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Download by: [McMaster University] Date: 03 November 2016, At: 03:24
Drug Development and Industrial Pharmacy
ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: http://www.tandfonline.com/loi/iddi20
Hydrophobic amino acids grafted onto chitosan:
a novel amphiphilic chitosan nanocarrier for
hydrophobic drugs
Marjan Motiei, Soheila Kashanian & Avat (Arman) Taherpour
To cite this article: Marjan Motiei, Soheila Kashanian & Avat (Arman) Taherpour
(2016): Hydrophobic amino acids grafted onto chitosan: a novel amphiphilic chitosan
nanocarrier for hydrophobic drugs, Drug Development and Industrial Pharmacy, DOI:
10.1080/03639045.2016.1254240
To link to this article: http://dx.doi.org/10.1080/03639045.2016.1254240
Accepted author version posted online: 02
Nov 2016.
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2. Hydrophobic amino acids grafted onto chitosan: a novel amphiphilic chitosan
nanocarrier for hydrophobic drugs
Marjan Motiei a
, Soheila Kashanian b, c
*, Avat (Arman) Taherpour d,e
a
Department of Biology, Faculty of Science, Razi University, Kermanshah, Islamic Republic of
Iran.
b
Faculty of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and
Nanotechnology Research Center (NNRC), Razi University, Kermanshah, Islamic Republic of
Iran.
c
Nano Drug Delivery Research Center, Kermanshah University of Medical sciences,
Kermanshah, Islamic Republic of Iran.
d
Organic Chemistry Department, Chemistry Faculty, Razi University, Kermanshah, Islamic
Republic of Iran.
e
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah,
Islamic Republic of Iran.
*Corresponding author, e-mail: kashanian_s@yahoo.com
Fax: +98 831 4274559
P.O. Box: 67149
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3. ABSTRACT
Objective: To develop a novel biocompatible amphiphilic drug delivery for hydrophobic drugs,
chitosan (CS) was grafted to a series of hydrophobic amino acids including L-alanine (A), L-
proline (P) and L-tryptophan (W) by carbodiimide mediated coupling reaction. Materials and
methods: Chemical characteristics of the modified polymers were determined and confirmed by
FT-IR, 1
H-NMR, and UV-vis spectroscopy and the degree of substitution was quantified by
elemental analysis. The modified polymers were used to form amphiphilic chitosan nanocarriers
(ACNs) by the conventional self-assembly method using ultrasound technique. The morphology
and size of ACNs were analyzed by scanning electron microscope (SEM) and Dynamic light
scattering (DLS). Results and discussion: The sizes of spherical ACNs analyzed by SEM were
obviously smaller than those of determined by DLS. The ACNs effectively surrounded the
hydrophobic model drug, letrozole (LTZ), and demonstrated different encapsulation efficiencies
(EE), loading capacities (LC), and controlled drug release profiles. The characteristics of ACNs
and the mechanism of drug encapsulation were confirmed by molecular modeling method. The
modeling of the structures of LTZ, profiles of A, P, and W grafted onto CS and the wrapping
process around LTZ was performed by quantum mechanics (QM) methods. There was a good
agreement between the experimental and theoretical results. The cell viability was also evaluated
in two cell lines compared to free drug by MTT assay. Conclusion: The hydrophobic portion
effects on ACNs’ characteristics and the proper selection of amino acid demonstrate a promising
potential for drug delivery vector.
KEYWORDS: amphiphilic nanocarrier; hydrophobic amino acids; letrozole; controlled release;
cytotoxicity; solubility
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4. 1. Introduction
Nanotechnology in the medicinal field has attracted considerable attention in solving complex
issues associated with conventional anti-cancer drugs such as hydrophobic structure, nonspecific
distribution, systemic toxicity, and low therapeutic index. Drug delivery nanosystems are used to
mask the intrinsic properties of the drug, protect it against chemical and biological degradation
and control drug release based on the physiochemical properties of the nanostructures [1, 2]. The
nanostructures can be synthesized from hydrophobic and amphiphilic polymers to load
anticancer drugs. Amphiphilic polymers are self-assembled systems consisting of hydrophobic
and hydrophilic portions that form core-shell nanostructures through intra and/or intermolecular
interactions. These nanostructures can be applied as delivery systems for hydrophobic drugs by
their ability to encapsulate hydrophobic drugs in their hydrophobic cores and by their ability to
separate the drug from the hydrophilic environment with their hydrophilic shells [3, 4]. The
stability of drug is closely related to the physicochemical properties of the microenvironment in
the delivery system, and to the interactions between the drug and the polymer [5].
The polymeric matrix can be of natural or synthetic origin. CS is a natural aminopolysaccharide
composed essentially of unbranched chain of glucosamine and N-acetylglucosamine that
obtained by extensive deacetylation of chitin [6]. CS is usually preferred in medicinal and
pharmaceutical fields because of its pH sensitivity, biocompatibility, biodegradability, low
immunogenicity, high cationic charge density and ease of chemical modification because of the
presence of amino and hydroxyl groups on its backbone [2, 7, 8].
Various hydrophobically modified CS with deoxycholic acid [9], stearic acid [10], linoleic acid
[11], and palmitic acid [12] have confirmed the synthesis of nanostructures by self-assembly in
aqueous media. Also, there are studies related to the preparation and application of amino acids
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5. grafted onto CS. Amino acids grafted CS have been used in beads for uptake of heavy metals
[13], immobilization of enzymes [14, 15] and binding low density lipoprotein [16]; in
nanocarriers for gene delivery [7, 17, 18, 19] and oral delivery of insulin [20]; and also in
synthesizing glycosaminoglycan membranes to promote chondrogenesis [21].
In this study, to improve assembly and entrapment of hydrophobic drugs, CS was modified with
three nonpolar amino acids with chemically hydrophobic side chain. These hydrophobic amino
acids tend to be located inside the particle to protect them from the aqueous environment. This
characteristic has been used to promote the formation of self-assembly nanostructures and
enhance their potential application as a drug delivery carrier.
