Carbohydrate Polymers 208 (2019) 345–355

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

The role of intermolecular interactions on the encapsulation of human T

insulin into the chitosan and cholesterol-grafted chitosan polymers
⁎
Safoura Salar1, Majid Jafari1, S. Fatemeh Kaboli, Faramarz Mehrnejad
Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, 14395-1561, Tehran, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Chitosan and its derivatives used in drug delivery investigations could contribute to improving peptide and
Human insulin protein drug delivery systems. Herein, the molecular dynamics (MD) simulation approach was applied to
Chitosan evaluate the important driving factors of the human insulin encapsulation into the chitosan and cholesterol-
Cholesterol-modified chitosan modified chitosan polymers. The MD results revealed that the native conformation of insulin was stabilized by
Molecular dynamics simulation
the chitosan polymers. In the present study, the effect of cholesterol moieties of modified chitosan was also
Binding free energy
Protein drug delivery
examined and the results indicated that the cholesterol components would decrease the tendency of chitosan
polymers to human insulin. Further analyses showed that the intermolecular interactions between the tyrosine,
phenylalanine, and acidic residues are important in the formation of the insulin-polymer complexes. Another
interesting finding was that the van der Waals, electrostatic, and CH-π interactions play key roles in the en-
capsulation process. Generally, in the case of human insulin, the MD simulation results would seem to suggest
that the chitosan nanoparticles could be the more suitable carrier than the cholesterol-grafted chitosan nano-
particles.

1. Introduction
Nowadays, protein drug delivery has attracted increasing attention
since it has a significant role in improving a diverse range of diseases
treatment like cancer, diabetes, inflammatory diseases, and viral in-
fections (Herrera Estrada & Champion, 2015). The intrinsic physico-
chemical properties of therapeutic proteins like poor stability, high
clearance rate, high molecular weight, and susceptibility to enzymatic
degradation are obstacles to their potential performance (Amidi,
Mastrobattista, Jiskoot, & Hennink, 2010; Leader, Baca, & Golan, 2008;
Scaletti et al., 2018; Yun, Cho, & Park, 2013). To minimize the draw-
backs related to the protein and peptide-based drugs, several strategies
have been applied such as penetrating peptides, virus-like particles,
supercharged proteins, and biopolymer based nano-delivery systems
(Fu, Tang, Hardie, Farkas, & Rotello, 2014; Mukhopadhyay, Mishra,
Rana, & Kundu, 2012). In the drug delivery systems, the organic
polymeric nanoparticles have drawn more attention than the other
nanoparticles because of their biocompatibility, stability, non-im-
munogenic nature, and the ability to deliver different peptides and
proteins (Derman, Mustafaeva, Abamor, Bagirova, & Allahverdiyev,
2015; Letchford & Burt, 2007; Loverde, 2014; Makadia & Siegel, 2011;
Mir, Ahmed, & ur Rehman, 2017; Tomic, Vidis-Millward, Mueller-
Zsigmondy, & Cardot, 2016; Turino et al., 2017; Zhang, Chang et al.,
2017). Among various polymeric nanoparticles, chitosan and its deri-
vatives hold great promise for delivery of peptides and proteins, owing
to their unique properties (Sarmento et al., 2007; Xiong et al., 2016).
Chitosan, a cationic copolymer, consists of N-acetyl D-glucosamine
and D-glucosamine, varying in molecular weight. It is a biodegradable,
hydrophilic, and mucoadhesive polymer which is able to open the tight
junctions between epithelial cells (Alpar & Groves, 2006; Sari et al.,
2016). In addition, it involves functional groups, OH and NH2, which
allow chemical modification of the molecule for synthesizing of new
chitosan derivatives (Hoven, Tangpasuthadol, Angkitpaiboon, Vallapa,
& Kiatkamjornwong, 2007; Luo & Wang, 2014; Yuan, Jacquier, &
O’Riordan, 2017; Zhang, Pan, Dong, & Li, 2017). Different chitosan and
its derivatives-based delivery systems have been developed to enhance
the therapeutic effect of proteins and peptides. For example, Noh et al.
(2014) developed a novel chitosan hydrogel system for delivery of GM-
CSF, a monomeric glycoprotein that functions as a cytokine, in order to
improve safety and reduce toxicity (Noh et al., 2014). Kim et al. (2012)
reported that dual encapsulation of bone morphogenetic protein-2
(BMP-2) and insulin-like growth factor-1 (IGF-1) with chitosan gel/
gelatin microsphere (MSs) could augment the osteoblastic activity of
bone cells (Kim et al., 2012). Moreover, an experimental study revealed

