molecules

Article
Development of Computational In Silico Model for Nano Lipid
Carrier Formulation of Curcumin
Omar Waleed Abduljaleel Albasri, Palanirajan Vijayaraj Kumar * and Mogana Sundari Rajagopal

Faculty of Pharmaceutical Sciences, Department of Pharmaceutical Technology, UCSI University, Jalan Menara
Gading, Taman Connaught, Cheras, Kuala Lumpur 56000, Malaysia
* Correspondence: [email protected]

Abstract: The oral delivery system is very important and plays a significant role in increasing the
solubility of drugs, which eventually will increase their absorption by the digestive system and
enhance the drug bioactivity. This study was conducted to synthesize a novel curcumin nano lipid
carrier (NLC) and use it as a drug carrier with the help of computational molecular docking to
investigate its solubility in different solid and liquid lipids to choose the optimum lipids candidate for
the NLCs formulation and avoid the ordinary methods that consume more time, materials, cost, and
efforts during laboratory experiments. The antiviral activity of the formed curcumin–NLC against
SARS-CoV-2 (COVID-19) was assessed through a molecular docking study of curcumin’s affinity
towards the host cell receptors. The novel curcumin drug carrier was synthesized as NLC using a
hot and high-pressure homogenization method. Twenty different compositions of the drug carrier
(curcumin nano lipid) were synthesized and characterized using different physicochemical techniques
such as UV–Vis, FTIR, DSC, XRD, particle size, the zeta potential, and AFM. The in vitro and ex
vivo studies were also conducted to test the solubility and the permeability of the 20 curcumin–NLC
formulations. The NLC as a drug carrier shows an enormous enhancement in the solubility and
permeability of the drug.

Keywords: SLNs; NLCs; curcumin; drug carrier; permeability; molecular docking

Citation: Albasri, O.W.A.; Kumar,

P.V.; Rajagopal, M.S. Development of 1. Introduction
Computational In Silico Model for
Since the early 1990s, pharmaceutical technology research groups have been paying
Nano Lipid Carrier Formulation of
increasing attention to lipid nanoparticles for diverse applications as carrier systems [1,2].
Curcumin. Molecules 2023, 28, 1833.
https://doi.org/10.3390/
The researchers looked at solid lipid nanoparticles (SLNs) and nanostructured lipid carriers
molecules28041833
(NLCs) [3]. Different NLCs formulations, including curcumin, were developed for oral use
in this work. NLCs are new colloidal carrier systems composed of solid and liquid lipids.
Academic Editor: Rita Cortesi These lipids are carefully combined to create particle–matrix mixtures [4]. Compared to
Received: 3 December 2022 pure solid lipids, the presence of liquid lipids in these mixtures produces depression with a
Revised: 3 January 2023 melting point. The resulting blends are solid at room and body temperature [5,6]. NLCs
Accepted: 17 January 2023 are a new kind of lipid nanoparticles that have the advantages of improving the drug
Published: 15 February 2023 loading and storage capacities within the particle matrix [7]. The medicine during storage,
presented as the loading capacity, was enhanced. Changing the lipid concentration of NLCs
by the incorporation of liquid lipid resulted in improving the physical stability. However,
drugs that have a higher solubility in oils than in solid lipids can be dissolved in the oil, and
Copyright: © 2023 by the authors. the role of the surrounding lipid is to provide the chemical stability for active compounds
Licensee MDPI, Basel, Switzerland. that are chemically sensitive [8].
This article is an open access article A variety of lipids, both solid and liquid, are used in the manufacturing process of
distributed under the terms and
SLNs and NLCs formulations [9]. Palmitic acid, sitosterol alcohol, glyceryl monostearate
conditions of the Creative Commons
(GMS), tripalmitate, and stearic acid are the solid lipids which are often used in SLNs
Attribution (CC BY) license (https://
and NLCs formulations [10]. Liquid lipids such as oleic acid, olive oil (containing ethyl
creativecommons.org/licenses/by/
palmitate), corn oil (containing apocarotenal), and grape seed oil (containing linoleic acid)
4.0/).

Molecules 2023, 28, 1833. https://doi.org/10.3390/molecules28041833 https://www.mdpi.com/journal/molecules

Molecules 2023, 28, 1833 2 of 23

are combined with solid lipids to make NLCs. Normally, tween 80 and poloxamer 188 are
typically used as surfactants [11].
Curcumin is mostly derived from the natural plant. The richest and most cost-effective
source of crude curcumin is Curcuma longa [12]. It has also been used to treat a range of
problems in various patients, including diabetes, liver disease, cancer, and rheumatoid
disease [13]. Curcumin is only sparsely soluble in water [14]. Chemically, it is an unstable
molecule with a limited biological half-life because it is poorly absorbed and rapidly
metabolized [15]. All these factors contribute to curcumin’s lower bioavailability when
taken as a whole [16]. It is categorized as GRAS (generally recognized as safe) by the
US Food and Drug Administration [17]. The most attractive and vital reason for the
therapeutic use of curcumin is its superior safety profile. It has been demonstrated that
curcumin has a very low toxicity [18]. The bioactivity of orally taken curcumin has been the
most fundamental basis for this apprehension. Thus, it can be used as an anti-inflammatory,
anti-microbial, anti-oxidant, anti-viral, and anti-cancer agent [19–21].
In the last two decades, much research and many experiments were carried out to
investigate the optimal characteristics of the drug [22]. The previous trials were not effi-
cient and expensive [23], thus virtual screening was applied as a new approach based on
structural information [24]. The method of virtual screening can be classified as structure-
based and ligand-based drug designing methods [25,26]. The first one describes molecular
docking while the second method deals with the relationship between the quantitative
structure’s activity and pharmacophore modeling [27,28]. The structural information in-
creases the range of molecular targets of proteins and protein–ligand complexes via certain
steps, starting with chemical synthesis techniques, purification, X-ray crystallography, and
nuclear magnetic resonance spectroscopy (NMR) [29].
Generally, the molecular docking method determines the interaction between the
ligand and target molecule [30,31]. It can also predict the ligand binding affinity in the
formation of a stable complex with protein in minimum free binding energy [32,33]. The
binding can occur via non-covalent interactions such as hydrogen bonds, ionic bonds,
hydrophobic, and van der Waals forces [34].
The current research proposed the development of NLC molecules to improve the
oral solubility and permeability of curcumin. The research also focused on developing
a computational molecular docking to study the solubility of curcumin in different solid
and liquid lipids to choose the optimum candidates for the NLCs formulation rather than
the frequent experimental method, which consumes much time, efforts, and high costs.
The molecular docking model was applied to study and enhance the antiviral activity of
curcumin against COVID-19 by studying the binding affinity to the host cell receptors of
the angiotensin-converting enzyme (ACE2) [35].