Amino acids are chiral molecules with a relatively low molecular weight and vary considerably
in their physicochemical properties due to their various side chains. A, the default amino acid
with an aliphatic hydrocarbon side chain, tends to cluster together and stabilize the structure by
means of hydrophobic interactions. P is a cyclic amino acid with a pyrrolidine side chain whose
nitrogen atom is part of a five-membered ring. The aromatic side chain (indole group) of W is
large and rigid structure and can have significant effect on the surrounding environment, even in
the absence of specific covalent or hydrogen bonds. W is a major constituent of the hydrophobic
cores inside proteins [22, 23].
This work has been focused on the development of novel amphiphilic nanocarriers based on
grafting hydrophobic amino acids onto the primary amino groups of CS through amide linkage
using 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide/Nhydroxyl-succinimide (EDC/NHS) [2,
6]. This chemical modification confers CS new interesting properties. The modified polymers
were characterized by FT-IR, 1
H NMR, UV-visible spectroscopy and elemental analysis as well,
in order to demonstrate the presence of the new bond and to determine the degree of amino acids
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6. grafted onto CS. The amphiphilic polymers were used to assess the effect of the amino acids on
self-assembled nanocarriers and their entrapment of hydrophobic drugs. The proof-of-concept of
these nanocarriers for hydrophobic drug delivery was investigated using LTZ as a model drug.
We hypothesized that due to the nature of the amino acid, these nanocarriers will exhibit
different characteristics. The chemical modification impact on the physico-chemical properties
of nanocarriers was investigated by SEM, FT-IR, in vitro drug release rate, and cytotoxicity
assay. Also, a simple molecular modeling was performed to obtain an in-depth understanding of
the characterization of ACNs and their encapsulation mechanism of LTZ.
2. Material and methods
2.1. Materials
CS (with a degree of deacetylation of 90.28% and a molecular weight of 100-300 kDa) was
obtained from MP Biomedicals, LLC (Solon, OH 44139). A, P, W and Polysorbate 80 (Tween
80), EDC, NHS, Dimethyl sulfoxide (DMSO), sodium pyruvate, sodium bicarbonate, Trypsin-
EDTA solution 0.25%, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)
were purchased from Sigma–Aldrich (St. Louis, MO, USA). Fetal Bovine Serum (FBS), L-
glutamine solution (200 mM), and penicillin/streptomycin solution (10000 U/mL / 10000
µg/mL) were prepared from Biochrom (Berlin, Germany) and Roswell Park Memorial Institute
(RPMI) 1640 from GIBCO Invitrogen (Grand Island, NY). Michigan Cancer Foundation-7
(MCF-7) and Pheochromocytoma 12 (PC-12) cell lines were purchased from Pasteur Institute,
Tehran, Iran. LTZ was a kind gift from Iran Hormone. Buffer substances and all other chemicals
or solvents were of analytical grade and purchased from Merck (Germany).
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7. 2.2. Preparation of modified CS
CS is comprised of two kinds of repeating units, glucosamine (Mw: 161) and N-acetyl-
glucosamine (Mw: 203). According to the degree of deacetylation, the average molecular weight
of the repeating units is 164.17 that is used in the calculation of the number of amino groups [6].
Amino acids (A, P and W) were grafted onto CS using EDC/NHS in stoichiometric amounts.
The amount of amino acids used is 0.1 equivalent/[NH2] of CS and the equal amount of EDC and
NHS is 1.5 equivalent/[COOH] of amino acid.
First, CS solution in acetic acid (2% v/v) was prepared in a bath sonication at ambient
temperature (ca. 25-28 °C) for 40 min and then adjusting pH to 5 using NaOH 1N. Thereafter,
amino acid was added to PBS (0.01 M, pH 6) under bath sonication at ambient temperature (ca.
25-28 °C) for 2 h. EDC and NHS were added to amino acid mixture under uniform stirring at
4°C for 30 min. The mixture was gradually added into CS solution dropwise under constant
stirring at 4 °C for 30 minutes and continued for 24 h at room temperature. After dialysis
(cellulose membrane with molecular weight cutoff 12 kDa) for 3 days against double deionized
water, the excess coupling reagents and unreacted amino acids were eliminated, then the
products were freeze-dried and stored at -20 °C for further assay such as FT-IR, 1
H NMR, UV-
vis spectroscopy and elemental analysis.
2.3. Preparation of ACNs and LTZ-loaded ACNs
ACNs were prepared according to a modified method described by Park et al. [24]. Amino acid
grafted CS solution was suspend under stirring at 25 °C for 24 h, followed by ultrasonication
(Ultrasonic Processor, Dr.Hielscher) at 90 W for 20 min to get an optically clear solution. The
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8. freshly prepared solution was subjected to SEM and DLS and the freeze-dried sample was used
for FT-IR.
LTZ-Loaded ACNs were prepared by suspension of amino acid grafted CS solution at 25 °C for
24 h. Then Tween 80 (7 mg/mL) was subsequently added to the solution and stirred at 45-50°C
temperature for 2 h until the mixture was homogeneous. Distinct concentrations of LTZ (0.2 and
0.3 mg/mL) were dissolved in CH2Cl2 and were gradually dropped into the aqueous solution
under ultrasound sonication in 90 w for 20 minutes. The freshly prepared ACNs solution was
subjected to LC, EE and release profile measurements. The sample for FT-IR analysis was
freeze-dried and stored at -20 °C.
2.4. Characterization
The FT-IR spectra of the products were recorded with a Bruker FTIR-6000 spectrometer in a
wavenumber range of 4000–400 cm-1
using KBr pellets. 1
H NMR spectra of the samples were
recorded on a Bruker, Avance II, 400 spectrometer using tetramethylsilane as an internal
standard and D2O as solvent at 25 °C. The degree of amino acid substitution was determined by
the elemental analysis (C, N, H) using a Euro Elemental Analyzer. Estimation of solubility was
evaluated by dissolving the freeze-dried CS derivative (2 mg/mL) in 2% acetic acid and
recording the transmittance of the solution on a UV-vis spectrophotometer (Philips PU 8620,
USA) at 600 nm. High transmittance reflects high solubility [25]. The sizes and size distributions
of ACNs were determined using a Malvern Zetasizer (model 3600, Malvern Instruments Ltd.,
Worcestershire, UK) equipped with a He–Ne laser operating at 4.0 mW and 633nm at a
scattering angle of 90° and the temperature of 20 °C. Surface and shape characteristics of
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9. nanocarriers were evaluated by means of a SEM (KYKY-EM3200) at an operating voltage of 25
kV.