⁎
Corresponding author.
E-mail address: Mehrnejad@ut.ac.ir (F. Mehrnejad).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.carbpol.2018.12.083
Received 8 October 2018; Received in revised form 4 December 2018; Accepted 24 December 2018
Available online 26 December 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
S. Salar et al. Carbohydrate Polymers 208 (2019) 345–355

Fig. 1. (a) The native conformation of human insulin is shown as a new cartoon model. Chain A and chain B are colored in green and gray, respectively. C6-C11, C7-
C28, and C20-C40 represent disulfide bonds between Cys6-Cys11, Cys7-Cys28, and Cys20-Cys40, respectively. The first frame of the trajectory of the chitosan (b),
chit_1chl (d), and chit_2chl (f) systems. (c), (e), and (g) show the last frame of the trajectory of the chitosan, chit_1chl, and chit_2chl systems, respectively. The
chitosan polymers and the cholesterol moieties are colored in magenta and blue, respectively (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article).

that N, N-Dimethyl-N-Octyl chitosan is a suitable delivery system for
oral delivery of insulin (Shamsa, Mahjub, & Mansoorpour, 2018). Silva
et al. (2018) offered a model describing the process of the nano-com-
plex formation between chitosan and insulin. The authors indicated
that the main driving force between chitosan and insulin is electrostatic
interaction (Silva et al., 2018).
The structural changes of protein adjacent to nanoparticles have
been measured by circular dichroism (CD) spectroscopy in most recent
studies. Agudelo, Nafisi, and Tajmir-Riahi, (2013) investigated the ef-
fect of chitosan nanoparticles on the secondary structure of
β‑Lactoglobulin by CD spectra. They exhibited that the presence of
chitosan had no major effect on the protein structure (a slight increase
of α-helix contents) (Agudelo et al., 2013). Li et al. (2012) reported a
decrease in the α-helical content and an increase in the β-strand content
of bovine serum albumin (BSA) upon the interaction with the 6-O-
cholesterol modified chitosan (Li et al., 2012). Furthermore, computa-
tional studies investigated the conformation of proteins after com-
plexation with nanoparticles. Yahyaei, Mehrnejad, Naderi-manesh, and
Rezayan, (2017) showed that the interaction of the cholesterol (CS)
modified chitosan nanogels with follicle-stimulating hormone (FSH) led

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Fig. 2. The number of hydrogen bonds between insulin and the (a) chitosan, (b)
chit_1chl, and (c) chit_2chl polymers.

to no major impact on the secondary structure of hormone throughout

Fig. 3. The total number of contacts between insulin and (a) chitosan, (b)
the 200 ns MD simulations (Yahyaei et al., 2017). Together, these stu- chit_1chl, and (c) chit_2chl.
dies outline that the effect of chitosan and its derivatives on the protein
structure is dependent on the type of protein and the properties of
and cholesterol-grafted chitosan polymers on the structure and dy-
nanoparticles like their molecular weight and physicochemical struc-
namics of human insulin. The research also examined the critical in-
tures (Alonso-Sande et al., 2006; Xu & Du, 2003; Yahyaei, Mehrnejad,
teractions between the peptide and the chitosan polymers during the
Naderi-manesh, & Rezayan, 2018; Zhang et al., 2010).
insulin-polymer complex formation. The binding pattern between the
Insulin, a peptide hormone, is used to treat diabetes and has two
peptide hormone and the cholesterol-modified chitosan is investigated
peptide chains, called chain A and chain B (Fig. 1 (a)). These two chains
for the first time in the present study. However, the focus of the re-
are joined together by two disulfide bridges, as well as, an in-
search was to provide basic information for comparison of chitosan and
tramolecular disulfide bond is formed in chain A (2015, Vinther et al.,
cholesterol-grafted chitosan as a potential human insulin carrier.
2013). Chain A is composed of 21 residues with 3 helices and 2 beta
Throughout the paper, the terms chit, chit_1chl, chit_2chl will refer to
strands. Further, Chain B has a length of 30 amino acids with 2 helices
chitosan, chitosan with 1 cholesterol moiety, and chitosan with 2
and 2 beta strands (Fávero-Retto, Palmieri, Souza, Almeida, & Lima,
cholesterol moieties, respectively.
2013). Despite its therapeutic effect, insulin is unstable in low pH and
easily is degraded by gastrointestinal enzymes (Mortazavian, Dorkoosh,
& Rafiee-Tehrani, 2014; Pan et al., 2002). Therefore, design and de- 2. Computational methods
velopment of a new generation of drug delivery systems –based on
polymeric nanoparticles that control the release of the peptide hormone The 3D structure of human insulin was taken from the RCSB protein
and protect it against enzyme degradation- have gained momentum data bank archive (PDB ID: 4EY9) (Fig. 1(a)) (Fávero-Retto, Palmieri,
during the last decades (Still, 2002). Souza, Almeida, & Lima, 2013). The pH of the environment in the
Taking into account the aforementioned matter, the current MD present study was 6.5, accordingly, the protonated states of human
simulation study was designed to investigate the effect of the chitosan insulin were obtained from the H++ server (Gordon et al., 2005). The
Chitosan and cholesterol-modified chitosan polymers with 1 and 2

