Preparation and characterization of a synthetic curcumin analog inclusion complex and preliminary evaluation of in vitro antileishmanial activity

Article abstract of DOI:10.1016/j.ijpharm.2020.119764

The aim of this work was to prepare and characterize inclusion complexes between a synthetic curcumin analog (dibenzalacetone, DBA) and beta-cyclodextrin (β-CD); and to evaluate their in vitro antileishmanial activity. DBA was synthetized and characterized by spectroscopic methods and the inclusion complexes were obtained by kneading and lyophilization (LIO) in 1:1 and 1:2 stoichiometries. Phase solubility and dissolution assays showed a 40-fold increase in the aqueous solubility of DBA and its complete dissolution from LIO 1:1 formulation after 120 min respectively. Solid-state characterization by differential scanning calorimetry and near infrared spectroscopy demonstrated the inclusion of DBA in the β-CD cavity at the molar ratios tested, with LIO 1:1 formulation being the most stable. Using nuclear magnetic resonance experiments, the protons inside the cavity of β-CD were the most affected after the inclusion of DBA molecule. The cellular viability of THP-1 macrophage cells treated with plain DBA, β-CD and DBA/CD inclusion complexes showed that the plain DBA and DBA/CD at 1:2 stoichiometry presented toxicity, while β-CD alone and DBA/CD at 1:1 stoichiometry showed no toxicity up to 640 μg mL^?1. The in vitro assay with free-living promastigotes demonstrated that plain DBA and β-CD had IC₅₀ of < 10 and > 320 μg mL^?1 respectively, while only inclusion complexes with 1:1 stoichiometry showed antiproliferative activity with IC₅₀ = 51.3 μg mL^?1. Using the amastigote intracellular forms, there was also a difference between the plain and β-CD complexed DBA with complexes of 1:1 and 1:2 stoichiometry presenting EC₅₀ = 66.3 μg mL^?1 and 58.9 μg mL^?1 respectively. The study concluded that DBA/CD at 1:1 molar ratio has the potential to decrease the intrinsic toxicity of plain DBA towards Leishmania host cells, which may be a therapeutic advantage in the application of these compounds.

Full text of DOI:10.1016/j.ijpharm.2020.119764

InternationalJournalofPharmaceutics589(2020)119764
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Preparation and characterization of a synthetic curcumin analog inclusion
complex and preliminary evaluation of in vitro antileishmanial activity
Luciana Matos Alves Pintoa,b, Oluwatomide Adeoyeb, Sérgio Scherrer Thomasia,
⁎
Ana Paula Franciscob, Manuela Colla Carvalheirob, Helena Cabral-Marquesb,
a Chemistry Department, Universidade Federal de Lavras, Campus Universitário s/n, C.P. 3037, 37200-900 Lavras/MG, Brazil
b Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
A R T I C L E I N F O
A B S T R A C T
Keywords:
The aim of this work was to prepare and characterize inclusion complexes between a synthetic curcumin analog
(dibenzalacetone, DBA) and beta-cyclodextrin (β-CD); and to evaluate their in vitro antileishmanial activity. DBA
was synthetized and characterized by spectroscopic methods and the inclusion complexes were obtained by
kneading and lyophilization (LIO) in 1:1 and 1:2 stoichiometries. Phase solubility and dissolution assays showed
a 40-fold increase in the aqueous solubility of DBA and its complete dissolution from LIO 1:1 formulation after
120 min respectively. Solid-state characterization by differential scanning calorimetry and near infrared spec-
troscopy demonstrated the inclusion of DBA in the β-CD cavity at the molar ratios tested, with LIO 1:1 for-
mulation being the most stable. Using nuclear magnetic resonance experiments, the protons inside the cavity of
β-CD were the most affected after the inclusion of DBA molecule. The cellular viability of THP-1 macrophage
cells treated with plain DBA, β-CD and DBA/CD inclusion complexes showed that the plain DBA and DBA/CD at
1:2 stoichiometry presented toxicity, while β-CD alone and DBA/CD at 1:1 stoichiometry showed no toxicity up
to 640 μg mL−1. The in vitro assay with free-living promastigotes demonstrated that plain DBA and β-CD had
IC50 of < 10 and > 320 μg mL−1 respectively, while only inclusion complexes with 1:1 stoichiometry showed
antiproliferative activity with IC50 = 51.3 μg mL−1. Using the amastigote intracellular forms, there was also a
difference between the plain and β-CD complexed DBA with complexes of 1:1 and 1:2 stoichiometry presenting
EC50 = 66.3 μg mL−1 and 58.9 μg mL−1 respectively. The study concluded that DBA/CD at 1:1 molar ratio has
the potential to decrease the intrinsic toxicity of plain DBA towards Leishmania host cells, which may be a
therapeutic advantage in the application of these compounds.