2.5. Determination of EE and LC
The content of LTZ-loaded ACNs was determined by UV–vis spectrophotometry. LTZ-loaded
ACNs solution was placed in a dialysis membrane bag with a molecular cut-off of 12 kDa and
into 100 mL of PBS solution (0.1 M, pH 7.4). The entire system was kept for 1 h at 37±0.5 °C
with continuous magnetic stirring (100 rpm). At appropriate time, 2 mL of the medium was
collected and the amount of LTZ was evaluated by UV–vis spectrophotometer set at 240 nm
versus a calibration curve prepared in the same buffer. EE and LC of LTZ were calculated from
Eqs. (1) and (2) respectively:
Eqs. (1) EE (%) = (Total LTZ − Free LTZ
Total LTZ⁄ ) × 100
Eqs. (2) LC (%) = (Total LTZ − Free LTZ
Nanocarrier Weights⁄ ) × 100
2.6. In vitro release study of LTZ-loaded ACNs
In vitro release study of LTZ-Loaded ACNs was performed by the dialysis bag diffusion
technique. LTZ-loaded ACNs solution was poured in a dialysis membrane bag, tied and placed
into 50 mL of PBS solution (0.1 M, pH 7.4). The entire system was kept at 37±0.5 °C with
continuous magnetic stirring (100 rpm). At appropriate time intervals, 1 mL of the release
medium was removed and 1 mL fresh medium PBS solution was added into the system. The
amount of LTZ in the release medium was evaluated by UV–vis spectrophotometry set at 240
nm versus a calibration curve prepared in the same buffer.
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10. 2.7. Computational methods
The modeling of the linkage between LTZ and the complex profiles of CS with A, P and W was
performed by quantum mechanics (QM) methods. The calculations of the modeling were
performed by Spartan ‘10 package [Spatran ’10-Quantum Mechanics Program: (PC/x86)
1.1.0v4. 2011, Wavefunction Inc., USA]. The structures of LTZ, profiles of A, P and W grafted
onto CS and LTZ-loaded ACNs were optimized by B3LYP/6-31G* method. In each model,
there are six aminoglycoside units which three of them have been functionalized by amino acids,
one by acyl group and two of them are free and not functionalized. The pattern and the sequences
for all of the amino acids grafted CS profile models were the same and the structures were
optimized by UHF/PM6//B3LYP/6-31G* methods. The modeling of wrapping process of amino
acids grafted CS profile models around LTZ was performed by molecular mechanics MMFF94
method and the obtained structures were optimized by UHF/PM6//B3LYP/6-31G* methods.
2.8. Cytotoxicity studies
MCF-7 and PC-12 were grown in RPMI 1640 supplemented with 10% (v/v) FBS, 1% Glutamine
and 1% Penicillin-Streptomycin. The cells were maintained in a humidified 5% CO2 and 95%
RH atmosphere at 37°C and divided after completed growth using Trypsin- EDTA solution
0.25% for viability assay.
In order to evaluate the effect of (A, P and W)-ACNs, LTZ and LTZ loaded-ACNs against
MCF-7 and PC-12 viability, 2104
and 104
cell/well were seeded and grown with 100 μl
completed RPMI 1640 24 h prior to treating cells in a 96-well plate, respectively. After the
adhesion phase, the medium was removed and cells were treated with 100 µL of (A, P and W)-
ACNs, LTZ and LTZ loaded-ACNs at different concentrations of (10, 30, 50 and 70 nM) in
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11. complete culture medium. After additional 24 h of incubation, viability assay of the samples was
evaluated using standard MTT assay. In the assay, 50 µL of MTT (0.5 mg/mL) was placed in the
medium and the plate was incubated at 37°C in CO2 incubator for 4 h. The medium was then
removed, 150 µl of solubilization buffer (DMSO) was added to each well to dissolve purple
formazan crystals. The absorbance of the solution was measured using a Microplate Reader
(LabSystems Multiskan, USA) at a wavelength of 570 nm.
2.9. Statistical analysis
Statistical analysis was performed using Excel program version 2010. All results were expressed
as mean±standard deviation (SD).
3. Results and Discussion
3.1. FT-IR analysis
The grafting of amino acids onto CS through the formation of an amide linkage between
carboxyl group of the amino acids and an amine group of CS was analyzed by FT-IR technique.
Fig. 1(A) shows FT-IR spectra of CS, A, A-CS, LTZ and LTZ-ACNs. FT-IR spectra of CS
powder show two peaks around 891 and 1157 cm-1
corresponding to saccharide structure and the
peaks at 1089, 1382 ,1601 ,1650 ,2878 and 3444 cm-1
were assigned to >CO-CH3 stretching
vibration, CH3 symmetrical deformation mode, amine, amide, C-H stretching vibration, and N-H
symmetric stretching vibration, respectively [26]. Simultaneously five characterization peaks of
A are observed at 1410 (CH3 bending), 2986 (CH stretching), 1528-1307 (NH3 bending) and
1354 (COO stretching) [27]. When CS and A peaks are compared with A-CS, the appearance of
CH3 bending absorbance peak at 1416 cm-1
, increasing of the absorbance peaks of carboxamide
I bands (C=O stretching) and II bands (N-H deformation) at about 1650 and 1601 cm-1
and also
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12. shifting to 1629 and 1534 cm-1
are indicative of successful graft of A on CS. LTZ shows major
peaks at 2228 cm-1
for C≡N stretching, 3114 cm-1
for sp2 CH stretching, 670-870 cm-1
for out-
of-plane CH bending [28]. In LTZ-loaded A-ACNs, the peak of 2230 is related to C≡N
stretching of LTZ shifted from 2228. The characteristic absorbance peak confirms the presence
of LTZ in LTZ-ACNs.