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Fig. 4. The number of contacts between insulin and (a) cholesterol of chit_1chl.
(b) and (c) the number of contacts of first and second cholesterols of chit_2chl
with insulin, respectively.
Fig. 5. (a) The number of water molecules in the first hydration shell of insulin.
(b) The helicity pattern of insulin in the water (purple line), chitosan (red line),
chit_1chl (blue line), and chit_2chl (green line) systems (For interpretation of
the references to colour in this figure legend, the reader is referred to the web
version of this article).
cholesterol moieties were designed and optimized by the HyperChem
software version 8.0 (HyperChem(TM) Professional 8.0 (2018)).
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Carbohydrate Polymers 208 (2019) 345–355
Fig. 6. The number of contacts between each residue of insulin in the presence
of (a) chitosan, (b) chit_1chl, and (c) chit_2chl polymers.
Chitosan with a degree of deacetylation of 50% was used for the current
study (Aiba, 1990). Moreover, 50% of the amine groups of the polymer
were protonated and the other 50% were deprotonated (Franca, Freitas,
& Lins, 2011). The partial charges of each type of the mentioned car-
bohydrate polymers were fixed by the automated force field topology
builder database (ATB) (Malde et al., 2011). Afterward, the topology
files for all the carbohydrate polymers were taken for the GROMOS96
54a7 force field. The MD simulations were conducted using the GRO-
MACS simulation package, version 5.1.2 (Abraham et al., 2015;
Berendsen, van der Spoel, & van Drunen, 1995). The carbohydrate
polymers (30 counts) were randomly inserted around human insulin
based on a previous simulation study (Yahyaei et al., 2018) (Fig. 1). The
SPC/E water model (Berendsen, Grigera, & Straatsma, 1987) and
100 mM sodium chloride were used to the dissolution of the peptide-
polymer complexes in a cubic box (box size 9.5 nm*9.5 nm*9.5 nm).
The periodic boundary conditions (PBC) and the steepest descent al-
gorithm were used for the energy minimization process. The systems
reached equilibrium under the NVT ensemble (constant number of
particles, volume, and temperature) (for 500 ps) and the NPT ensemble
(constant number of particles, pressure, and temperature) (for 1000 ps).
The pressure and temperature of the systems were regulated by the
semi-isotropic Parrinello–Rahman algorithm (Parrinello & Rahman,
1981) and the Nose-Hoover algorithm (Hoover, 1985; Nosé & Klein,
1983), respectively. The Particle Mesh Ewald (PME) algorithm (Darden,
York, & Pedersen, 1993) was applied to handle the long-range elec-
trostatic interactions. A value of 1.0 was used for both short-range
electrostatic cutoff and short-range van der Waals cutoff. The free en-
ergy calculations were performed using the g_mmpbsa tool (Kumari,
Kumar, Lynn, & Lynn, 2014). The main MD simulations were carried
out for 200 ns with a time step length of 100 ps.
3. Results and discussion
3.1. Insulin contacts with the polymers and cholesterol moieties
As noted by Zhang et al. (2012), hydrogen bonds (H-bonds) have a
S. Salar et al. Carbohydrate Polymers 208 (2019) 345–355

Fig. 7. The two-dimensional free energy landscape based on RMSD and Rg of insulin in the (a) water, (b) chitosan, (c) chit_1chl, and (d) chit_2chl systems.