Beta-cyclodextrin
Dibenzalacetone
Curcumin
Leishmania major
Leishmaniasis
1. Introduction
the registration and purchase of drugs to treat leishmaniasis, may lead
to an insufficient stock of drugs to treat patients in endemic areas
Leishmaniasis is a neglected disease transmitted by phlebotomine
sandflies. Depending on the species of the parasite, it can cause cuta-
neous lesions or visceral symptoms, often leading to death. The coun-
tries most affected by the disease are Brazil, India, Ethiopia, Kenya,
South Sudan, Somalia, and Sudan, representing more than 90% of new
cases. Many factors have been linked to the spread of the disease, in-
cluding the high incidence of adverse effects of first-choice drugs
(pentavalent antimonials) and high toxicity related to second-choice
drugs (amphotericin B and pentamidine). The emergence of strains
resistant to available treatments may also influence the increased in-
cidence of the disease. In addition, problems related to drug adminis-
tration, with daily injections, contribute to poor adherence to treatment
regimen. Other factors, such as the absence of public policies that favor
(Oliveira et al., 2011; WHO, 2020).
Presently, 1 billion people are at risk and there are 20,000–30,000
deaths due to visceral leishmaniasis registered per year (DNDi, 2020)
and the treatment for this disease is still a challenge due to drug toxicity
and several side effects. While the use of combination therapies has
become a common approach to overcome the resistance acquired by
parasites to certain drugs, new strategies to fight against this disease are
required to enhance therapeutic effectiveness, improve the quality of
life of the affected population and, if possible, lower costs (Chappuis
et al., 2007). Drug delivery and nanomedicine approaches, and the
discovery of new molecules (from natural or synthetic sources) are of
utmost importance for safely combatting leishmaniasis (Gutiérrez et al.,
2016; Vijayakumar and Das, 2018).
⁎ Corresponding author.
E-mail address: hcmarques@ff.ulisboa.pt (H. Cabral-Marques).
https://doi.org/10.1016/j.ijpharm.2020.119764
Received 29 April 2020; Received in revised form 8 August 2020; Accepted 10 August 2020
Availableonline25August2020
0378-5173/©2020ElsevierB.V.Allrightsreserved.

L.M.A. Pinto, et al.
InternationalJournalofPharmaceutics589(2020)119764
There are reports in the literature that curcumin (Das et al., 2008;
Shahiduzzaman and Daugschies, 2011) and its derivatives (Changtam
et al., 2010; Chauhan et al., 2018) have antiparasitic activity. Active
curcuminoid analogs exhibited antileishmanial activity against pro-
mastigotes of L. major and amastigotes of L. mexicana (Changtam et al.,
2010). Also, an antiproliferative effect of dibenzalacetone (DBA), an
analog of curcumin, was reported on the intracellular amastigotes of L.
donovani (Chauhan et al., 2018). Thus, in this work, the physico-
chemical properties and antileishmanial activity of an inclusion com-
plex between a curcuminoid compound (dibenzalacetone) and β-cy-
clodextrin (β-CD) were evaluated. The hypothesis was that the use of β-
CD could improve the aqueous solubility and hence, the activity of the
tested molecule. Dibenzalacetone (DBA), a curcumin derivative, was
synthesized and inclusion complexes were prepared and characterized
for subsequent testing in free-living promastigotes and intracellular
amastigotes of Leishmania sp. Since DBA is a molecule easily and in-
expensively obtained, the results for its use in cases of leishmaniasis
may be extremely relevant.
shaking for 300 min (5 h) in triplicate. A control with plain DBA was
performed. At specific times, aliquots were withdrawn and the absor-
bances were monitored at λmax = 230 nm. The absorbances were
normalized to compare the different conditions, as follows:
Normalized Absorbance = Absorbance of DBA at time t/Absorbance
of DBA at time zero
The inclusion kinetics plots were analyzed using a linear regression
of the data: absorbance vs time, ln absorbance vs time and 1/absorbance
vs time, to obtain the order and the kinetic constant of complexation
(Moraes et al., 2007a; Rodrigues et al., 2011).