Fig. 1(B) shows the FT-IR spectra of CS, P, P-CS, LTZ and LTZ-ACNs. There are six peaks of
P at 1085 (ring stretching), 1560 (NH bending), 1378 (OH bending), 1624 (C=O stretching),
1254 (C-O stretching), 2984-3008 (CH2 stretching) and 1293 (C-H bending) [29]. When CS and
P peaks are compared with P-CS, due to the overlapping of peaks corresponding to ring
stretching vibrations of P and the >CO-CH3 stretching vibration of the original CS, a new peak
appears at about 1077 cm-1
. The presence of two peaks at 1634 cm-1
and 1539 cm-1
can also
indicate the successful graft of P on CS. Eventually, the peak of 2230 confirms the presence of
LTZ in LTZ-ACNs.
Fig. 1(C) shows the FT-IR spectra of CS, W, W-CS, LTZ and LTZ-ACNs. When CS and W
peaks are compared with W-CS; the appearance of new peak at 1554 cm-1
, the disappearance of
O-H stretching peak at 3048 cm-1
and the shifting of the broadened peak of N-H stretching at
3402 cm-1
to a much sharper peak at 3398 cm-1
indicate that the synthesis of W-CS has been
achieved successfully [30]. In LTZ-loaded W-ACNs, the peak of 2230 is also related to C≡N
stretching of LTZ. All the characteristic absorbance peaks confirm that the synthesis of LTZ-
ACNs has been achieved successfully.
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13. 3.2. 1
H-NMR analysis
Fig. 2 shows 1
H NMR spectra of CS and CS derivatives in D2O to confirm the chemical reaction
between CS and amino acids. The proton assignment of CS is as follows (ppm): 1.9 ppm (the
three acetyl protons of N-acetyl glucosamine), 3.01 ppm (the H-2 proton of the glucosamine or
N-acetyl glucosamine), 3.55-3.74 ppm (the ring protons of H-3, 4, 5, and 6) [7]. The discussion
of computational and modeling (DFT method) shows that two signals of P (3.62 ppm (-CH- near
carboxyl group) and 2.75 ppm (–CH- near amino group)) and also W (4.18 ppm (-CH2-) and
3.31 ppm (–CH-)) are shielded by the strong signals of CS. The proton signals at 1.2 ppm (-CH2-
in P-CS), 7.14 ppm (aromatic ring in W-CS), 1.04 and 1.81 ppm have appeared in the 1
H NMR
spectra of the amino acids grafted CS. The methyl group of A due to the near chiral center
appeared as a multiple signal at 1.04 ppm and the same phenomenon was observed in other CS
derivatives spectra. The multiple signals at 1-1.81 ppm belong to the -CH2- group next
to the chiral center (CH2-C*) with two diastereotopic hydrogens. Therefore, the spectra of amino
acids grafted CS show not only the similar characteristic resonance peaks, but also new proton
signals indicating the successful synthesis of the amino acids grafted CS and the formation of
self-assembled structures with hydrophobic core [31]. Additionally, the degree of N-acetylation
of CS (8.5%) was determined by the ratio of integral intensity of the three acetyl protons of N-
acetyl glucosamine to the sum of integral intensities of the ring protons of H-2, 3, 4, 5, 6 [32].
3.3. Elemental analysis
The degrees of amino acid substitution were determined by elemental analysis (C, N, H). The
degrees of A, P and W substitution onto CS were estimated to be 27.5%, 30.08% and 17.61%,
respectively. The differences might be due to the intramolecular reaction occurring between the
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14. amino group and activated carboxyl group of A and W [7, 20]. Due to special structure and the
absence of normal backbone NH in P, the degree of substitution was higher than those of A and
W.
3.4. Water solubility
There are some factors affecting the solubility of CS such as: protonation of amino groups [33],
degree of deacetylation, crystallinity [34], and introduction of chemical modifiers [33, 35]. The
degree of solubility should be governed by a balance among these key factors. In constant
deacetylation degree of CS, removal of the two hydrogen atoms of the amine and the
introduction of specific chemical modifiers on CS produce polymers with improved solubility
due to intense distortion of inter and intramolecular hydrogen bonds in the ordered backbone of
CS [8]. The chemical nature of the grafted amino acids onto CS will also have an important
impact on the modified polymer solubility.
According to Table 1, it is evident that there are significant amount of free amino groups in the
derivatives to preserve certain degrees of solubility. Therefore, degree of substitution is
compatible with water solubility. The water solubility of aliphatic, aromatic and five membered
amino acids such as A, P and W at neutral pH are 14.3, 61.2 and 1.3 S/mass (%), respectively
[36]. It is expected that the hydrophobic groups will reduce the polymer solubility. In contrast,
the amino acid A improves the water solubility of the grafted polymer. The small side chain of A
with no chemical reactivity and its random distribution along the CS backbone has effectively
disturbed the formation of the crystalline domain and the hydrogen bond among the functional
groups of CS. W-CS shows the lowest degrees of solubility than all other polymers which is in
agreement with the W water solubility. The aromatic side chain of W is large and rigid enough to
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15. impact the conformation of the surrounding structure and the possible 𝜋 − 𝜋 stacking of the
aromatic side chains will lead to the formation of hydrophobic core and reduction of solubility
[23]. Interestingly, P-CS didn’t have any significant change in the solubility comparing to CS
which is probably due to the distinctive structure of P. Although its side chain is chemically
hydrophobic but its polarity depends on its location and can behave as a polar or nonpolar
residue and this is why it does not affect the solubility of the polymer [23].
3.5. Particle size and morphology
Size distribution and surface morphology are based on the chemical nature of grafted moieties
and the degree of substitution on CS [37, 38, 39]. The (A, P and W)-ACNs analyzed by DLS
exhibited relatively narrow hydrodynamic diameter in the range of 247±32.57, 123.2±33.1 and
261.95±45.61 nm as indicated by relatively low PDI values in the range of 0.369±0.056,
0.331±0.013 and 0.4±0.076, respectively. As shown in Fig. 3, SEM images of all ACNs
demonstrate regular distribution and spherical shape with no aggregation between the prepared
nanocarriers. (A, P and W)-ACNs have the diameter around 156.8, 64.19 and 158.3 nm,
respectively. The ACNs characteristics especially variable sizes are due to the amphiphilic
structure of grafted polymers. The hydrophobic portions are located in the interior of the
nanocarriers to cluster together via hydrophobic interactions which reducing their contact with
water [23]. A is a default amino acid with a small aliphatic hydrocarbon side chain, W has a
large and rigid indole side chain and P is a unique amino acid due to a rigid pyrrolidine side
chain and the absence of normal backbone NH for hydrogen bonding [22, 23]. All these intrinsic
differences of the amino acids and their degrees of substitution will significantly affect the power
of interactions and eventually cause different ACNs sizes and morphologies.