Fig. 8. The secondary structure contents of insulin as a function of MD simulation time in the (a) water, (b) chitosan, (c) chit_1chl, and (d) chit_2chl systems.

crucial role in the complex formation between 6-O-cholesterol modified
chitosan and bovine serum albumin (Li et al., 2012). To gain more
insight into the mechanism of the peptide encapsulation into the
polymers, the number of H-bonds between each insulin residue and the
carbohydrate polymers was analyzed (Fig. 2, only residues with H-
bonds are shown in the figure). As shown in Fig. 2, the median number
of H-bonds in the insulin-chitosan complex is more than that of in the
insulin-chit_1chl and insulin-chit_2chl complexes. This is because the
hydrophobic cholesterol moieties in chit_1chl and chit_2chl tend to
aggregate with each other during the MD simulation times (Panels (d)-
(g) in Fig. 1). Hence, the chit_1chl and chit_2chl polymers were not
exposed by cholesterol to the peptide hormone. The results are in
complete accordance with a previous simulation study that highlights
the higher trend of cholesterols moieties of cholesterol-modified chit-
osan chains to each other (Yahyaei et al., 2017). In general, the results
have indicated that the polar (glutamine and asparagine), acidic (glu-
tamate), and basic (lysine and histidine) residues of human insulin in
the chitosan system involved in the formation of H-bonds more than the
other systems (Fig. 2). The results also may be explained by the fact that
the peptide hormone encapsulated by the chitosan polymers, was more

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Fig. 9. The contribution energy of each insulin residue to the total binding energy in the (a) chitosan, (b) chit_1chl, and (c) chit_2chl systems. The contribution energy
of the positively charged (d) and the negatively charged (e) residues of insulin in the total binding energy. (f) The van der Waals (vdW), non-polar solvation (SASA),
electrostatic interactions, and polar solvation energies between insulin and the carbohydrate polymers. NB energy, PB energy, and TB energy represent the non-polar
binding energy, polar binding energy, and total binding energy, respectively. In panels (d), (e), and (f) the red, blue, and green bars indicate the chitosan, chit_1chl,
and chit_2chl systems, respectively. NB energy = vdW energy + SASA energy, PB energy = Elec energy + PS energy, and TB energy = NB energy + PB energy. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

stable than the peptide hormone in the cholesterol-modified chitosan
polymers (Fig. 1(c)).
In the following section, the normal distribution for the number of
contacts between the carbohydrate polymers and insulin was also
computed (Fig. 3). As revealed by Fig. 3, the average number of con-
tacts between the chitosan polymers and the peptide hormone is more
than this probability in the other polymers, which is due to the higher
trend of the chitosan chains to insulin. Consequently, compared with
the chit_1chl and chit_2chl systems, the probability of the peptide en-
capsulation into the chitosan system is more plausible to happen. In
other words, it can be seen that the normal distribution plots moved left
along the x-axis with increasing the cholesterol moieties (Fig. 3). These
results match well with the previous observations and confirm that
more cholesterol moieties would decrease the tendency of the chitosan
polymers to insulin.
To gain more information about the interactions of polymers and
insulin, the normal distribution for the number of contacts between the
peptide hormone and each cholesterol of the chit_1chl and chit_2chl
polymers was also calculated (Fig. 4). As indicated in Fig. 4, the average
number of contacts of first and second cholesterols in the chit_2chl
system was 18.26 and 75.49, respectively, while this value was 51.95
for the cholesterol molecule in the chit_1chl system. These findings
indicated that increasing the cholesterol portions could not enhance the
number of cholesterol contacts with the peptide hormone. In other
words, the mentioned contacts are not specific because of a different
polymer organization around human insulin.
3.2. Changes in the first hydration shell of insulin
The effect of the polymer chains on the first hydration shell (FHS) of
insulin was examined by calculating the median number of water mo-
lecules around the peptide hormone during the MD simulation times
(Fig. 5(a)). Previous studies have shown that the total number of water
molecules in the hydration shell of the proteins is altered by different
factors (Timasheff, 2002). Accordingly, the distribution and order of
water in the FHS of proteins could be disturbed adjacent to other mo-
lecules. As represented in Fig. 5 (a), the least amount of water in the
FHS of insulin was present in the chitosan system, suggesting that the
surface of the peptide hormone was surrounded by the chitosan poly-
mers with more probability than the other systems (Fig. 1(c)).
These results confirm our findings in the H-bonds analysis and
contribute additional evidence that suggests the chitosan polymers
surround the peptide better than two other polymers.
As mentioned, the hydration layer of proteins can be affected by
adjacent molecules (Fogarty & Laage, 2014). However, when a protein
is surrounded by other molecules, it does not mean that those molecules
would change the protein function. For example, displacing hydration
shell of a protein with polymers cannot change the protein function
since the polymers act in the same way as the hydration layer (Gallat
et al., 2012). Our observations represented that the chitosan polymers
could perturb the first hydration shell of human insulin but nearly the
native conformation of the peptide hormone remains unchanged. The
secondary structure contents of human insulin in pure water did not