2.4. Preparation of the inclusion complexes
Inclusion complexes were produced in 1:1 and 1:2 stoichiometry
using the kneading (KN) and lyophilization (LIO) methods. Briefly, in
the KN method DBA and β-CD were weighed and placed in a mortar
with a small amount of absolute ethanol. The paste was homogenized
for 30 min with a pestle and then kept in a desiccator to dry. In the LIO
method the compounds were weighed, and DBA was dissolved in ab-
solute ethanol, while β-CD was dissolved in ultrapure water. The so-
lutions were mixed and rotaevaporated. After rehydration the sample
was freeze dried. Physical mixtures (PM) were prepared with the simple
contact of the compounds.
2. Methodology
2.1. Molecular modeling
The structure of DBA was constructed, and optimization was per-
formed with density functional theory (DFT) in vacuum, using B3LYP
method and 6-31G basis set, at the Gaussian 09W software. The purpose
of the minimization of the molecule was to analyze possible interactions
with the cavity of cyclodextrin, based on its polarity and shape.
2.5. Characterization of the inclusion complexes
2.5.1. Differential scanning calorimetry
Calorimetric assays were performed in
a DSC Q200 – TA
2.2. Synthesis and characterization of DBA
Instruments. 3–5 mg samples of plain DBA, plain β-CD, DBA/CD in-
clusion complexes at 1:1 and 1:2 molar ratios, prepared by KN and LIO,
as well as PM used as a control, were weighed and placed in aluminum
pans with a hole. Scans were performed from 30 to 300 °C with a
heating rate of 10 °C/min. The atmosphere was N2 with a flow rate of
50 mL/min.
0.5 mL of acetone and 1 g of NaOH were added to a 1:1 water:-
ethanol mixture. Then, the obtained solution was poured in 0.9 mL of
benzaldehyde and stirred with a glass stick for 30 min. Yellow crystals
of dibenzalacetone were obtained, filtered under vacuum, washed and
recrystallized with ethanol. The final product was filtered again to be
characterized by Fourier transform infrared spectroscopy, using a
2.5.2. Near infrared spectroscopy
Bruker Alpha II Fourier Transform Infrared Spectrometer and by 1H, 13
C
The procedure was performed in a NIR-ATR ABB - Model TLA 2000.
The same samples prepared for DSC were mounted in a glass blade and
and HMQC nuclear magnetic resonance, recorded in a Bruker Ultra-
Shield 300 MHz spectrometer using DMSO‑d6 or CDCl3 as solvent.
the experiment was set for a resolution of 8 cm−1, 75 scans min−1
,
range from 4,000 to 10,000 cm−1, using the software GRAMS AI 7.0.0.
2.3. Phase solubility studies and inclusion kinetics
2.5.3. 1H nuclear magnetic resonance
Phase solubility studies were carried out using the protocol adapted
from (Higuchi and Connors, 1965). Excess amounts of DBA (6 mM)
were added to solutions containing increasing amounts of β-CD
(0–12 mM) in triplicate and stirred for 48 h at 37 °C. Samples were
centrifuged (14,000 rpm/20 min), filtered and the absorbances were
recorded at 230 nm.
The objective of the experiment was to observe if there are any
changes in the chemical shifts of the protons of plain DBA or β-CD, after
the complexation.
1H NMR spectra of the samples: plain DBA, plain β-CD and DBA/CD
inclusion complex, were recorded at 20 °C in DMSO‑d6 solution using a
Bruker Ultra-Shield 300 MHz spectrometer. Chemical shifts (δ) were
reported in parts per million (ppm) relative to the residual solvent peak.
The association constant (Ka) was determined using the following
equation (Higuchi and Connors, 1965), where S0 is the intrinsic solu-
bility of DBA:
2.6. Dissolution test
slope
S0 (1 slope)
Ka =
Samples of plain DBA and DBA/CD inclusion complexes at 1:1 and
1:2 molar ratios were placed in erlenmeyers flasks and shaken
(200 rpm) for 120 min at 37 °C in triplicate. Aliquots of 2 mL were
withdrawn each 5 min and after filtration the absorbances were read at
230 nm. The volume of the samples was replaced with ultrapure water
after each estimation (Haroun and El-Halawany, 2010).