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16. 3.6. Effect of LTZ concentration on EE and LC
The physico-chemical characteristics of core, shell and drug, drug concentration and interactions
between drug and ACNs are the most factors with great impact on EE and LC [40, 41]. The
specific chemical structure of drug molecule and the hydrophobic portion of ACNs suggest that
various interactions including hydrophobic interactions and hydrogen bonding will take place in
the drug-ACN complex [41, 42]. Table 2 shows EE and LC in (A, P and W)-ACNs. According
to the results, higher concentration of LTZ increases EE and LC in A-ACNs and W-ACNs.
These data confirm the presence of larger hydrophobic reservoirs in (A and W)-ACNs for higher
entrapment of LTZ. In P-ACNs due to the smaller size of hydrophobic pocket, high
concentration of LTZ leads to higher LC and lower EE. This data demonstrates that varying the
structure of the hydrophobic portion, the degree of substitution and the drug ratio to ACNs play a
key role in controlling the EE and LC [43].
3.7. In vitro release studies
Fig. 4 shows the release profiles of LTZ and LTZ loaded ACNs with 0.2 and 0.3 mg/mL of drug
in PBS (pH 7.4) at 37 °C as a function of time. LTZ at a concentration of 0.2 and 0.3 mg/ml was
completely released within 6 h. Nevertheless; the release profile of LTZ from ACNs is a two-
step biphasic process, an initial burst release followed by a slower and steady release into the
medium. ACNs with 0.2 mg/mL and P-ACNs with 0.3 mg/mL of LTZ reach the equilibrium
after 48 h and (A and W)-ACNs with 0.3 mg/mL of LTZ need 72 h to reach the equilibrium. It
can be concluded that the rate and amount of drug release in amino acids grafted ACNs are
interdependent to the drug concentration and also the chemical structure of amino acids.
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17. The release occurred at high rates in (A and W)-ACNs with 0.2 mg/mL of LTZ and P-ACNs
with 0.3 mg/mL of LTZ. The slow release of drug from ACNs could be attributed to the kind of
interaction between LTZ and the amino acids. This phenomenon can be explained by various
factors including hydrophobicity, mobility/rigidity, hydrogen bonding, steric factor and π–π
interaction that may hold the drug inside the hydrophobic core [41, 44]. The fact that P-ACNs
have slower drug release rate at 0.2 mg/mL of LTZ is an indication that the hydrophobic
interaction between the pyrrolidine side chain and LTZ is stronger than other amino acids.
However, at higher drug concentration the diffusion rate is higher. This could be explained by
two reasons: (1) Due to the higher ratio of surface/volume, the hydrophobic reservoirs are
saturated completely and the remaining drugs entrapped and/or adsorbed physically in pinholes
and cracks of the particles, (2) According to Fig. 6(B), P structure shows different spatial
orientations in LTZ-P-CS. Two of three conformations form a hydrophobic core to interact with
LTZ. Another one is in opposite direction that may form more superficial core which can adsorb
LTZ and may cause the high diffusion rate of the drug molecules out of the nanocarriers. (A and
W)-ACNs have similar surface to volume ratio and approximately equal release rate at lower
concentration of LTZ but at higher concentration, the amount of LTZ released in W-ACNs was
higher than that of A-ACNs. According to Yoksan et al, it is expected that the high LC and EE of
A-ACNs provide a fast release rate and concomitantly high amount of released LTZ [19], but
these results clearly indicate that the interactions between the drug and amino acid side chain
have affected on the release profile. The fact that A-ACNs have higher LC and EE and its
cumulative release percentage is 65.4% compared to W-ACNs with 79.1% after 72 h, we can
conclude that A with a small aliphatic hydrocarbon side chain has a stronger hydrophobic
interaction with the drug molecules than W with a large aromatic side chain.
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18. 3.7. Computational discussion and modeling
The calculated molecular length of the optimized structure of the CS profile (with six amino
glycoside units) was 26.47Å. The results of the calculations have shown that the molecular
lengths of the amino acids grafted CS profiles would be shorter after the formation of amino
acid-CS complex profiles with LTZ. The molecular lengths of (A, P and W)-CS profiles were
30.44, 35.31 and 38.33Å, respectively (Fig. 5(A-D)). The results of the calculations have also
shown that the molecular lengths of (A, P and W)-CS profiles become shorter (26.22, 23.68 and
32.12Å, respectively) during the self-assembly process around LTZ. The differences in the
molecular lengths of the amino acids-CS profiles before and after wrapping around LTZ are:
4.22, 11.63 and 6.21Å, respectively. The calculations have shown that P-CS profile has the
largest change before and after wrapping process around LTZ (Fig. 5 and 6). The steric
constraints of the structures due to the different forms of the amino acids, π-π stacking of the
aromatic groups, the Van der Walls interactions and the hydrogen bonds formation can play key
role for the structural properties of the complexes during the wrapping around process in this
modeling. But, hydrogen bond interaction seems to be the most important interaction between
LTZ and amino acids-CS profiles. As shown in Fig. 6, one W (at the end of the W-CS profile)
and also one P (at the middle of the P-CS profile) in LTZ-(P, W)-CS do not have any hydrogen
bond linked to LTZ during the wrapping around process. They have just been distorted and
oriented outside their profiles to allow (P and W)-CS to wrap around LTZ molecule well. These
molecules could be responsible for adsorbing some of the LTZ molecules through different
mechanisms than the wrapping around process. The presence of these molecules outside the
profile models correlates with higher release rate of LTZ. It is important to note that during the
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19. wrapping around process the two factors i.e. steric constraint and hydrogen bonds formation play
more important role than π-π stacking between the aromatic rings of LTZ and W-CS complex
profile (Fig. 6(C)).