2. Results and Discussion

2.1. Molecular Dynamic Study for Curcumin Solubility and Interaction with Candidate Lipids
The docking study of curcumin with lipids was performed through PyRx software.
Eleven types of lipids: solid lipids (GMS, stearic acid, palmitic acid, sitosterol alcohol, and
tripalmitate) and liquid lipids (oleic acid, apocarotenal, ricinoleic acid, ethyl palmitate,
linoleic acid, and omega-3) were utilized to perform the docking study for the solubility
and interaction prediction between curcumin and the selected lipids. These were previously
tested and evaluated through laboratory experiments (solubility and partition study of
curcumin with lipids). Then, a comparison of the obtained results from the docking study
with the one obtained from the laboratory experiment to specify the degree of accuracy,
validity, and reliability for the applied docking system for solubility anticipation between
the curcumin and lipids was made.
For apocarotenal (obtained from corn oil) CID 5478003, the types of bond interaction
between apocarotenal and curcumin were examined. The results represent one hydrogen
bond as a carbon–hydrogen bond (distance 3.507) and six hydrophobic pi–alkyl bonds
(distance range 3.849–4.897). For omega-3 (obtained from soybean oil) CID 71306824, the
Molecules 2023, 28, 1833 3 of 23

types of bond interaction between omega-3 and curcumin were examined. The results
represent one hydrogen bond as a conventional hydrogen bond (distance 2.398), one
hydrogen bond as a carbon–hydrogen bond (distance 3.537), and three hydrophobic pi–
alkyl bonds (distance range 5.066–5.472). For linoleic acid (obtained from grape seeds
oil) CID 5280450, the types of bond interaction between linoleic acid and curcumin were
examined. The results represent one hydrophobic pi–sigma (distance 3.573) bond and three
hydrophobic pi–alkyl bonds (distance range 3.792–4.982). For ethyl palmitate (obtained
from olive oil) CID 12366, the types of bond interaction between ethyl palmitate and
curcumin were examined. The results represent one hydrophobic pi–sigma (distance 3.536)
bond and four hydrophobic pi–alkyl bonds (distance range 3.658–5.498). For ricinoleic acid
(obtained from castor oil) CID 14030006, the types of bond interaction between ricinoleic
acid and curcumin were examined. The results represent three hydrophobic pi–alkyl bonds
(distance range 3.879–5.082) as shown in Figure 1.

Figure 1. 3D molecular interaction between curcumin (CUR) with different liquid lipids (highlighted
in yellow) for solubility prediction. The formed bonds are described as black short lines.

Oleic acid shows the types of bond interaction between oleic acid and curcumin. The
results represent four hydrogen bonds (distance range 3.464–3.514) and twelve hydrophobic
pi–alkyl bonds (distance range 3.911–5.433). Figure 2 represents the 3D interaction between
the curcumin molecule and the oleic acid molecule with bond types and distances.

Figure 2. 3D interaction between curcumin molecule (gray) and oleic acid oil (yellow). The formed
bonds are described as black short lines.

Considering solid lipids (SL), the types of bond interaction between Cetostearyl alcohol
and curcumin were examined. The results represent four hydrophobic pi–alkyl bonds
(distance range 4.015–4.306), while the types of bond interaction between stearic acid and
curcumin were examined. The results represent four hydrophobic pi–alkyl bonds (distance
range 3.827–5.029). The types of bond interaction between palmitic acid and curcumin
were examined, and the results represent two hydrogen bonds (distance 3.545 and 3.643)
and four hydrophobic pi–alkyl bonds (distance range 3.654–5.101). The types of bond
Molecules 2023, 28, 1833 4 of 23

interaction between tripalmitin and curcumin were examined and the results represent
two hydrogen bonds as conventional hydrogen bonds (distance 2.664 and 2.701) and two
hydrophobic pi–alkyl bonds (distance 3.917 and 4.729), as presented in Figure 3.

Figure 3. 3D molecular interaction between curcumin (CUR) with different solid lipids (highlighted
in yellow) for solubility prediction. The formed bonds are described as black short lines.

As observed in Table 1, the types of bond interaction between GMS and curcumin
represent six hydrogen bonds as conventional hydrogen bonds, one hydrophobic pi–sigma
(C–H) bond, and one hydrophobic pi–alkyl bond (pi-orbital). Figure 4 represents the 3D
and 2D interaction between the curcumin molecule and GMS molecule, respectively; the
bonds type and distances were also described.

Table 1. Types of bonds for curcumin–GMS interaction.

Distance Category Type

2.44011 Hydrogen bond Conventional hydrogen bond
2.27678 Hydrogen bond Conventional hydrogen bond
2.99625 Hydrogen bond Conventional hydrogen bond
3.46712 Hydrophobic Pi–sigma
5.46068 Hydrophobic Pi–alkyl
2.44011 Hydrogen bond Conventional hydrogen bond
2.27678 Hydrogen bond Conventional hydrogen bond
2.99625 Hydrogen bond Conventional hydrogen bond

The scale of root means square deviation (RMSD) clustering for the solubility construal
docking for GMS was set to nine and was used to determine how well scoring combinations
pose and calculate the ligand (CUR) in the lipid site (GMS). Discovery Studio software
was used to interpret the obtained docking results. Molecular docking insights revealed
that CUR bound to GMS via six hydrogen bonds, one pi–sigma C–H hydrophobic bond,
and one pi–alkyl hydrophobic bond. The values for the binding energy of each lipid with
curcumin were obtained and used as an indicative tool to expect the stability of the formed
formulations and was approximately −8.6 Kcal/mol for GMS, which was significantly
lower than the other compared ten lipids (range from −6.5 to −1.8).
Molecules 2023, 28, 1833 5 of 23

Figure 4. 3D and 2D molecular interaction between curcumin molecule (gray) and GMS (yellow).

The lowest binding energy of GMS has the highest binding affinity to curcumin and
indicates that the ligand binds strongly to the receptor, which proves that GMS has the
highest solubility tendency for curcumin. BIOVIA Discovery Studio software confirmed
the interaction between CUR and GMS. It was reported that conventional hydrogen bond-
ing shows the strongest interaction between molecules and the results showed that six
conventional H-bonding were generated between CUR as the H-donner and GMS as the
H-accepter in the type of OH–H bond, which shows fundamental solubilizing services in a
biomolecular structure [36].
Hydrogen bonds perform as facilitators to CUR–GMS binding and offer an inordinate
chance for the solubilization of CUR in GMS since it can endorse the ligand binding
affinity among the donor and acceptor. It was conveyed that hydroxyl groups of GMS
play a crucial role in binding through hydrogen bonding generation and solubilizing CUR.
Therefore, this demonstrated that CUR could bind and have an affinity to GMS through
strong conventional hydrogen bonding [37]. In addition, the shortest distance of these
conventional hydrogen bonds compared to another type of bond present (less than 3000 ◦ A)
proves that the presence of such bonds gives a higher binding affinity and solubility
between curcumin and GMS molecules [38]. These findings were not reported with the
other compared 10 lipids. Moreover, the presence of a pi–alkyl bond in the interaction
between curcumin and GMS provides the stability for the system, and the presence of the
pi–sigma bond provides an extra stability to the interaction between the molecules [39]. On
the other hand, oleic acid was found to be the best liquid lipid candidate to be mixed with
GMS for the NLCs formulation since the high compatibility between these two lipids was
found experimentally and due to the obtained docking results, which showed that oleic
acid was superior compared to other liquid lipids in demonstrating a lower binding energy
of −6.5 kcal/mol. Moreover, the presence of four C–H bonds in the oleic acid–curcumin
interaction allows solubility [40].
A comparison between the predicted solubility of curcumin in different solid and
liquid lipids obtained from the molecular docking study (through calculating the binding
energy) and the actual solubility results obtained from laboratory experiments (by calculat-
ing the amount of curcumin dissolved in these lipids) was conducted to see how far this
computational docking system can be recommended for choosing the optimum solid and
liquid lipid. The results were promising and showed a similar order of solubility (from
highest to lowest) for both solid and liquid lipids, indicating the accuracy of the docking
study applied, as shown in Table 2.
Molecules 2023, 28, 1833 6 of 23

Table 2. Predicted versus actual results of curcumin solubility in different lipids.