The standard Gibbs free energy (ΔG°) was calculated using the
equation described in sequence (Patel and Patel, 2010; Yadav et al.,
2009), where S0 and Ss are the solubility of DBA in absence and in
presence of β-CD respectively:
S0
SS
G0
=
2.303RTlog
2.7. Anti-leishmaniasis assays
The inclusion kinetics experiment was run at 37 °C and was used to
estimate the time to reach the complexation equilibrium. DBA and β-
CD, at 1:1 and 1:2 molar ratios, were placed in ultrapure water and left
2.7.1. Assessment of cell viability in differentiated THP-1 monocytic cells
THP-1 cell line derived from an acute monocytic leukemia patient,
were grown in RPMI-1640 medium supplemented with 10% inactivated
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L.M.A. Pinto, et al.
InternationalJournalofPharmaceutics589(2020)119764
fetal bovine serum (iFBS), 100 U/mL penicillin and 100 mg/mL strep-
tomycin and incubated at 37 °C in a humidified atmosphere of 5% CO2.
A cell suspension at 3 × 105 cell mL−1 in RPMI medium containing
50 ng mL−1 of PMA [phorbol 12-myristate 13-acetate (Sigma-Aldrich,
Spain)] to promote differentiation into macrophages was prepared and
cells were seeded (200 µL) into 96-well plates and incubated at 37 °C,
5% CO2 for 48 h to allow differentiation. Cells were washed and ex-
posed to the test formulations of plain DBA, plain β-CD and DBA/CD
inclusion complexes at 1:1 and 1:2 molar ratios, previously prepared
and diluted in RPMI medium at concentrations of 5, 10, 20, 40, 80 and
160 μg mL−1 in respect to DBA content. Plates were incubated at 37 °C,
5% CO2 for 24 h. After washing with PBS, a 0.5 mg mL−1 solution of
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
was added in each well (50 µL) and incubated for 3 to 4 h (37 °C, 5%
CO2). At that time, DMSO (200 μL/well) was added to each well to
solubilize the formazan-MTT crystals and plates were shaken for
15 min. The plates were read on a microplate reader at 570 nm.
Fig. 1. Energy minimization of the DBA molecule using the software Gaussian
09W.
respectively).
The first band observed in FTIR was in the range of
3000–3100 cm−1, indicating the presence of a sp2 carbon connected to
hydrogen. An intense peak at 1649 cm−1 indicates the presence of the
conjugated carbonyl and at 1589 cm−1 is the signal of the double bond
C]C. At 1494 cm−1 is the CeC stretching vibrations of the aromatic
ring and the CeH bending vibration appeared at 760 and 596 cm−1, as
described in the literature (Periasamy et al., 2014).
2.7.2. Assessment of antileishmanial activity against promastigote cultures
A Leishmania major (MHOM/SA/85/JISH118 strain, kindly pro-
vided by Dr. Simon Croft from London School of Hygiene and Tropical
Medicine, UK) promastigotes suspension at 2 × 106 promastigote mL−1
in SCN medium was prepared. 100 μL of promastigotes were seeded
into the middle 60 wells of the 96-well plates. Stock solutions of DBA,
plain β-CD and DBA/CD inclusion complexes at 1:1 and 1:2 molar ratios
were prepared and serially diluted in SCN medium. 100 μL of the di-
luted solutions at 10, 20, 40, 80, 160 and 320 μg mL−1 were added to
each well and incubated.
1H NMR assignments of DBA was recorded in DMSO‑d6 and also in
CDCl3 solvent for unambiguous confirmation of the structure
(Supplementary material S2 – a, b and c) and are in accordance with the
literature (Chauhan et al., 2018). One doublet in δH 7.79 ppm and
another in δH 7.37, with a coupling constant of 15 Hz, indicates the
presence of the trans double bond connection, the main link obtained in
this reaction. 13C NMR chemical shifts were described in Supplemen-
tary material S2 - d and e, with seven carbons. Cross peaks obtained by
HMQC NMR demonstrated that proton Ha is linked to C2, Hd-meta is
linked to C6, Hd-para is linked to C7 and Hc is linked to C5 (Supple-
mentary material S2 - f).