Fig. 7 (A-C) shows the calculated energy diagrams of the amino acid-CS complex profiles with
LTZ. The free formation energies (ΔG°f) and the energy differences were calculated and
compared in Fig. 7. The free formation energies (ΔG°f) of (A, P and W)-CS complex profiles
with LTZ were -1239.40, -828.16 and -808.12 kJ mol-1
, respectively. The calculations show that
P-CS profile has the biggest ΔG°f after the wrapping process around LTZ. The differences of
ΔG°f of LTZ-P-CS with LTZ-A-CS and LTZ-W-CS were 411.24(98.31) and 431.28(103.10) kJ
mol-1
(kcal mol-1
), respectively. The differences of ΔG°f of LTZ-A-CS and LTZ-W-CS is
20.04(4.79) kJ mol-1
(kcal mol-1
) that is almost equal to a hydrogen bond energy. The ΔG°f
calculation results indicate that the stability sequence of the amino acid-CS complex profiles
with LTZ is: LTZ-P-CS > LTZ-A-CS ≥ LTZ-W-CS (Fig. 7). It is worth to note that the LTZ
release from amino acid-CS complex profiles is inverse compared to the calculated ΔG°f .
Therefore, the stability sequence and the sequence of LTZ release from amino acid-CS complex
profiles are: LTZ-W-CS ≥ LTZ-A-CS> LTZ-P-CS. The results show a good agreement between
the experimental and the theoretical results.
3.8. Cytotoxicity studies
The biocompatibility of ACNs, LTZ and LTZ-loaded ACNs at different concentrations were
evaluated against cultured MCF-7 and PC-12 cell lines by MTT assay. The MTT assay is based
on cleavage of soluble yellow tetrazolium rings and formation of insoluble purple formazan
crystals by mitochondrial enzyme in viable cells. Therefore, the amount of formazan formed is
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20. directly proportional to the number of viable cells [45]. Fig. 8 shows IC50 values of ACNs, LTZ
and LTZ (0.3 mg/mL)-loaded ACNs tested at 24 h. These results clearly suggest that the activity
of LTZ-loaded ACNs against MCF-7 cell line was greater than that of the free drug (Fig. 8(A)),
although PC-12 cell line displayed higher resistance to both free LTZ and LTZ-loaded ACNs
(Fig. 8(B)). MTT assay with 70 nM free LTZ, LTZ-loaded A-ACNs, P-ACNs and W-ACNs
reduced the cell growth by 63.3%, 46.4%, 35.5% and 46.7%, respectively. The results are in
good harmony with the release rate of LTZ, so that the higher release rate of LTZ- loaded P-
ACNs after 24 h leads to the highest reduction in cell growth. The data also clearly showed that
there is no obvious toxicity by any concentration of the synthesized ACNs which makes them
potential safe and effective drug-delivery carriers.
4. Conclusion
In the present study, a novel type of amphiphlic nanocarriers based on CS and hydrophobic
amino acids (A, P and W) was successfully fabricated using EDC/NHS for the controlled
delivery of hydrophobic drugs in a simple and cost-effective process. The covalent linkage
between CS and amino acids was confirmed by FT-IR and 1
H NMR and the amount of amino
acids grafted was determined by elemental analysis. Our results also demonstrated that the
solubility of CS was improved by the modification with A, by which A-CS exhibited high
solubility in 2% acetic acid. The size, morphology and structure of the ACNs were examined by
SEM and FT-IR. The drug-release study showed that the ACNs provided controlled release of
the entrapped hydrophobic model drug, LTZ, and the release behavior was influenced greatly by
the interactions between hydrophobic moiety of ACNs and hydrophobic drug. The affinity
between the drug and nanocarrier may have contributed to the main mechanism determining
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21. drug release. The theoretical calculations and modeling confirmed that the LTZ release from
amino acids-CS complex profiles changed inversely with the calculated ΔG°f and the stability
sequence. These data confirmed also the experimental results. Cytotoxicity studies also showed
the greater activity of LTZ-loaded ACNs against MCF-7 cell line than that of the free drug,
higher resistance to both free LTZ and LTZ-loaded ACNs against PC-12 cell line and no obvious
cytotoxicity of ACNs against the cell lines. Furthermore, these results indicate that the ACNs
would be a potential safe and efficient delivery system to control the release of hydrophobic
drug.
References
1. Sun T, Zhang YS, Pang B, Hyun DC, Yang M, Xia Y. Engineered nanoparticles for drug
delivery in cancer therapy. Angewandte Chemie International Edition. 2014;53:12320-64.
2. Prabaharan M, Grailer JJ, Steeber DA, Gong S. Stimuli‐ Responsive Chitosan‐ graft‐
Poly (N‐ vinylcaprolactam) as a Promising Material for Controlled Hydrophobic Drug Delivery.
Macromolecular bioscience. 2008;8:843-51.
3. Liu K-H, Chen B-R, Chen S-Y, Liu D-M. Self-assembly behavior and doxorubicin-
loading capacity of acylated carboxymethyl chitosans. The Journal of Physical Chemistry B.
2009;113:11800-7.
4. Liu C-G, Chen X-G, Park H-J. Self-assembled nanoparticles based on linoleic-acid
modified chitosan: Stability and adsorption of trypsin. Carbohydrate polymers. 2005;62:293-8.
JU
ST
AC
C
EPTED

22. 5. Razmimanesh F, Amjad-Iranagh S, Modarress H. Molecular dynamics simulation study
of chitosan and gemcitabine as a drug delivery system. Journal of molecular modeling.
2015;21:1-14.
6. Tsai SP, Hsieh CY, Hsieh CY, Wang DM, Huang LLH, Lai JY, Hsieh HJ. Preparation
and cell compatibility evaluation of chitosan/collagen composite scaffolds using amino acids as
crosslinking bridges. Journal of applied polymer science. 2007;105:1774-85.
7. Layek B, Singh J. Amino acid grafted chitosan for high performance gene delivery:
comparison of amino acid hydrophobicity on vector and polyplex characteristics.