Predicted Solubility Actual Solubility

Solid Lipids
(kcal/mol) (mg/g)
GMS −8.6 3.58 ± 0.03
Palmitic acid −4.8 3.28 ± 0.06
Tri Palmitin −2.9 3.25 ± 0.30
Stearic acid −2.4 2.62 ± 0.12
Cetosteryl −1.8 2.06 ± 0.13
Liquid lipids
Oleic acid −6.5 4.61 ± 0.13
Castor oil −5.3 3.52 ± 0.52
Olive oil −5.1 3.48 ± 0.34
Soybean oil −4.7 1.36 ± 0.26
Corn oil −4.4 1.06 ± 0.15
Grape seeds oil −3.9 0.59 ± 0.64

2.2. Formulation of Curcumin-Loaded Nanostructured Lipid Carriers

The influence of the process parameters, homogenization pressure, and speed is very
important for the production of NLC by the hot high-pressure homogenization (HHPH)
method [41]. The correct choice of both the pressure and speed would directly affect the
size of the nanoparticles [42]. For this purpose, the pressure range was taken in between 500
and 1000 bars, and the speed range was 10,000–15,000 rpm. To optimize the formulation of
curcumin NLC, the dependent variables including the entrapment efficacy (EE%) particle
size, ploy dispersity index (PDI), and zeta potential were evaluated to choose the best
formula. Table 3 shows the results of the formula obtained concerning the EE% P size,
PDI, and zeta potential. The outcomes showed that the particle size of the best selected
formulas (F1,F2,F4,F12, and F19) was less than 200 nm, which lies within the acceptable
range [43], the PDI was found to be ≤0.3, indicating a unimodal or a uniform mono
dispersion size distribution [44], the zeta potential results were more than 40, indicating a
good stability [45], and the EE% results were high, meaning that the majority of the drug
added during the formulation process was entrapped, resulting in less loss of valuable
active medicinal ingredients [46].

Table 3. Formulations results of EE%, particle size, PDI, and zeta potential.

F. Code EE% P. Size (nm) PDI Zeta (-)

F1 84.23 ± 1.35 99.64 ± 8.64 0.192 ± 0.01 42.3 ± 0.01
F2 80.11 ± 0.60 100.29 ± 5.16 0.172 ± 0.03 41.8 ± 0.13
F3 56.37 ± 2.13 164.06 ± 3.49 0.694 ± 0.06 23.4 ± 0.11
F4 83.41 ± 0.29 99.86 ± 4.16 0.164 ± 0.29 41.8 ± 0.34
F5 61.62 ± 3.17 190.64 ± 2.31 0.524 ± 0.17 29.1 ± 0.06
F6 70.09 ± 0.84 159.10 ± 3.19 1.135 ± 0.34 26.4 ± 0.08
F7 55.25 ± 3.73 305.04 ± 1.05 0.961 ± 0.19 24.7 ± 0.46
F8 49.19 ± 2.49 341.23 ± 2.43 0.659 ± 0.03 22.9 ± 0.61
F9 45.64 ± 0.51 251.11 ± 1.38 0.654 ± 0.24 20.4 ± 0.94
F10 60.73 ± 0.28 284.32 ± 0.67 0.829 ± 0.06 30.4 ± 0.31
F11 65.34 ± 0.61 268.06 ± 4.13 0.675 ± 0.14 34.9 ± 0.62
Molecules 2023, 28, 1833 7 of 23

Table 3. Cont.

F. Code EE% P. Size (nm) PDI Zeta (-)

F12 83.08 ± 0.13 123.29 ± 5.03 0.197 ± 0.28 41.8 ± 0.09
F13 68.09 ± 1.28 146.16 ± 1.29 0.829 ± 0.07 29.1 ± 0.37
F14 52.43 ± 1.42 193.43 ± 3.45 0.753 ± 0.03 32.5 ± 0.13
F15 62.39 ± 3.24 227.16 ± 4.07 0.761 ± 0.31 29.7 ± 0.16
F16 81.06 ± 1.06 116.22 ± 3.18 0.219 ± 0.62 41.8 ± 0.08
F17 49.43 ± 2.67 237.24 ± 5.10 0.894 ± 0.37 28.1 ± 0.27
F18 52.57 ± 2.61 249.70 ± 0.94 0.691 ± 0.09 20.6 ± 0.31
F19 82.31 ± 0.27 110.13 ± 0.07 0.168 ± 0.15 39.7 ± 0.82
F20 43.51 ± 3.16 312.13 ± 1.49 0.694 ± 0.20 25.1 ± 0.19

2.3. Compatibility Study of the Excipients Used in the NLCs Formulations

2.3.1. Fourier Transforms Infrared Spectra
Curcumin’s characteristic FTIR peaks were identified at 3502 cm−1 , indicating the
presence of the phenolic OH group. The prominent peak at 1629 cm−1 was identified
as a mixed peak of (C=C) and (C=O) character vibrations. The symmetric aromatic ring
stretching vibrations (C=C ring) have another high peak at 1602 cm−1 . The strong 1512 cm−1
peak is attributed to (C=O) olefinic C–H bending vibrations at 1344 cm−1 , while an enol
C-O band was produced at 1280 cm−1 , a C–O–C peak at 1114 cm−1 , and benzoate trans-
C–H vibration at 962 cm−1 . Figure 5 represents the FTIR spectrum of GMS and oleic acid.
In the FTIR spectra of the prepared curcumin NLCs-F1, there was no discernible change
in curcumin’s distinctive peaks (Figure 6). Furthermore, there was no evidence of peak
shifting.

Figure 5. FTIR spectra of (A) GMS and (B) oleic acid, plot of %Transmittance vs. 1/cm.

Figure 6. FTIR for pure curcumin (A), the optimized formulation of curcumin–NLC F1 (B), which
shows no chemical interaction with curcumin major peaks, plot %Transmittance vs. 1/cm.
Molecules 2023, 28, 1833 8 of 23

The lack of additional peaks/broadening of the peaks suggests that the process param-
eters did not cause the curcumin to be stressed, resulting in degradation. Curcumin also
had no chemical interactions and was confirmed to be compatible with the formulation
excipients [47,48].