2.7.3. Antileishmanial screening against intracellular cultures
After seeding and differentiation of the THP-1 cells in a 96-well
microplate, the infection of the transformed THP-1 cells with
Leishmania infantum (MHOM/MA/67/ITMAP263 strain, isolated in
Morocco, was a kind gift from Prof. Ana Tomás from i3S, Universidade
do Porto, Portugal) promastigotes was performed with 2.5 × 105
parasites/well. The plates were incubated at 37 °C, 5% CO2 for 24 h to
allow the parasites to infect the differentiated THP-1 cells. Then the
infected macrophages were treated with the test formulations serially
diluted in RPMI with 2% FBS and the plates were incubated at 37 °C,
5% CO2 for 24 h. The parasite-rescue-transformation-assay (Jain et al.,
2012) was performed with controlled lysis of L. infantum amastigotes-
infected macrophages. Plates were incubated at 26 °C for 72 h for
transformation of rescued amastigotes to promastigotes. MTT assay was
done for the quantitative analysis of transformed promastigote.
The mechanism of reaction involves the reaction of one molecule of
acetone with two molecules of benzaldehyde. Two aldolic condensa-
tions occur in the sequence (Fig. 2).
3.3. Phase solubility and inclusion kinetics
The phase solubility diagram for DBA at increasing concentration of
β-CD is presented in Fig. 3. An AL type profile was clearly observed, and
the solubility increased approximately 40-fold with the concentration
of β-CD. This could be attributed to the formation of soluble DBA/CD
inclusion complexes at 1:1 stoichiometry (Periasamy et al., 2014).
The association constant (Ka) was determined as 1158 L mol−1
which indicates the formation of a very stable complex at the tem-
perature tested. Reports in the literature described that Ka values be-
tween 200 and 5000 L mol−1 are considered more suitable to improve
the solubility and stability of molecules with low aqueous solubility
3. Results and discussion
3.1. Theoretical
DBA is a symmetrical and totally conjugated molecule. Besides the
carbon to oxygen polar bond, due to the electronegativity difference,
the molecule has a large apolar structure, thus overcoming any possible
polar interaction in the central ketone portion.
O
O
OH
O
O
The optimization of the molecule (Fig. 1) shows that the ketone
group has remarkably more tendency to attract electrons (red) than the
aromatic rings (green). Although all the electronic density is spread
along the axis toward the aromatic rings. This characteristic turns the
molecule almost insoluble in water. The cavity of the cyclodextrins
could accommodate the rings of the molecule in a 1:1 or 1:2 DBA/CD
molar ratios, thus indicating that a stable inclusion compound could be
formed and an increase in the water solubility can be reached.
H
OH
O
O
OH
O
H
O
3.2. Synthesis and characterization of DBA
The pale-yellow powder of DBA, obtained in the synthesis, was
characterized by FTIR and NMR (Supplementary material S1 and S2
Fig. 2. Proposed mechanism of reaction for the synthesis of DBA.
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L.M.A. Pinto, et al.
0.075
InternationalJournalofPharmaceutics589(2020)119764
20
15
10
5
PM 1:2
PM 1:1
KN 1:2
KN 1:1
0.060
0.045
LIO 1:2
LIO 1:1
0.030
y = a + b*x
Mean
Equation
Plot
Weight
Intercept
DBA
CD
Instrumental
0.00433
0.00499
7.40334
0.99179
0.98364
0.97955
0.015
0
Slope
Residual Sum of Squares
Pearson's r
R-Square(COD)
Adj. R-Square
0.000
-5
0
2
4
6
8
10
12
0
50
100
150
200
250
300
350
[ -CD] mM
Temperature oC
Fig. 3. Phase solubility diagram obtained for DBA in the presence of β-CD at
Fig. 4. Thermograms obtained by differential scanning calorimetry of β-CD,
DBA, PM and DBA/CD inclusion complexes obtained by LIO and KN at 1:1 and
1:2 stoichiometries.
37 °C.
(Patel and Patel, 2007).
The ΔG° is a thermodynamic parameter that can be related to the
transfer of molecules from the aqueous solution toward the cavity of β-
CD and gives information about the spontaneity of the process at con-
stant pressure and temperature. Values of ΔG° obtained for DBA were
all negative and increased as the concentration of β-CD was raised
(Table 1). This was indicative that the process was spontaneous and
that the environment inside the cavity of β-CD was more favorable for
DBA molecules than water, as the concentration of β-CD increased.
For the inclusion kinetics analysis, when included in the cyclodex-
trin cavity, the guest molecule undergoes changes in its spectroscopic
characteristics, like the intensity and the maximum absorption peak. In
solution, there is a balance between the associated and dissociated form
of the molecule to be complexed, according to criteria of polarity and
steric effects (Chaves et al., 2010; Grillo et al., 2008; Rodrigues et al.,
2011) that can be monitored by UV–Vis.