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Fig. 10. Radial distribution functions (RDF) analysis for all residues of insulin
in the chitosan (a), chit_1chl (b), and chit_2chl (c) systems.
change over the MD simulation time, which was because of the crucial
roles of the hydration layer in retaining the native conformation of the
peptide hormone. Probably, the polymers conserve the native structure
of human insulin in a similar way to the hydration layer. Although the
polymers did not remarkably change the insulin structure, the entropic
contribution of water molecules released from the first hydration shell
of the peptide hormone cannot be ignored. It has been shown that the
water molecules in the hydration layer of proteins have an ordered
structure and display an unfavorable entropy (Sheu & Yang, 2010). Zou,
Habermann-rottinghaus, and Murphy, (1998) have proved that the
entropy would increase in the presence of urea around non-polar
groups of proteins since the molecule is larger than water and could
replace several water molecules from the hydration layer (Zou et al.,
1998). Insulin is a hydrophobic peptide hormone and the water
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Carbohydrate Polymers 208 (2019) 345–355
molecules in its hydration shell can be substituted by the carbohydrate
polymers and this process may increase the entropy.
As explained earlier, numbers of hydrogen bonds between chitosan
and insulin are remarkably more than the other systems and this could
justify the less distribution of water molecules around insulin in the
chitosan system. Besides, this claim can be confirmed with the number
of contact analysis between each residue of the peptide and the poly-
mers in all systems (Fig. 6). As discussed above, the number of water
molecules around the chitosan is less than that of the other systems.
Fig. 6 depicts that in the chitosan system, the residues of insulin make
more contacts with the polymer chains than the other systems. These
results are coincident, and illustrate that the interactions between
chitosan and insulin could be stronger than the other polymers.
3.3. Insulin stability in the polymers
To provide more information about the stability of the peptide
hormone in the presence of the carbohydrate polymers, we computed
the free energy landscape (FEL) based on the Cα root mean square
deviation (RMSD) and the radius of gyration (Rg) of the peptide hor-
mone (Fig. 7). The contour plot shows that in the water system, when
the Rg of insulin is approximately 1.02 or1.03 nm, the conformation of
the peptide hormone could be stable and ΔG equals 0 (See the black
points in Fig. 7(a)).
In comparison to the aqueous solution, the Rg analysis shows no
remarkable change in the presence of chit_2chl. The RMSD values of
insulin in the water and chit_2chl systems are approximately the same
as each other with the values between 0.25 nm and 0.4 nm. The cor-
relation between the water system and the chit_2chl system is inter-
esting because the interactions of chit_2chl with the peptide hormone
may become lower with increasing the cholesterol moieties. Therefore,
chit_2chl had no significant effect on the insulin conformation.
Compared to the water system, the smallest values (≅ 0.05–0.06 nm) of
RMSD were observed in the chitosan system. As a result, this decrease
in the peptide fluctuations revealed that chitosan can keep insulin in the
most stable conformation, which is in compliance with previous studies
(Dhanasekaran, Rameshthangam, Venkatesan, Singh, & Vijayan, 2018;
Li et al., 2012). Interestingly, there were also differences in the RMSD
and Rg values of the chit_1chl system with those in the water system. In
the presence of chit_1chl, the most stable structure of insulin (ΔG = 0)
was obtained when Rg ≅ 1. 85 nm and RMSD ≅ 0.2 nm (Fig. 7(c)).
He et al. (2017) indicated that the secondary structure of insulin
released from the CS/TPP/insulin nanoparticles did not undergo any
change (He et al., 2017). Therefore, Dictionary secondary structure of
proteins (DSSP) (Kabsch & Sander, 1983) was also calculated to identify
the changes trend in the secondary structure contents of the peptide
hormone (Fig. 8).
Considering the results of the control system (the aqueous solution),
a comparison between the three polymer systems shows that no no-
ticeable changes were observed in the insulin secondary structure
contents, whereas in the presence of chit_2chl, the first alpha helix,
residues I2-C5, of the peptide was unstable over the approximately
130 ns–170 ns.
To further validate the MD results, we obtained the percentage of
helicity per residue during the simulation times (Fig. 5(b)). As reflected
in Fig. 5(b), in the chit_2chl system (green line), the pattern of helicity
for the first alpha helix was different compared to that of in the water
system and this finding was in excellent agreement with the DSSP re-
sults. Thereupon, the secondary structure contents of insulin indicate no
notable change in the presence of chit, chit_1chl, and chit_2chl.
3.4. Binding energies and CH-π interactions between insulin and the
polymers
To identify the key residues involved in the encapsulation process,
the contributions of each polypeptide residue to the total binding free
S. Salar et al. Carbohydrate Polymers 208 (2019) 345–355