2.3.2. Differential Scanning Calorimetry

At 180.55 ◦ C, pure curcumin’s melting endothermic peak was observed that complied
with similar findings [49,50]. Moreover, other formula excipients like lipids, ethanol,
poloxamer 188, and tween 80 showed a thermogram that proves the compatibility of
curcumin with all excipients used for the formulation. By detecting the fluctuation of the
temperature and energy during the phase transition, DSC was an approach for investigating
the crystallization or amorphous behavior of medication in pure and NLCs. The melting
process for PRE reached a maximum peak temperature of 60.93 ◦ C. The melting peak
for the CUR, on the other hand, was not visible in the thermogram of the lyophilized
CUR–NLCs with the presence of an oleic acid peak at 240 ◦ C. The CUR entrapment in
hydrophobic voids of GMS could explain the lack of a distinctive endothermic peak in
the DSC thermogram of NLCs. Figure 7 showed the DSC thermogram of the selected
formula F1 in comparison to pure curcumin. The removal of CURs endothermic peak in
CUR–NLCs powder shows that the drug was molecularly disseminated in the lipid matrix
and transformed from a crystalline to an amorphous state [47,51].

Figure 7. DSC studies of pure curcumin (A), the prepared curcumin NLCs-F1 (B) with the absence
melting peak of curcumin at 180 ◦ C confirming amorphous state plot of mW vs. temp.

2.3.3. X-ray Diffraction Spectra (XRD)

The XRD patterns of pure drug and freeze-dried drug-loaded NLCs F1 were inves-
tigated. Curcumin peaks were absent or had a very low or negligible intensity in NLCs,
indicating a decline in freeze-dried curcumin’s crystallinity. NLCs with broad and low-
intensity peaks have a weak crystalline character. This was due to curcumin’s trapping
within the NLCS. The XRD analysis of the curcumin shows that the pure substance has
a very high crystallinity with an intense sharp peak. The crystallinity of the drug was
significantly reduced in NLCs, indicating curcumin entrapment within the lipidic core.
This may have been achievable because of curcumin’s high solubility in the solid lipid
(GMS) which was utilized [52]. Figure 8 shows the XRD analysis of the selected formula F1
in comparison to pure curcumin.

Figure 8. Powder X-ray diffraction studies of pure curcumin (A), and curcumin NLCs-F1 (B) presenting
significant lowering intensity due to high amorphization, the plot of Counts vs. Theta-2 Theta (deg).
Molecules 2023, 28, 1833 9 of 23

2.4. Evaluation of Curcumin–NLC Formulation

2.4.1. Determination of Drug Content and Incorporation of Efficacy
The NLCs were broken using methanol, and the encapsulated drug was recovered for
examination. The curcumin incorporation efficacy of the formulations investigated was
moderate to high, ranging from 45.32 ± 0.28% to 83.45 ± 0.13% (w/w) with the highest
content found in the optimum F1 84%, which was considered an acceptable percent [53,54].
The % medication concentration in all the NLCs formulations ranged from 85.67 ± 2.34 to
95.68 ± 2.38.

2.4.2. Particle Size and Poly-Dispersity Index (PDI)

The PDI of curcumin-loaded optimized NLCs revealed a system that was display-
ing substantial polydispersity. It displays the PDI of 0.192 and the particle size of the
F1 formulation (99.64 nm). Because of their non-ionic nature, polyhydroxy surfactants
stabilize the formulation by generating a spatial exclusion, resulting in low and nearly
PDI with the highest particle dispersion [55]. A PDI score of 0.10 to 0.40 indicates that the
system is considerably poly-dispersed [56]. Moreover, the low particle size provides a high
surface area of contact with the dissolution medium, resulting in the enhanced solubility of
curcumin [57].

2.4.3. Zeta Potential

The results of the zeta potential for curcumin-loaded optimized NLCs F1 was
−42.30 ± 0.01, indicating a good stability behavior of the colloids. Surfactant, lipid, or
combined surfactant and lipid may be responsible for the surface charge. Tween 80 and
poloxamer 188 were utilized as the surfactants and stabilizer in the manufacture of NLCs.
Tween 80 is a nonionic surfactant, which means it does not affect the particle surface charge
(zeta potential). Poloxamer 188, on the other hand, is a nonionic block linear copolymer-
type stabilizer, and its negative charge contributes to the particle polarity. The lipid phase
of the NLCs was made up of GMS and oleic acid. GMS is a long-chain fatty acid glycerol
ester that does not affect the surface charge, whereas oleic acid is a medium-chain fatty acid
whose negative surface charge related to the carboxyl group may contribute to the zeta
potential [58]. As a result, a zeta potential of more than −40 mV reflects a good stability
behavior of the prepared colloid system [59].

2.4.4. EE Percent and Drug Loading

When the amount of GMS was raised in curcumin-NLCs, the EE improved. However,
it was dropped when the oleic acid concentration was reduced, and the EE was shown to
be lower. This may be related to lipid precipitation, which happens during the creation
of particles. When NLCs are cooled after being made, the lipids recrystallized, resulting
in a drug-free core or a core with a reduced drug content. As a result, a lipid rise beyond
a certain point causes a poor EE. Tween 80 was also found to have a substantial effect.
The EE of the formulation rose as the concentration of tween 80 was increased. The EE of
the NLCs dispersion was found to be F1, F2, F4,and F12, and F19 had a percentage EE of
84.23 ± 1.35, 80.11 ± 0.60, 83.41 ± 0.29, 83.08 ± 0.13, and 82.31 ± 0.27%, respectively. A
factorial design was applied to optimize the prepared NLCs and assess their critical quality
attributes through EE [60]. Figure 9 shows that the increase in the lipid concentration and
media time sonication will increase the EE. The response surface central composite was
developed (CCD) in design expert software (Version 10.0.1, Stat-Ease Inc., Minneapolis,
MN, USA) was used to design the experiment, which fitted to the quadradic model using
Factorial Design-Expert software [61].
Molecules 2023, 28, 1833 10 of 23

Figure 9. Counter and response surface plots of entrapment efficiency (stirring time and lipid
concentration), n = 6.

2.5. In Vitro Drug Release

The curcumin release potential from the lipid particles was tested for 24 h. A spec-
trophotometric approach was used to analyze each sample in triplicate. In vitro tests were
conducted to compare the release properties of pure curcumin and the NLCs (Figure 10).
The time taken for an aliquot time interval of medication to be released showed that the
dissolution rate was increased in NLCs. Formula F1 had a burst release pattern at first, with
more than 50% of the medication being released within 6 h, whereas pure curcumin took
more than 24 h to dissolve 50% of the curcumin. When compared to other formulations,
NLCs formulation F1 had the smallest particle size when compared to pure curcumin and
the other formulations F2, F4, F12, and F19, which showed a higher release rate in the
dissolution media. As a result, this formulation was chosen for further testing [62,63].
The in vitro drug release research was conducted at 37 ◦ C for 24 h in a phosphate
buffer (pH 6.8). Because curcumin is a lipophilic medication, it was added to the receptor
medium to keep the sink conditions constant. The data clearly show that NLCs released
the medication significantly more efficiently than a simple curcumin suspension. This can
be ascribed to the phospholipidic nano formulations’ nanosized particles. Nanoparticles
have a larger surface area, which improves the particle interaction with the dissolving
liquid. Due to the release of curcumin adsorbed on the surface of nanoparticles, all of
the curcumin-loaded NLCs showed a burst drug release over the first 4 h. Following
that, the release of curcumin from the nanoparticles showed a regulated release pattern,
with roughly 85% of the medication being released in the F1 formulation for up to 12 h,
compared to ordinary curcumin. It could be attributed to the fact that curcumin, which
is contained deep within the lipidic core and must travel along a longer diffusion path to
reach the surface than a medication encapsulated near the surface, is hydrophobic [64]. The
increased drug release from the F1 formulation compared to the other formulations (F2,
F4, F12, and F19) could be attributed to the much smaller particle size, which increases the
surface area and, as a result, the drug release rate [65,66].