Table 2
Enthalpy, onset temperature and maximum temperature obtained by DSC.
Enthalpy (J g−1
)
Tonset (°C)
Tmax (°C)
β-CD
DBA
312.4
77.52
110.83
108.64
265.41
82.86
50.25
105.22
243.26
48.01
239.50
71.16
−51.49
226.10
−2.768
242.30
220.6
LIO 1:1
246.49
107.77
102.77
228.20
103.23
225.94
105.40
226.06
109.17
228.38
LIO 1:2
KN 1:1
72.49
−0.9476
284.50
2.326
224.06
72.91
KN 1:2
PM 1:1
PM 1:2
219.91
75.12
310.50
5.725
217.05
81.30
290.2
5.150
220.69
In this work, after approximately 30 min, the tested solutions
reached equilibrium in both stoichiometries (data not shown), thus
being a relatively short time for preparing the inclusion complexes. No
changes were observed in the λmax, only in its intensity.
3.4. Characterization of the inclusion complex
3.4.1. Differential scanning calorimetry
After analysis of the kinetic profile, a second-order kinetics was
determined, with R2 = 0.99392 and 0.99119 for stoichiometries 1:1
and 1:2 respectively. Thus, when doubling the concentration of the
guest molecule, the tendency is for the complexation rate to increase by
DBA presents a sharp endothermic peak at 108 °C related to the
melting point of the molecule (Fig. 4 and Table 2). An exothermic
transition at 265 °C appears and is probably due to the decomposition of
the molecule. On the other hand, β-CD has a unique endo transition at
110 °C related to the loss of water molecules from the cavity (Carvalho
et al., 2018; Martins et al., 2020).
22. The kinetic constants calculated were
k = 0.18557 and
0.25942 h−1 for molar ratios 1:1 and 1:2 respectively. These values are
in accordance with values of the literature for molecules considered
hydrophobic such as bupivacaine (Moraes et al., 2007b) and prazi-
quantel (Chaves et al., 2010; Rodrigues et al., 2011). The results suggest
that, in both molar ratios, there was a tendency for the DBA molecule to
interact with the cavity of β-CD and the equilibrium was quickly
achieved.
It was observed that LIO 1:1 decreases the transition temperature
around 100 °C, because the water molecules will leave the cavity be-
fore, to accommodate the guest molecule. This is thermodynamic more
favorable to occur as the molecule of DBA is less polar than water. The
enthalpy observed is in accordance with this statement (Table 2). In LIO
1:2, as the proportion of β-CD is higher, the DBA molecules, when in-
serted into the cavity, do not displace all the water molecules. However,
the exothermic peak observed above 250 °C is completely suppressed in
LIO 1:2 and get smaller in LIO 1:1, denoting that, as the molecule is
protected by the β-CD, the decomposition does not occur in this tem-
perature. A pronounced decrease was also observed in the enthalpy
related to LIO 1:1, more than LIO 1:2, corroborating the discussion that
at 1:1 stoichiometry the complexation is preferred.
Table 1
Standard Gibbs free energy (ΔG°) for solubilization of
DBA in aqueous solution of β-CD.
[β-CD] mM
ΔG° (KJ mol−1
)
3
−6.21
−7.79
−8.64
−9.04
−9.44
6
KN and PM (1:1 and 1:2) have a very similar profile, with no sig-
nificant decrease in the maximum temperature peak (Tmax), as ob-
served for LIO 1:1. In this case, both KN and PM do not seem to be a
good method to prepare the DBA/CD inclusion complexes as it was also
8
10
12
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L.M.A. Pinto, et al.
InternationalJournalofPharmaceutics589(2020)119764
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
Table 4
1H NMR chemical shifts of DBA in DMSO‑d6 before and after the complexation
with β-CD, at 20 °C.
LIO 1:2
LIO 1:1
KN 1:2
KN 1:1
PM 1:2
PM 1:1
Hydrogen position
DBA chemical shift
(ppm)
DBA/CD chemical shift
(ppm)
Δδ (ppm)
CD
Ha (alfa)
7.371
7.791
7.824
7.475
7.365
7.785
7.820
7.469
−0.006
−0.006
−0.004
−0.006
DBA
0.0
Hb (beta)
Hc (Ar orto)
Hd (Ar meta, para)
4000
5000
6000
7000
8000
9000
10000
Wavenumber (cm-1)
Fig. 5. Near infrared spectra of DBA, β-CD, PM and DBA/CD inclusion com-
more affected after the inclusion were the H5 and H3, indicating that
the molecule of DBA is inserted near them.
plexes obtained by KN and LIO at 1:1 and 1:2 stoichiometries.