Biomacromolecules. 2013;14:485-94.
8. Chen Y, Li J, Li Q, Shen Y, Ge Z, Zhang W, Chen S. Enhanced water-solubility,
antibacterial activity and biocompatibility upon introducing sulfobetaine and quaternary
ammonium to chitosan. Carbohydrate Polymers. 2016;143:246-53.
9. Li G, Song P, Wang K, Xue Q, Sui W, Kong X. An amphiphilic chitosan derivative
modified by deoxycholic acid: preparation, physicochemical characterization, and application.
Journal of Materials Science. 2015;50:2634-42.
10. Moreno PM, Santos JCdC, Gomes CP, Varela-Moreira A, Costa A, Leiro V, Mansur HS,
Pêgo AP. Delivery of Splice Switching Oligonucleotides by Amphiphilic Chitosan-Based
Nanoparticles. Molecular pharmaceutics. 2015.
11. Wang B, He C, Tang C, Yin C. Effects of hydrophobic and hydrophilic modifications on
gene delivery of amphiphilic chitosan based nanocarriers. Biomaterials. 2011;32:4630-8.
12. Bossio O, Gómez-Mascaraque LG, Fernández-Gutiérrez M, Vázquez-Lasa B, San
Román J. Amphiphilic polysaccharide nanocarriers with antioxidant properties. Journal of
Bioactive and Compatible Polymers: Biomedical Applications. 2014;29:589-606.
JU
ST
AC
C
EPTED

23. 13. Ishii H, Minegishi M, Lavitpichayawong B, Mitani T. Synthesis of chitosan-amino acid
conjugates and their use in heavy metal uptake. International journal of biological
macromolecules. 1995;17:21-3.
14. Yi S-S, Noh J-M, Lee Y-S. Amino acid modified chitosan beads: improved polymer
supports for immobilization of lipase from Candida rugosa. Journal of Molecular Catalysis B:
Enzymatic. 2009;57:123-9.
15. El-Ghaffar MA, Hashem M. Chitosan and its amino acids condensation adducts as
reactive natural polymer supports for cellulase immobilization. Carbohydrate Polymers.
2010;81:507-16.
16. Fu G, Shi K, Yuan Z, He B, Liu B, Shen B, Wang Q. Preparation of tryptophan modified
chitosan beads and their adsorption of low density lipoprotein. Chinese Science Bulletin.
2003;48:2303-7.
17. Morris VB, Sharma CP. Folate mediated histidine derivative of quaternised chitosan as a
gene delivery vector. International journal of pharmaceutics. 2010;389:176-85.
18. Morris VB, Sharma CP. Folate mediated in vitro targeting of depolymerised
trimethylated chitosan having arginine functionality. Journal of colloid and interface science.
2010;348:360-8.
19. Yoksan R, Akashi M. Low molecular weight chitosan-g-l-phenylalanine: preparation,
characterization, and complex formation with DNA. Carbohydrate Polymers. 2009;75:95-103.
20. Abbad S, Zhang Z, Waddad AY, Munyendo WL, Lv H, Zhou J. Chitosan-Modified
Cationic Amino Acid Nanoparticles as a Novel Oral Delivery System for Insulin. Journal of
biomedical nanotechnology. 2015;11:486-99.
JU
ST
AC
C
EPTED

24. 21. Zhu H, Ji J, Lin R, Gao C, Feng L, Shen J. Surface engineering of poly (D, L‐ lactic
acid) by entrapment of chitosan‐ based derivatives for the promotion of chondrogenesis. Journal
of biomedical materials research. 2002;62:532-9.
22. Berg JM, Tymoczko JL, Stryer L, Clarke ND. Biochemistry, 2002. New York: W H
Freeman.
23. Murray RK. Harper's illustrated biochemistry: McGraw-Hill Medical New York; 2012.
24. Park JH, Cho YW, Chung H, Kwon IC, Jeong SY. Synthesis and characterization of
sugar-bearing chitosan derivatives: aqueous solubility and biodegradability. Biomacromolecules.
2003;4:1087-91.
25. Chan P, Kurisawa M, Chung JE, Yang Y-Y. Synthesis and characterization of chitosan-g-
poly (ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery.
Biomaterials. 2007;28:540-9.
26. Salimon J, Salih N, Abdullah BM. Improvement of physicochemical characteristics of
monoepoxide linoleic acid ring opening for biolubricant base oil. BioMed Research
International. 2011;2011.
27. Rodríguez-Lazcano Y, Maté B, Gálvez O, Herrero VJ, Tanarro I, Escribano R. Solid L-α-
alanine: Spectroscopic properties and theoretical calculations. Journal of Quantitative
Spectroscopy and Radiative Transfer. 2012;113:1266-75.
28. Dey SK, Mandal B, Bhowmik M, Ghosh LK. Development and in vitro evaluation of
Letrozole loaded biodegradable nanoparticles for breast cancer therapy. Brazilian Journal of
Pharmaceutical Sciences. 2009;45:585-91.
29. Mary YS, Ushakumari L, Harikumar B, Varghese HT, Panicker CY. FT-IR, FT-raman
and SERS spectra of L-proline. Journal of the Iranian Chemical Society. 2009;6:138-44.
JU
ST
AC
C
EPTED

25. 30. Xiang Y, Si J, Zhang Q, Liu Y, Guo H. Homogeneous graft copolymerization and
characterization of novel artificial glycoprotein: Chitosan‐ poly (L‐ tryptophan) copolymers
with secondary structural side chains. Journal of Polymer Science Part A: Polymer Chemistry.
2009;47:925-34.
31. Jiang G-B, Quan D, Liao K, Wang H. Novel polymer micelles prepared from chitosan
grafted hydrophobic palmitoyl groups for drug delivery. Molecular pharmaceutics. 2006;3:152-
60.
32. Kasaai MR. Determination of the degree of N-acetylation for chitin and chitosan by
various NMR spectroscopy techniques: A review. Carbohydrate Polymers. 2010;79:801-10.
33. Ahmed TA, Aljaeid BM. Preparation, characterization, and potential application of
chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery.
Drug design, development and therapy. 2016;10:483.