Figure 10. Comparison of cumulative % drug release NLC formulas (F1, F2, F4, F12, and F19) and
pure curcumin suspension versus time (h), mean ± SD (n = 6).

2.6. Morphological Analysis

The AFM image was obtained with a scan rate of 0.5 Hz over a selected area in the
dimension of 3 × 3 µm. The force applied to the surface was roughly adjusted by a set-point
Molecules 2023, 28, 1833 11 of 23

of 16 nm and an amplitude of 26.57 nm. An atomic force microscopy examination in 3D

imaging presented a round spherical shape of the prepared nanoparticles [67]. The particle
size in the AFM micrographs of nanoparticles is somewhat bigger than in the dynamic
light scattering micrographs, which is likely owing to the flattening of the NLCs during
the drying phases in the sample preparation for AFM imaging (Figure 11) [68]. The phase
image confirmed the stability of the NLCs formulation due to the bright surfaces, which
indicates positive phase shift–repulsive forces between the tip and samples, suggesting the
good hydration of the lipid [15,69]. Curcumin-loaded nanoparticles had a similar size of
99.64 ± 8.64 nm. The attendance of a spheroidal shape, closer to a disc than a sphere, is
confirmed by the reduced nanoparticle height reported by AFM. As previously stated, this
structure validates the frequency of the polymorphic form of lipids, which is linked to a
high loading capacity and a low tendency to expulse the encapsulated drug from the lipid
matrix [70].

Figure 11. Atomic force microscopy image of optimum curcumin–NLC F1 determining the morphol-
ogy of the prepared nano lipids (size area 3 × 3 µm).

2.7. In Vitro Gut Permeation Study

Figure 12 depicts the intestinal permeability of a curcumin suspension and curcumin-
NLCs. The permeability of a curcumin suspension and curcumin NLCs through the rat gut
was reported to be 6.83 ± 1.53 (Papp of 0.488 × 10−5 cm/s) and 42.52 ± 3.15 mcg/cm2 /h
(Papp of 6.84 × 10−5 cm/s), respectively, after 8 h. At p ≥ 0.05, there was a substantial
difference in the permeability between the curcumin NLCs formulation and curcumin sus-
pension. Smaller particle sizes and the incorporation of permeation enhancers (poloxamer
188 and tween 80) resulted in an increased drug penetration via curcumin NLCs, according
to these findings [71].

Figure 12. Intestinal permeation study representing permeation of curcumin suspension and
curcumin-NLCs versus time (h), mean ±SD (n = 6).

2.8. Molecular Docking Studies of the Anti-Viral Activity of Curcumin NLC against SARS-CoV-2
(COVID-19)
The docking scores of the binding affinity between the different host cell receptors
and curcumin in the curcumin–NLC complex are tabulated in Table 4. A comparison was
applied for the obtained docking results of the curcumin interaction with the host cell
Molecules 2023, 28, 1833 12 of 23

receptors of ACE 2 and the curcumin interaction with NLC in the curcumin–NLC–host
cell receptor complex to study the influence of the formed curcumin–NLC on the binding
affinity for the target host cell receptors. First, the PyRx software was applied for the
docking of curcumin with the NLC components and it was then explored through the
Discovery Studio visualizer. Again, the obtained micelles were subjected to docking with
the three host cells through the same process as described earlier.

Table 4. The docking scores of curcumin with different targets (host cell receptors) in the curcumin–
NLC complex.

Target Binding Energy (kcal/mol)

ACE 2 for ACE2 Receptors–Curcumin in Complex
7KMB −9.1
7KNB −8.6
7KNH −8.4

The anti-viral potential of curcumin against COVID-19 using different targets was
investigated. The results showed a significant reduction in the binding energy, indicating
an enhancement in the binding affinity of the ligand toward the targets. The curcumin
interaction with NLC in a complex has shown an interaction potential and exhibited
−6.8 kcal/mol binding energy and formed one conventional H-bond with C: ASP994, one
carbon–hydrogen bond C: GLY999, two pi–sigma C: THR998 and A: THR998, and five pi–
alkyl bonds B: VAL991, C: PHE970, A: TYR756, B: ARG995, and C: ARG995. Tables 5 and 6
epitomize the results of the data obtained from the active residues, bond length (A0), bond
types, and bond categories involved in the molecular interactions of curcumin with NLC
and curcumin with different receptors (7KMB,7KNB, and 7KNH) in the curcumin–NLC–
receptor complex.

Table 5. The active residues, bond length (A0), bond types, and bond categories involved in the
molecular interactions of curcumin with NLC in a complex.

Bond Length (A0) Bond Category Bond Type

2.90749 Hydrogen bond Conventional hydrogen bond
3.5423 Hydrogen bond Carbon hydrogen bond
3.2796 Hydrophobic Pi–Sigma
3.8657 Hydrophobic Pi–Sigma
5.31932 Hydrophobic Alkyl
5.12768 Hydrophobic Pi–alkyl
4.84944 Hydrophobic Pi–alkyl
4.1316 Hydrophobic Pi–alkyl

Table 6. The active residues, bond length (A0), bond types, and bond categories involved in the
molecular interactions of curcumin with ACE2 receptors (7KMB, 7KNB, and 7KNH) in a complex.

Bond Length (A0) Bond Category Bond Type

Curcumin–7KMB receptor in a complex
2.58816 Hydrogen bond Conventional hydrogen bond
2.03326 Hydrogen bond Conventional hydrogen bond
1.99813 Hydrogen bond Conventional hydrogen bond
2.08162 Hydrogen bond Conventional hydrogen bond
Molecules 2023, 28, 1833 13 of 23

Table 6. Cont.

Bond Length (A0) Bond Category Bond Type

2.21662 Hydrogen bond Conventional hydrogen bond
2.95087 Hydrogen bond Conventional hydrogen bond
4.14296 Electrostatic Pi–anion
5.1296 Hydrophobic Pi–Pi Stacked
5.35067 Hydrophobic Pi–alkyl
Curcumin–7KNB receptor in a complex
2.07633 Hydrogen bond Conventional hydrogen bond
2.07392 Hydrogen bond Conventional hydrogen bond
2.71198 Hydrogen bond Conventional hydrogen bond
2.63368 Hydrogen bond Conventional hydrogen bond
2.60406 Hydrogen bond Conventional hydrogen bond
3.01875 Hydrogen bond Conventional hydrogen bond
3.71325 Hydrogen bond Carbon hydrogen bond
4.66974 Hydrophobic Amide–Pi Stacked
4.4217 Hydrophobic Alkyl
3.88384 Hydrophobic Pi–alkyl
4.12778 Hydrophobic Pi–alkyl
Curcumin–7KNH in a complex
2.68924 Hydrogen bond Conventional hydrogen bond
2.75846 Hydrogen bond Conventional hydrogen bond
2.39584 Hydrogen bond Conventional hydrogen bond
2.44114 Hydrogen bond Conventional hydrogen bond
2.66824 Hydrogen bond Conventional hydrogen bond
3.40111 Hydrogen bond Carbon hydrogen bond
3.56468 Hydrogen bond Carbon hydrogen bond
3.74797 Hydrogen bond Carbon hydrogen bond
4.00134 Electrostatic Pi–anion
4.55541 Hydrophobic Alkyl