On the other hand, the protons of DBA were affected diffusely
(Table 4), being the most affected Ha, Hb and the aromatics in meta and
para positions. So, probably, as the molecule is symmetric, it is inserted
facing the aromatic rings towards the outer face of β-CD and affecting
the H6 proton of β-CD. Although the results give an indication that DBA
is interacting inside β-CD cavity, further studies using 2D ROESY NMR
could help to elucidate the geometry of the inclusion complex formed.
confirmed by the NIR experiments.
3.4.2. Near infrared spectroscopy
It was observed in the spectra of LIO 1:1 and 1:2 that the char-
acteristic peaks of DBA (~8750 and 6000 cm−1) were suppressed, and
the peak for β-CD (~6800 cm−1) decreased (Fig. 5). However, for PM
and KN at 1:1 and 1:2 molar ratios, a similar profile was obtained, with
the main peaks of DBA and β-CD remaining in the spectra (Jug et al.,
2005). So, the results support that PM and KN techniques are not good
to incorporate the guest molecule, as already determined by DSC.
This indicates that the best procedure to prepare the DBA/CD in-
clusion complexes was LIO and it was the one chosen for the biological
tests.
3.5. Dissolution test
The in vitro dissolution profiles of DBA before and after complexa-
tion with β-CD is shown in Fig. 6. For complexes prepared by kneading
at 1:1 and 1:2 molar ratios, the observed profile was similar to those
obtained for plain DBA, with very low release. So, for these samples, the
complexation using the KN technique did not seem to be an advantage.
On the other hand, the complexes obtained by lyophilization ex-
hibited the fastest dissolution rates, and the tendency to reach a plateau
(Wang et al., 2011), demonstrating a zero-order kinetic. The required
times for 50% release rate from LIO 1:1 and 1:2 were 40 and 60 min,
respectively. This demonstrates that LIO1:1 has a slow release rate than
LIO 1:2.
3.4.3. Nuclear magnetic resonance
The experiment gave us an idea of how the inclusion of the DBA
molecule occurs inside the cavity of β-CD.
Nine peaks were assigned for the molecule of β-CD (Table 3) and are
in agreement with the literature (Marques et al., 1990). The protons
The sample LIO 1:1 reached the maximum cumulative release of the
guest molecule after the equilibrium at 120 min, while LIO 1:2 released
around 75% of DBA at the same time interval. This indicates that, at
Table 3
1H NMR chemical shifts of β-CD in DMSO‑d6 before and after the complexation
with DBA, at 20 °C.
H1
100
80
60
40
20
0
LIO 1:1
LIO 1:2
KN 1:1
KN 1:2
DBA
HO
H3
H2
H5
H4
HO
H6
Hydrogen
position
β-CD chemical shift
(ppm)
DBA/CD chemical shift
(ppm)
Δδ (ppm)
0.062
H2
Superimposed with
water
3.268
H4
3.301
3.378
3.542
3.574
3.629
4.468
4.819
5.750
5.695
0.077
0.090
0.089
0.081
0.033
0.014
0.055
0.041
H5
3.632
H3
3.663
H6
3.548
0
20
40
60
80
100
120
OH6
H1
4.435
Time (min)
4.833
OH2
OH3
5.695
Fig. 6. Dissolution profile of DBA alone and the inclusion complexes obtained
5.654
by kneading and lyophilization method at 1:1 and 1:2 molar ratio.
5

L.M.A. Pinto, et al.
InternationalJournalofPharmaceutics589(2020)119764
140
a)
(Fig. 7a). It was observed that DBA alone and DBA/CD at 1:2 molar
ratio presented toxicity for these cells, with CC50 = 12.7 and
71.51 μg mL−1, respectively, while β-CD alone and DBA/CD at
1:1 molar ratio showed no toxicity up to 640 μg mL−1. So, the complex
at 1:1 stoichiometry was not damaging the host cells, in all the con-
centrations tested.