Fig. 11. The CH-π contacts between the CH groups of the pyranose rings of chitosan and the aromatic amino acids. (a)- (h) indicate H26, H31, F22, F46, Y14, Y19,
Y37, and Y47, respectively. The chitosan polymers, amino acid side chains, and hydrogen atoms are colored in magenta, green, and silver, respectively (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

energies were calculated (panels (a) to (c) in Fig. 9).
As described in a previous study (Fávero-Retto et al., 2013b), insulin
is a hydrophobic peptide consisting of 51 amino acids with a hydro-
phobic core. Fig. 9 shows that most of the insulin hydrophobic residues
tend to interact with the carbohydrate polymers. The C7, T8, I10, Y14,
Q15, N18, Y19, C20, V23, Y37, L38, G41, and F46 residues in the
chitosan system, the L13, Y14, L16, N18, V23, N24, Q25, L27, V33,
Y37, L38, F45, and F46 residues in the chit_1chl system, and the S9, I10,
Y14, Y19, V23, and L38 amino acids in the chit_2chl system, all with
ΔG < −10 kJ /mol, are important residues that have contributed to the
interactions with the carbohydrate polymers. The most striking result to
emerge from the data is that, in all three polymer systems, the residues
with the phenyl ring in their side chain including the phenylalanine and
(/or) tyrosine (4-hydroxyphenylalanine) residues play important roles
in the encapsulation of the peptide hormone by the carbohydrate
polymers. In addition, we computed the binding free energy of the basic
and acidic residues, separately. Chitosan is a positively charged
polymer, and as demonstrated in Fig. 9(d), the electrostatic repulsions
are the dominant forces between the basic residues and the
carbohydrate polymers. It needs to be noted although G1 and F22 are
not basic residues, they are the N-terminal amino acids of the peptide
chains with a free amine group contributed to the electrostatic repul-
sions. Moreover, notable electrostatic attractive forces contribute to the
interactions of the negatively charged residues with the carbohydrate
polymers (Fig. 9(e)). Although the N21 and T51 residues are not acidic
residues, they contributed to the electrostatic attractive interactions.
The reason for this is the amino acids are located at the C-terminal
region of the peptide chains. Therefore, they can interact with the -NH3
groups of chitosan by their free carboxyl group.
As represented in Fig. 9(f), the van der Waals interactions are more
favorable in the chitosan system than the other polymers. One un-
anticipated finding was that chit_2chl with more cholesterol moieties
had less van der Waals interactions with the peptide than the other
systems. These results suggested that the cholesterols tendency to ab-
sorb each other was remarkably greater than their desire to absorb
insulin. Overall, the cholesterol-cholesterol complex formation was
more probable to occur than the cholesterol-insulin association (Fig. 1).
Moreover, the electrostatic interaction energies in chitosan were more