For the 7KMB receptor, the curcumin interaction with the receptor in a complex
represents a significant enhancement in the binding affinity through an energy reduction
to −9.1 kcal/mol; the interaction also formed an additional four H-bonds compared to
the two H-bonds formed for curcumin with NLC, indicating an increase in the curcumin
affinity towards the receptor. The formed conventional H-bond showed a great increase
through six formed bonds F: TYR202, F: SER511, F: SER511, F: ARG514, F: LYS562, and F:
GLY395. Moreover, pi–anion, pi–pi stacked, and pi–alkyl were formed with F: ASP206, F:
TYR202, and F: TYR202, respectively (Figure 13).
For the 7KNB receptor, interestingly, the curcumin interaction with the receptor in
a complex represents a strong enhancement of the binding affinity through an energy
reduction to −8.6 kcal/mol and formed a new extra five H-bonds, indicating a high increase
in the curcumin affinity towards the receptor. The interaction showed six conventional
H-bonds C: THR998, A: ARG995, A: ARG995, A: ARG995, B: ARG995, and B: VAL991, one
carbon–hydrogen bond C: ASP994, one amide pi-stacked bond A: ASP994:C, O;ARG995,
and 3 pi–alkyl bonds C: VAL991, A: ARG995 and B: ARG995 (Figure 14).
Molecules 2023, 28, 1833 14 of 23

Figure 13. 2D interaction of curcumin–NLC with ACE 2 binding receptor (7KMB) in a complex.

Figure 14. 2D interaction of curcumin–NLC with ACE 2 binding receptor (7KNB) in a complex.

For the 7KNH receptor, a study of the curcumin interaction with the receptor in a
complex represents an enhancement in the binding affinity through an energy reduction
to −8.4 kcal/mol and formed new additional six H-bonds and carbon–hydrogen bonds,
indicating a high increase in the curcumin affinity towards the receptor. The interaction
showed five conventional H-bonds C: GLN755, B: ASN969, B: ARG995, B: ARG995, and B:
GLY971, three carbon–hydrogen bonds C: ASP994, B: ALA972, and B: ILE973, one pi–anion
bond C: GLU990, and one pi–alkyl bond C: LEU752 (Figure 15).
The results in Table 6 show a significant tendency of the binding affinity of curcumin
to the three binding receptors of ACE2 when formulated as an NLC due to the low binding
energy and the increased H-bonding [72]. Consequently, these increase the antiviral
property of curcumin against SARS-CoV-2 and disrupt SARS-CoV-2 binding to ACE2
binding receptors and prevent their entry into the cell. It is anticipated that enhancing the
curcumin solubility through the NLCs formulation will provide a suitable concentration of
the medicine for its absorption and bioavailability, which enhances the curcumin’s anti-viral
activity in the treatment of COVID-19 [73].

Figure 15. 2D interaction of curcumin–NLC with ACE 2 binding receptor (7KNH) in a complex.

3. Materials and Methods

3.1. Materials
Curcumin, stearic acid, palmitic acid, and sitosterol alcohol were obtained from Sigma-
Aldrich, St. Louis, MO, USA. Glyceryl monostearate (GMS), poloxamer 188, and tween 80
were obtained from Research-lab Fine Chem Industries, Mumbai, India. Oleic acid and
tripalmitin were kindly received as gifts from TCS Chemicals, Mumbai, India.
Molecules 2023, 28, 1833 15 of 23

3.2. Molecular Dynamic Study for Curcumin Solubility and Interaction with Solid and
Liquid Lipids
The interaction of curcumin (CUR) lipids for the solubility prediction [SDF, PDB]
was studied. Low molecular weight curcumin was derived from the PubChem website
(https://pubchem.ncbi.nlm.nih.gov/, accessed on 3 January 2023) and downloaded as
SDF and converted to pdb format using Discovery Studio Software (version- 19.1.0.18287).
Eleven lipid structures including solid lipids (GMS, tripalmitin, stearic acid, palmitic acid,
and sitosterol alcohol) and liquid lipids (oleic acid, apocarotenal, ethyl palmitate, omega-3,
ricinoleic acid, and linoleic acid) were also downloaded as SDF from the PubChem website
and prepared for docking through the same process as curcumin. All the structures of the
selected compounds that aided as molecules for modeling studies were adjusted using the
Auto-Dock software (PyRx software version 0.8, python.exe) before docking. Using PyRx
software, the lipid molecules were subjected to auto-dock to build a macromolecule.
The ligand curcumin was dragged to the software in SDF format and subjected to
minimizing energy (E = 234.8) and was saved as the pdbq format. After selecting each lipid
molecule involved (which was used in experimental laboratory studies in this project) and
ligand, run vina was applied to study the interaction between lipid and ligand curcumin
through PyRx software. The docking outcomes were scrutinized to recognize and assess
the binding capability between these compounds. Discovery Studio software was used to
analyze the type of bond interaction between lipid and curcumin and we chose the best
fit interaction for the optimum solubility. Auto-dock vina in PyRx software is the most
preferable software for molecular docking. The molecular docking approach can be used to
model the interaction between small molecules and proteins at the atomic level, allowing
for the characterization of the behavior of small molecules in the binding site of target
proteins, as well as elucidating the fundamental biochemical processes [74,75]. Discovery
Studio software is an agglomeration to transcribe small molecules and macromolecule
systems. It is developed by Dassault Systems BIOVIA (Accelrys) [76]. Discovery Studio is a
single unified, graphical interface for advanced drug design and protein modeling research.
This software provides a plethora of viewers for display plots and graphical representations
of data [77]. In the present study, there was an investment in this software to predict the
interaction and solubility between hydrophobic drugs such as curcumin and lipids.

3.3. Formulation of Curcumin-Loaded Nanostructured Lipid Carriers

A hot high-pressure homogenization method was used to make NLCs. The first step
was to mix 200 mg of glycerol monostearate with 200 mg of oleic acid at 80 ◦ C. Then,
10 mg of curcumin was mixed at the same temperature, dispersing a solution of poloxamer
188 (1% w/v) and tween 80 (1% w/v). The surfactant phase was achieved by heating the
lipids utilized in the study over their melting point. For 15 min, a machine that stirred at
15,000 rpm mixed the lipids and surfactants. It was homogenized three times at 1000 bar
pressure and 80 ◦ C. A nanoemulsion was made and when it cooled, it turned into a mixture
of lipid nanoparticles in water. Different formulas were prepared for the NLCs production
using high-pressure homogenization and ultra-sonication to prepare 20 formulas, as shown
in Table 7. A total of 10 mg of curcumin was used in all the formulas with different lipids
percent (GMS and oleic acid) using different amounts of surfactants (poloxamer 188 and
tween 80) along with a different stirring time.

Table 7. The composition of the prepared NLC.