DBA
-CD
DBA/CD1:1
DBA/CD1:2
120
100
80
Tests with free-living promastigotes of L. major (Fig. 7b), showed
inhibition of the parasite’s growth for plain DBA and DBA/CD at
1:2 molar ratio, with IC50 < 10 μg mL−1. The effects of curcumin on
promastigotes parasites have been demonstrated earlier in reference
strains of L. major, L. tropica and L. infantum (Saleheen et al., 2002),
with IC50 = 4.5, 5.7 and 5.9 μM respectively which are in good
agreement with the value obtained for plain DBA. Plain β-CD had
IC50 > 320 μg mL−1 being unable to kill the parasites. DBA/CD at
1:1 molar ratio showed antiproliferative activity, with an
60
40
20
0
IC50 = 51.3 μg mL−1
.
10
100
1000
For the intracellular assay (Fig. 7c) it was observed that β-CD
showed no toxicity for the parasites, with EC50 > 100 μg mL−1, while
plain DBA destroys the amastigotes with IC50 of 4.4 μg mL−1. DBA/CD
1:1 and 1:2 presented EC50 of 66.3 and 58.9 μg mL−1 respectively.
Plain β-CD did not kill neither the parasites nor the macrophage
host cells, being therefore a safe carrier for DBA.
[DBA] g/mL
140
b)
DBA
-CD
DBA/CD 1:1
DBA/CD 1:2
120
100
80
60
40
20
0
The selectivity index (SI) is considered as the highest exposure to
the drug that produces the desired efficacy with no toxicity to that
exposure. SI values were determined using the relationship CC50 against
THP-1/EC50 against each parasite strain. It appears from results display
in Table 5 that only DBA/CD at 1:1 molar ratio showed potentially high
SI for both Leishmania strains. This is mainly due to the lack of cyto-
toxicity demonstrated against the host cells (in this case THP-1).
DBA/CD at 1:1 molar ratio kills the free living or intracellular
parasites at higher concentrations than DBA alone, however no toxicity
was observed for the host macrophage cell, even in the highest con-
centrations. Therefore, the adjustment of the dosage, can be made with
more security, since higher concentrations can be administered, with no
host cell damage.
10
100
[DBA] g/mL
Plain DBA is toxic for Leishmania. sp in concentrations less than
10 μg mL−1 both for free living and intracellular parasites, but from
around 12 μg mL−1 it is also toxic for the host cells, being not safe
above this concentration.
140
120
100
80
c)
DBA/CD at 1:2 molar ratio also fights the free living or intracellular
parasites, as well as 1:1. But, in this case, it presents toxicity for the host
cells from around 71 μg mL−1 and could not be considered as safe.
Differences between 1:1 and 1:2 inclusion complexes may be due to
the distinct behavior observed in the dissolution profiles (item 3.5). At
1:1 stoichiometry DBA seemed to be more available for interaction with
the infected cells, since it can be more easily released from the cavity of
one molecule of β-CD than at 1:2 stoichiometry.
60
40
DBA
-CD
DBA/CD 1:1
DBA/CD 1:2
20
Table 5
0
Cytotoxicity in THP-1 cells (CC50, μg mL−1), activity against L. major promas-
tigotes and L. infantum amastigotes (EC50, μg mL−1) and selectivity index values
of DBA formulations.
1
10
100
[DBA] g/mL
Fig. 7. (a) Cellular viability of THP-1 macrophages. (b) Viability of free-living
promastigotes of L. major. (c) Viability of intracellular amastigotes of L. in-
fantum.
THP-1
L. major
L. infantum
CC50
EC50
SIa
EC50
SIa
DBA
13.2
< 10
51.3
> 1.3
> 12.48
> 1.8
na
4.4
3.0
1:1 molar ratio the DBA molecule had a slower release compared to
1:2 molar ratio.
DBA/CD 1:1
DBA/CD 1:2
β-CD
> 640
71.5
66.3
58.9
> 100
> 9.6
1.2
< 10
> 320
> 640
na
a
3.6. Biological tests
Selectivity index (SI) was calculated by the ratio between the CC50 (μg
mL−1) and EC50 (μg mL−1) values of DBA formulations. CC50 and EC50 values
were obtained in experiments against THP-1 cells, L. major promastigotes and L.
infantum amastigote-infected THP-1 cells.
Using the MTT endpoint, THP-1 macrophage cells were tested in
order to verify if the compounds would harm the cells of the host
6

L.M.A. Pinto, et al.
InternationalJournalofPharmaceutics589(2020)119764
4. Conclusion
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Luciana de Matos Alves Pinto: Conceptualization, Methodology,
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Oluwatomide Adeoye: Validation, Investigation, Writing - review &
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