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Fig. 12. The CH-π interactions between the CH groups of the pyranose rings of polymer and the aromatic residues in the chit_1chl and chit_2chl systems, respectively.
(a)- (d) represent F22, F46, Y14, and Y37 in the presence of the chit_1chl polymers, respectively. (e) and (f) indicate H31 and Y14 in the chit_2chl system,
respectively.
than those in chit_2chl. The MD results also indicated that the total
binding energy for chitosan, chit_1chl, and chit_2chl was −1562.73,
−1553.6, and −608.409, respectively. It is indicative of the more
tendency of chit_2chl polymers to each other rather than to insulin, that
is, in good agreement with the earlier results. The results in Fig. 9(f)
also illustrated that the polar binding energy plays no crucial role in the
encapsulation process of insulin to chit_2chl. Meantime, in all systems,
the non-polar binding energy was important to form the insulin-
polymer complexes.
All in all, based on what was mentioned above, it can be concluded
that the chitosan nanoparticle systems have more interactions with
insulin as compared with the other systems and have a strong tendency
to the peptide hormone. Therefore, in the case of insulin, the chitosan
nanoparticles could be the more suitable carrier than the cholesterol
modified chitosan nanoparticles since the hydrophobic cholesterol
moieties have more tendency to each other.
Dhanasekaran et al. (2018) showed that the hydrophobic interac-
tions and electrostatic attractions between chitosan and insulin are
responsible for encapsulation of protein by polymer chains
(Dhanasekaran et al., 2018). Pan et al. (2002) revealed this fact that the
association of insulin with the chitosan nanoparticles is driven by
electrostatic interactions (Pan et al., 2002). In another major study, Li
et al. (2012) found that hydrophobic interactions, hydrogen bonding,
and electrostatic interactions considerably contribute to the complex
formation between the 6-O-cholesterol modified chitosan (O-CHCS)
nanoparticles and bovine serum albumin (BSA) (Li et al., 2012). The
evidence presented in this section of the study suggests that although
different types of interactions are involved in the process of the insulin
encapsulation, the van der Waals and electrostatic interactions play key
roles in the insulin-chitosan complex formation. In order to further
explore this issue, the radial distribution function (RDF) analysis was
performed in the investigation (Fig. 10). As shown in Fig. 10, the ac-
cumulation and distribution of the polymer chains around the peptide
hormone are more probable to be at a distance of approximately 0.35-
0.6 nm. Accordingly, the hydrophobic, van der Waals, and electrostatic
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Carbohydrate Polymers 208 (2019) 345–355
interactions are favorable in the insulin-polymer complexes formation.
These data support the aforementioned results obtained from the free
binding energy calculation.
Along the critical roles of hydrogen bonds on recognizing the car-
bohydrate polymers by proteins, the CH-π interactions are speculated to
be pivotal in the formation of protein-carbohydrate complexes
(Asensio, Ardá, Cañada, & Jiménez-Barbero, 2013; Hudson et al.,
2015). The CH-π interactions engage two or three CH groups of sugar
pyranose and aromatic ring of amino acids (Asensio et al., 2013).
However, we aimed to evaluate the roles of the CH-π interactions in the
binding of human insulin to the chitosan polymers (Fig. 11 and Fig. 12).
As can be seen in Fig. 11 and Fig. 12, all types of the insulin aromatic
residues could make the CH-π interactions with the carbohydrate
polymers. Nonetheless, this kind of interaction in the chitosan system
was more than those of in the chitosan_1chl and chitosan_2chl systems.
It seems that the mentioned interactions have a crucial role in the
formation of the insulin-chitosan complex. Asensio et al. (2013) have
shown that the CH-π interactions could occur on various orientations of
the pyranose and aromatic surfaces (Asensio et al., 2013). Our MD re-
sults also illustrated that these types of interactions could form on the
different orientations of the ring surfaces.
4. Conclusions
In the present study, we have investigated the effect of the chitosan
and cholesterol-grafted chitosan polymers on the structure and dy-
namics of human insulin. In addition, the aim of the research was to
assess the significant interactions involved in the encapsulation process
of the peptide hormone by the carbohydrate polymers. It was demon-
strated that, generally, the native conformation of insulin did not re-
markably change in the presence of the chitosan and cholesterol-mod-
ified chitosan polymers. The second major finding was that increasing
the cholesterol contents of the chitosan derivatives could decrease the
interactions between the carbohydrate polymer and the peptide hor-
mone. Moreover, it was proved that the aromatics residues including
S. Salar et al.
phenylalanine and tyrosine, as well as, the negatively charged residues
have key roles in the formation of the insulin-polymer complexes. The
findings of this MD simulation research also suggested that the main
factors controlling the process of the insulin encapsulation were the van
der Waals, electrostatic, and CH-π interactions. In general, it seems that
the chitosan polymers could be the more suitable carrier than the
cholesterol-modified chitosan polymers for human insulin.
Competing financial interests
The authors declare no competing financial interests.
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