F. Code Drug (mg) Solid Lipid % Liquid Lipid (%)

F1 10 1 1
F2 10 2 2
F3 10 3 3
F4 10 4 4
Molecules 2023, 28, 1833 16 of 23

Table 7. Cont.

F. Code Drug (mg) Solid Lipid % Liquid Lipid (%)

F5 10 5 5
F6 10 1 2
F7 10 2 4
F8 10 3 6
F9 10 4 8
F10 10 5 10
F11 10 2 1
F12 10 4 2
F13 10 6 3
F14 10 8 4
F15 10 10 5
F16 10 3 1
F17 10 6 2
F18 10 9 3
F19 10 12 4
F20 10 15 5
Solid lipid and liquid lipid = 1:1, 1:2, 2:1, and 3:1%.

3.4. Compatibility Study of the Excipients Used in the NLCs Formulations

For the estimation of the compatibility among the component of the NLCs formulation,
FT-IR, DSC, and XRD studies were carried out. In this study, the spectra of all the individual
ingredients and the NLCs formulation were taken. The IR spectra were taken by an FT-IR
instrument (OPUS, Bruker, ALPHA, Billerica, MA, USA) in a region of 400 to 4000 cm−1 and
with a number of scanning of 32. A few amounts of the powder samples (further grinded for
homogenization) were placed in thin KBr disks and compressed using a minipresss machine
to prepare them for reading. Differential scanning calorimeter (ARKS TA, PerkinElmer,
DSC 4000, Waltham, MA, USA) curves were collected. For the calibration of the instrument,
indium was used. Approximately 10 mg of the powder sample was taken in an alumina
crucible and placed in the DSC. Then, over a temperature range of 20 ◦ C to 520 ◦ C within a
nitrogen rich environment, it was heated at a rate of 10 ◦ C/min. The DSC thermograms
were then visually analyzed [78]. An X-ray diffractometer was used to analyze the samples
during a period of 2 h with an angle of 10◦ to 80◦ . The X-ray diffraction (XRD) examination
of the NLCs was carried out using SPEC, Bruker model D8 ADVANCE X-ray diffractometer
(Hamburg, Germany), using Cu-K radiation as the source of X-rays at a 40 kV working
voltage and a 250 W electricity set was employed. At a 5◦ /min scanning speed and 0.02◦
step size, the samples were scanned in the range of 3–50◦ ± 2◦ .

3.5. Evaluation of Curcumin NLC Formulation

3.5.1. Drug Content
When 10 mg equivalent NLCs were dispersed for 15 min in triton X-100 (0.1% in
methanol) and centrifuged at 15,000 rpm for 30 min, the amount of medicine in the dis-
persion was assessed. Additionally, the visible spectrum at 425 nm was found after being
diluted with methanol. A Shimadzu-LC 2030 HPLC and a standard graph of the drug
produced in methanol were used to measure the drug concentration. The HPLC system
is equipped through a Shimadzu LC-10AD VP pump, a C18 Agilent column (Inertsil,
150 mm × 4.66 mm, 5–VIS detector), and a flow rate of 1 mL/min; the mobile phase has
been degassed. It is made up of an acetonitrile, water, and methanol (50:10:40) ratio. The
Molecules 2023, 28, 1833 17 of 23

detector wavelength was set at 425 nm. The injection volume was 10 µL. Methanol was
used as the diluent.The lipid-specific NLCs blanks were all the same [79].

3.5.2. Particle Size and Particle Size Distribution (PDI)

The polydispersity index (PDI) and mean particle size were measured using the
dynamic light scattering of NLCs F1-NLCs F20 (DLS Zetasizer, Nano ZS Malvern, Worces-
tershire, UK). Non-invasive backscatter technology was used to detect light scattering
at 173◦ (detector angle), measuring the particle size in a range of approximately 0.6 nm
to 6 µm. A foldable polycarbonate capillary cell was used to conduct the measurement
in a 50 V applied field. Before the analysis, to achieve a homogeneous dispersion, the
dispersions were diluted (1:200) in 0.22 m filtered double-distilled water. Three different
batches of curcumin NLCs were used to determine the mean particle size and PDI for
each concentration. The mean particle size standard deviation of three determinations was
obtained for each sample. The entire analysis was conducted at a temperature of 30 ◦ C.

3.5.3. Zeta Potential (ζ)

The zeta potential, also known as the surface charge potential, was estimated using the
Horiba nanoparticle size analyzer (SZ-100 nanoparticle series). Diluted NLC dispersions
were injected into the probe using an 80 mV electric field electrophoretic cell. All measure-
ments were made in triplicate at 25 ◦ C. The zeta potential was then directly determined
from the equation by using the Smolochowski equation as follows [80,81].

ζ = εµ/η

where ζ is the zeta potential, µ is the electrophoretic mobility, ε is the electric permittivity
of the liquid, and η is the viscosity of the liquid.

3.5.4. Entrapment Efficiency and Drug Loading

The Entrapment efficiency EE can be calculated using either the encapsulated or the
unloaded bioactive. This can be done with a hydrophilic bioactive by centrifugation for
10 min at 13,000 rpm, which yields an unloaded bioactive filtrate, which can be used to
determine the EE.

(weight o f drug added − f ree drug)

% Entrapment e f f iciency = × 100
(weight o f drug added)

3.6. In Vitro Drug Release Study

In vitro drug release studies on pure curcumin and curcumin NLCs were carried
out to determine the best formulation for further study. The dialysis bag’s molecular
weight threshold of 12–14 KDa was used to determine the in vitro drug release from the
formulations [35,82]. In this experiment, 10 mg of pure curcumin and the selected best five
NLCs formulas were inserted into a dialysis bag, which was suspended in a 250 mL beaker
filled with 100 mL of synthetic intestinal fluid (pH 6.8, phosphate buffer, tween 80) and
placed on a magnetic stirrer that spun at 100 revolutions per minute and was maintained at
a temperature of 37 ◦ C. Regularly, new dissolution medium samples were taken from the
dissolution medium and replaced. A volumetric flask filled with 10 Ml of methanol was
treated with 1 mL of the filtrate after it was filtered through Whatman filter paper No. 1.
As needed, additional dilutions were made. A spectrophotometric technique was used to
analyze the materials. Calculations were made with the Korsmeyer and Peppas model and
the PCP Disso Version 2.08 programmed [83–88] to determine the time needed for 50 and
85% drug release, respectively.
Molecules 2023, 28, 1833 18 of 23

3.7. NLCs Morphology Determination by AFM

Atomic force microscopy (AFM) was used to obtain high-resolution 3D images and it
provides more information about a sample’s mechanical and electrical properties, such as
the stiffness and adhesion strength. The samples were not coated. AFM was used to study
the morphology and particle size of the formed NLCs [89]. The studies were conducted in
contact mode in the air at an ambient temperature (25 ◦ C). On a tiny mica disc, droplets
of the final suspension (20 µL) were deposited and rinsed with Milli-Q water and dried
under a nitrogen flow after 5 min. The measurements were carried out in non-contact
mode using the high-resonance AFM cantilever (ACTA probe, n = 330 kHz) on an XE-100
instrument [90].