34. Sogias IA, Khutoryanskiy VV, Williams AC. Exploring the factors affecting the
solubility of chitosan in water. Macromolecular Chemistry and Physics. 2010;211:426-33.
35. Zhu D, Cheng H, Li J, Zhang W, Shen Y, Chen S, Ge Z, Chen S. Enhanced water-
solubility and antibacterial activity of novel chitosan derivatives modified with quaternary
phosphonium salt. Materials Science and Engineering: C. 2016;61:79-84.
36. Haynes WM. CRC handbook of chemistry and physics: CRC press; 2014.
37. Choochottiros C, Yoksan R, Chirachanchai S. Amphiphilic chitosan nanospheres: factors
to control nanosphere formation and its consequent pH responsive performance. Polymer.
2009;50:1877-86.
JU
ST
AC
C
EPTED

26. 38. Demina T, Akopova T, Vladimirov L, Zelenetskii A, Markvicheva E, Grandfils C.
Polylactide-based microspheres prepared using solid-state copolymerized chitosan and d, l-
lactide. Materials Science and Engineering: C. 2016;59:333-8.
39. Lepeltier E, Loretz B, Desmaële D, Zapp J, Herrmann J, Couvreur P, Lehr C-M.
Squalenoylation of Chitosan: A Platform for Drug Delivery? Biomacromolecules. 2015;16:2930-
9.
40. Wu M, Guo K, Dong H, Zeng R, Tu M, Zhao J. In vitro drug release and biological
evaluation of biomimetic polymeric micelles self-assembled from amphiphilic deoxycholic acid–
phosphorylcholine–chitosan conjugate. Materials Science and Engineering: C. 2014;45:162-9.
41. Woraphatphadung T, Sajomsang W, Gonil P, Treetong A, Akkaramongkolporn P,
Ngawhirunpat T, Opanasopit P. pH-Responsive polymeric micelles based on amphiphilic
chitosan derivatives: Effect of hydrophobic cores on oral meloxicam delivery. International
journal of pharmaceutics. 2016;497:150-60.
42. Ding J, Chen L, Xiao C, Chen L, Zhuang X, Chen X. Noncovalent interaction-assisted
polymeric micelles for controlled drug delivery. Chemical Communications. 2014;50:11274-90.
43. Kamari A, Aljafree N, Yusoff S. N, N-dimethylhexadecyl carboxymethyl chitosan as a
potential carrier agent for rotenone. International journal of biological macromolecules.
2016;88:263-72.
44. Yokoyama M. Polymeric micelles as drug carriers: their lights and shadows. Journal of
drug targeting. 2014;22:576-83.
45. Shukla S, Jadaun A, Arora V, Sinha RK, Biyani N, Jain V. In vitro toxicity assessment of
chitosan oligosaccharide coated iron oxide nanoparticles. Toxicology Reports. 2015;2:27-39.
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27. Legends
Table Legends
Table 1. Solubility of amino acids grafted CS
Table 2. EE and LC of ACNs in PBS solution (0.1 mol/L with pH7.4)
Table 3. Cumulative release of ACNs in LTZ concentration of 0.2 and 0.3 mg/mL after 48 and
72 hour, respectively.
Figure Legends
Fig. 1. FT-IR spectra of A-ACNs (A), P-ACNs (B) and W-ACNs (C).
Fig. 2. 1
H NMR spectra of CS, A-CS, P-CS and W-CS.
Fig. 3. SEM images of A-ACNs (A), P-ACNs (B) and W-ACNs (C).
Fig. 4. Release profiles of LTZ and LTZ loaded ACNs with drug concentration of 0.2 mg/mL
(A) and 0.3 mg/mL (B).
Fig. 5. The optimized structures of CS profile (A), A-CS complex profile (B), P-CS complex
profile (C), W-CS complex profile (D) and LTZ (E).
Fig. 6. The optimized structures of LTZ-A-CS complex profile (A), LTZ-P-CS complex profile
(B) and LTZ-W-CS complex profile (C).
Fig. 7. The energy diagrams of LTZ-A-CS complex profile (A), LTZ-P-CS complex profile (B)
and LTZ-W-CS complex profile (C). The free formation energies (ΔG°f) and the energy
differences were calculated and compared.
Fig. 8. The effect of ACNs (A, P and W), LTZ and LTZ-loaded ACNs (LTZ-A, LTZ-P and LTZ-
W) concentration on viability of MCF-7 cell line (A) and PC-12 (B) after MTT assay.
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28. Table 1
Polymer Transmittance (%)
CS 63.71
A-CS 69.97
P-CS 63.02
W-CS 52.01
Table 2
A-ACNs P-ACNs W-ACNs
LTZ (mg/mL) 0.2 0.3 0.2 0.3 0.2 0.3
LC (%) 12.3±0.65 18.14±0.25 12.39±0.68 17.069±0.28 12.71±1.33 17.33±0.17
EE (%) 84.86±4.52 89.63±1.23 86.17±4.76 84.81±1.39 84.27±2.26 86.07±0.85
Table 3
LTZ Concentration (mg/mL) A-ACNs P-ACNs W-ACNs
0.2 47.2±2.7 37.9±1.01 44.9±4.67
0.3 65.4±1.11 77.1±0.33 79.1±1.52
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29. Fig. 1.
(A) (B)
(C)
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30. Fig. 2.
Fig. 3.
(C)(A) (B)
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31. Fig. 4.
0
20
40
60
80
100
0 20 40 60 80 100
CumulativeRelease(%)
Time (h)
(B) LTZ A-ACNs P-ACNs W-ACNs
0
20
40
60
80
100
0 20 40 60 80 100
CumulativeRelease(%)
Time (h)
(A) LTZ A-ACNs P-ACNs W-ACNs
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32. Fig. 5.
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33. Fig.6.
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34. Fig. 7.
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35. Fig. 8.
0
20
40
60
80
100
120
LTZ A LTZ-A P LTZ-P W LTZ-W
Viability(%)
(A) 10 30 50 70
0
20
40
60
80
100
120
LTZ A LTZ-A P LTZ-P W LTZ-W
Viability(%)
(B) 10 30 50 70
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