doi.org/10.2174/
1568026617666170719171728.
Fujii, H., Takada, T., Nemoto, K., Abe, F., Fujii, A., Takeuchi, T., 1989. In vitro
immunosuppressive properties of spergualins to murine T cell response. J. Antibiot.
(Tokyo) 42, 788–794. https://doi.org/10.7164/antibiotics.42.788.
Sen, S.E., Prestwich, G.D., 1989. Trisnorsqualene alcohol, a potent inhibitor of vertebrate
squalene epoxidase. J. Am. Chem. Soc. 111, 1508–1510. https://doi.org/10.1021/
ja00186a062.
¨
Gaudin, A., Song, E., King, A.R., Saucier-Sawyer, J.K., Bindra, R., Desmaele, D.,
Couvreur, P., Saltzman, W.M., 2016. PEGylated squalenoyl-gemcitabine
nanoparticles for the treatment of glioblastoma. Biomaterials 105, 136–144. https://
doi.org/10.1016/j.biomaterials.2016.07.037.
Shapiro, H.M., 2000. Membrane potential estimation by flow cytometry. Methods 21,
271–279. https://doi.org/10.1006/meth.2000.1007.
Silverstein, R.M., Webster, F.X., Kiemle, D.J., 2005. Spectrometric identification of
organic compounds, 7th ed. Jhon Wiley and Sons Inc.
Gumustas, M., Sengel-Turk, C.T., Gumustas, A., Ozkan, S.A., Uslu, B., 2017. Effect of
Polymer-Based Nanoparticles on the Assay of Antimicrobial Drug Delivery Systems,
Multifunctional Systems for Combined Delivery, Biosensing and Diagnostics. Elsevier
Inc. https://doi.org/10.1016/b978-0-323-52725-5.00005-8.
Slobbe, L., 2012. Drugs that act on the immune system: Immunosuppressive and
immunostimulatory drugs, 1st ed, Side Effects of Drugs Annual. Elsevier B.V. https://
doi.org/10.1016/B978-0-444-59499-0.00038-6.
Gusperimus | C17H37N7O3 - PubChem [WWW Document], n.d. URL https://pubchem.
ncbi.nlm.nih.gov/compound/55362#section=Experimental-Properties (accessed
10.23.19).
Takeuchi, T., Iinuma, H., Kunimoto, S., Masuda, T., Ishizuka, M., Takeuchi, M.,
Hamada, M., Naganawa, H., Kondo, S., Umezawa, H., 1981. A new antitumor
antibiotic, spergualin: Isolation and antitumor activity. J. Antibiot. (Tokyo) 34,
1619–1621. https://doi.org/10.7164/antibiotics.34.1619.
´
´
˜
Gutierrez Pulido, H., de la Vara Salazar, R., 2008. Analisis y diseno de experimentos,
Segunda. ed. McGRAW-HILL/INTERAMERICANA.
Thangaraj, U.D., Rajagantham, M., Govindarajulu, K., Arulsamy, J.P., 2017. Synthesis,
crystal structure and vibrational spectral analysis of guanidinium hydrogen L-
aspartate single crystal. J. Phys. Sci. 28, 27–47. https://doi.org/10.21315/
jps2017.28.1.3.
Haque, M.A., Jantan, I., Harikrishnan, H., Ghazalee, S., 2019. Standardized extract of
Zingiber zerumbet suppresses LPS-induced pro-inflammatory responses through NF-
κB, MAPK and PI3K-Akt signaling pathways in U937 macrophages. Phytomedicine
54, 195–205. https://doi.org/10.1016/j.phymed.2018.09.183.
Wang, L., Gao, X., Gao, P., Deng, W., Yu, P., Ma, J., Guo, J., Wang, X., Cheng, H.,
Zhang, C., Yu, C., Ma, X., Bingfeng, L.V., Lu, Y., Shi, T., Ma, D., 2006. Cell-based
screening and validation of human novel genes associated with cell viability.
J. Biomol. Screen. 11, 369–376. https://doi.org/10.1177/1087057106286654.
Zhao, C.X., He, L., Qiao, S.Z., Middelberg, A.P.J., 2011. Nanoparticle synthesis in
microreactors. Chem. Eng. Sci. 66, 1463–1479. https://doi.org/10.1016/j.
ces.2010.08.039.
Heald, C.R., Stolnik, S., Kujawinski, K.S., Matteis, C. De, Garnett, M.C., Illum, L., Davis, S.
S., Purkiss, S.C., Barlow, R.J., Gellert, P.R., 2002. Poly(lactic acid)-Poly(ethylene
oxide) (PLA-PEG) Nanoparticles: NMR Studies of the Central Solidlike PLA Core and
the Liquid PEG Corona. Langmuir 18, 3669–3675 https://doi.org/https://doi.org/
10.1021/la011393y.
14