3.8. In Vitro Gut Permeation Study

The experimental protocol was approved by the Institutional Animal Ethical Com-
mittee (IAEC) with approval number CPCSEA/IAEC/JLS/16/9/21/13. Experiments on
a fasted Wistar rat’s (weighing 250–300 g) small intestine were conducted in a labora-
tory setting. An intestinal segment was removed from a dead rat after euthanasia using
CO2 inhalation and it was cut into lengths of 6–7 cm. Curcumin-NLCs and curcumin
suspension were added to the Krebs Ringer solution used to flush the stomach, and the
other end of the syringe was used to inject 1 mL of each formulation into the Sac. In a
beaker heated to 37 ◦ C, the sac was submerged in an oxygenated Krebs Ringer solution
(20 mL). The sink was maintained by removing and replacing aliquots of 1 mL every 0.5,
1, 1.5, 2, and 8 h with an equivalent volume of warm Krebs Ringer solution heated to
37 ◦ C. Each aliquot’s medication concentration was measured using HPLC (n = 3). The
HPLC system is equipped through a Shimadzu LC-10AD VP pump, a C18 Agilent column
(Inertsil, 150 mm × 4.66 mm, 5–VIS detector), and a flow rate of 1 mL/min; the mobile
phase has been degassed. It is made up of an acetonitrile, water, and methanol (50:10:40)
ratio. The detector wavelength was set at 425 nm. The injection volume was 10 µL. The
samples were measured after diluting suitably with methanol. Each formulation’s flow
and permeability coefficients were calculated [91]. The Curcumin permeability coefficient
(Papp) was computed from the mucosal to the serosal direction using the equation:
 cm   DQ 
Papp = ( AxCO)
sec dt
The DQ/dt is the rate of drug permeation from the tissue, A is the cross-sectional part
of the tissue, and Co is the initial curcumin concentration in the donor compartment at t = 0.

3.9. Molecular Docking Studies of the Anti-Viral Activity of Curcumin against SARS-CoV-2
(COVID-19)
The molecular docking was conducted on the Lenovo ThinkPad T440p using the
PyRx-Virtual Screening Tool. The structure of curcumin (sdf file format) was downloaded
from the official website of the National Center for Biotechnology Information PubChem
(https://pubchem.ncbi.nlm.nih.gov/, accessed on 3 January 2023). The energy minimiza-
tion (optimization) was performed by a Universal Force Field (UFF). The 3D structure of
curcumin–NLC constituents GMS, oleic acid, poloxamer 188, and tween 80 were obtained
from drawing these chemical structures through ChemSchetch software and transferred
from 2D to 3D and saved (as.mol file format). Then, they were converted to PDB format
using the Discovery Studio visualizer 2019.
These structures were prepared for docking through PyRx software. The three host
cell receptor structures of ACE2 (PDB ID: 7KMB, 7KNB, and 7KNH) were obtained from
the RCSB PDB site (https://www.rcsb.org/, accessed on 3 January 2023). The receptor
structures, with the aid of the Discovery Studio Visualizer 2021, were optimized, purified,
and prepared for molecular docking. Autodock vina 1.1.2 in PyRx 0.8 was used to perform
the molecular docking studies. A large number of glycosylated S proteins cover the surface
of SARS-CoV-2 and bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2),
Molecules 2023, 28, 1833 19 of 23

mediating a viral cell entry. Once the virus enters the cell, the viral RNA is released.
Polyproteins are translated from the RNA genome, and the replication and transcription
of the viral RNA genome occurs via the protein cleavage and assembly of the replicase–
transcriptase complex. Viral RNA is replicated, and structural proteins are synthesized,
assembled, and packaged in the host cell, after which viral particles are released [92]. For
molecular docking, both ligands (PDBQT files), as well as the targets, were selected. The
active binding receptors were examined and lightened using the BIOVIA Discovery Studio
Visualizer (version-19.1.0.18287).

3.10. Statistical Analysis

The results are expressed as the mean ± standard deviation (SD) (n = 6), and the sta-
tistical analysis was performed with GraphPad Prism 8 (GraphPad Software, Inc., La Jolla,
CA, USA). The significance difference between the experimental groups was evaluated by
the one-way ANOVA. The counter and response surface plots of the entrapment efficiency
(sonication time and lipid concentration) was evaluated using a quadric model (factorial
design).

4. Conclusions
The computational molecular docking denotes that the binding energy for GMS was
very low (−8.6 Kcal/mol), which indicates the highest binding affinity to curcumin and a
strong binding to the receptor with the highest solubility tendency for curcumin. Therefore,
glyceryl monostearate (GMS) was chosen as the best solid lipid with the highest drug
solubility among the screened solid lipids. Oleic acid was selected as a liquid lipid for the
NLCs formulation due to its high binding affinity to curcumin and compatibility with GMS
as well. The major absorption peaks of the FTIR with a prominent peak were observed at
1629 cm−1 as identified by the prepared NLC-curcumin. The reduced melting point in the
DSC detection and the XRD results shows a lower peak intensity of the curcumin–NLC
formula, indicating the amorphization of the curcumin crystals.
The low particle size of the novel NLC-curcumin provides a high surface area of
contact with the dissolution medium, which enhanced the solubility of curcumin. The
optimum NLCs formulas had an entrapment efficacy percent (EE%) = 84.23 ± 1.35. The
AFM image confirmed the spherical shape and the low particle size of the prepared NLC.
The novel NLC-curcumin shows an increase in the drug release from the best formula
compared to the other formulations. This could be attributed to the much smaller particle
size, which increases the surface area and, as a result, the drug release rate was increased
with the suspension of curcumin-NLCs. The permeability and penetration release rate in
the intestine and the gut was reported after 8 h and it was found to be 6.83 ± 1.53 and
42.52 ± 3.15 mg/cm2 /h, respectively, due to the small size of NPs and to the presence of
permeation enhancers (poloxamer 188 and tween 80). The future perspectives of the study
include the requirements of determination of the industrial feasibility of the proposed
delivery system, scale-up, and pilot plant studies.

Author Contributions: Conceptualization, P.V.K. and O.W.A.A.; methodology, O.W.A.A.; software,

O.W.A.A.; validation, P.V.K. and O.W.A.A.; formal analysis, O.W.A.A.; investigation, P.V.K., M.S.R.
and O.W.A.A.; resources, O.W.A.A.; data curation, O.W.A.A. writing—original draft preparation,
O.W.A.A.; writing—review and editing, P.V.K., M.S.R. and O.W.A.A.; visualization, O.W.A.A.; super-
vision, P.V.K.; project administration, P.V.K. and O.W.A.A.; funding acquisition, P.V.K. and O.W.A.A.
All authors have read and agreed to the published version of the manuscript.
Funding: This project was supported by the Fundamental Research Grant Scheme (FRGS) (Reference
code: FRGS/1/2021/ SKK0/UCSI/02/5), Ministry of Higher Education (MOHE), and the Research
Excellence and Innovation Grant (Project Code: REIG-FPS-2020-052) under Centre of Excellence in
Research, Value Innovation, and Entrepreneurship (CERVIE), UCSI University, Malaysia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Molecules 2023, 28, 1833 20 of 23

Data Availability Statement: Not applicable.

Acknowledgments: Many thanks to research colleagues for their kindness and support during the
research work.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not applicable.

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