Carbohydrate Polymers 194 (2018) 89–96

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

Fabrication of lecithin-gum tragacanth muco-adhesive hybrid nano-carrier T

system for in-vivo performance of Amphotericin B
Tooba Jabri, Muhammad Imran, Shafiullah, Komal Rao, Imdad Ali, Muhammad Arfan,
⁎
Muhammad Raza Shah
International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan

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

Keywords: Nano-carriers are excellent systems for improving bioavailability of poor aqueous soluble drugs. This study
Lecithin focuses fabrication of lecithin-gum tragacanth muco-adhesive hybrid NPs for enhancing Amphotericin B (AmpB)
Gum tragacanth oral bioavailability. AmpB loaded lecithin NPs were synthesized through solvent diffusion method. Green
Muco-adhesive synthesis of stable muco-adhesive gum tragacanth (GT) gold NPs was confirmed through UV–vis spectro-
Hybrid nanoparticles
photometer and FT-IR. AmpB loaded lecithin NPs hybrid with GT gold NPs were characterized for shape, size,
Amphotericin B
polydispersity index (PDI), zeta potential, drug entrapment efficiency and drug-excepients interactions using
Bioavailability
atomic force microscope (AFM), zetasizer, UV–vis spectrophotometer and FT-IR respectively. In-vivo bioavail-
ability of AmpB loaded in NPs was investigated in rabbits. AmpB loaded muco-adhesive NPs were found
polydispersed with 358.3 ± 1.78 nm mean size and −19.9 ± 0.51 mV zeta potential. They entrapped
78.91 ± 2.44% AmpB and enhanced its oral bioavailability in animals. Results reveal the hybrid NPs as efficient
carriers for enhancing AmpB oral bioavailability in controlled manner.

1. Introduction
Hydrophobicity of drug molecules decreases their gastric intestinal
absorption and subsequent bioavailability, upon oral delivery, thus
limiting their therapeutic efficacy (Chen, Khemtong, Yang, Chang, &
Gao, 2011). AmpB, a polyene antifungal, is one of the most active drugs
against systemic fungal infections. It has also been evaluated against
leishmaniasis and has exhibited promising clinical potentials. Results
are highly encouraging when AmpB is given to immune-compromised
cancer patients, suffering from meningeal or opportunistic systemic
fungal infections (Bates et al., 2001; Javed et al., 2015). Being from
Biopharmaceutical Classification System (BCS) class IV drugs, it is poor
aqueous soluble and poor permeable, leading to its instability in gastric
acidic environment and ultimately limited oral bioavailability
(Alamanos & Drosos, 2005; Silva, Barratt, Chéron, & Egito, 2013), thus
resulting in inferior therapeutic efficacy of the drug. Moreover, cur-
rently available commercial parenteral dosage forms of AmpB have
been associated with nephrotoxicity and erratic systemic bioavailability
(Bates et al., 2001). Improving the oral bioavailability of AmpB through
developing an efficient novel nano-carrier system is highly desirable.
Improving oral bioavailability of drugs without changing their
chemical nature has been a challenging task for formulation scientists.
Nano-carriers based drug delivery vehicles are offering promising so-
lutions to such problems (Javed et al., 2016). They are unique phar-
maceutical vehicles for addressing the problems associated with drug
molecules due to their physicochemical properties. Various nano-car-
rier systems based on synthetic polymers have been reported previously
for enhancing the oral bioavailability and anti-parasitic activity of
AmpB and reducing its hemolytic activity and nephrotoxicity. However,
such formulations usually result in lower drug loading and anti-para-
sitic activity (Italia, Yahya, Singh, & Kumar, 2009; Jain, Jatana,
Chakrabarti, & Kumar, 2010; Serrano et al., 2015). Therefore, devel-
opment of biocompatible and biodegradable nanoparticulate systems is
highly desired for improving the oral bioavailability of such drugs.
Lecithin, a hydrophobic mixture of naturally occurring phospholipids,
is widely applied in the food and pharmaceutical industries and is
considered a safe and biocompatible excipient. Being a kind of phos-
pholipids, lecithin functions as crucial component of the cell mem-
brane. It maintains membrane fluidity and works as an absorption en-
hancer to facilitate drug absorption. Therefore, lecithin-based
formulations improve the oral bioavailability of poor oral absorbed
drugs (Chen et al., 2016; Jin et al., 2013; Yanyu, Yunmei, Zhipeng, &
Qineng, 2006). Moreover, they offer greater opportunities for their
surface modifications with various ligands for achieving improved

⁎
Corresponding author at: H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, Karachi University, Karachi, 74200, Pakistan.
E-mail addresses: toobaasif137@gmail.com (T. Jabri), imranbjr.khan@gmail.com (M. Imran), shafi_ullah34@yahoo.com (Shafiullah), komalrao665@yahoo.com (K. Rao),
imdadchem26@gmail.com (I. Ali), m_arfan@upesh.edu.pk (M. Arfan), raza.shah@iccs.edu (M.R. Shah).

https://doi.org/10.1016/j.carbpol.2018.04.013
Received 22 December 2017; Received in revised form 2 April 2018; Accepted 3 April 2018
Available online 05 April 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
T. Jabri et al.
therapeutic efficacy of the encapsulated drugs (Chen et al., 2016; Javed
et al., 2016).
Recently different drug delivery systems have been designed for
improving the efficiency of orally delivered drugs through overcoming
gastrointestinal barriers. Various hydrogels and carriers based on bio-
degradable natural biopolymers and polysaccharides have been devel-
oped. Naturally occurring polysaccharides have got greater scientific
interests for pharmaceutical and biomedical applications due to their
lower toxicity, higher biocompatibility, and economic benefits (Gao
et al., 2014). GT is an anionic branched polysaccharide with a mole-
cular weight of 850 kD and is obtained from stems and branches of
different species of Astragalus. It is highly acid-resistant, tasteless, and
odorless water soluble mixture. It contains two fractions termed as
‘Tragacanthic acid’ and “Tragacanthin” (Rao et al., 2017). It is biode-
gradable and nontoxic and is widely used as stabilizer, thickener,
emulsifier and suspending agent in food, pharmaceutical and cosmetic
industries (Phillips & Williams, 2009). Interestingly, a recent study has
reported GT as a promising muco-adhesive biopolymer material and
can be effective for oral delivery of proteins and peptides (Nur,
Ramchandran, & Vasiljevic, 2016).
Various approaches have been investigated for maximizing the
physicochemical properties and in-vivo performance of poor water so-
luble drugs. The presence of a mucus layer that covers the surfaces of a
variety of organs has been capitalized for developing muco-adhesive
dosage forms. Mucins present in mucus are directly involved in adhe-
sion phenomena. Thus, mucus causes the drugs to remain in the ad-
ministration site for more prolonger times, increasing the local or sys-
temic bioavailability of the administered drugs (das Neves, Bahia,
Amiji, & Sarmento, 2011; Sosnik, das Neves, & Sarmento, 2014). The
surface coating of AmpB loaded lecithin NPs with muco-adhesive GT
gold NPs is an innovative idea for enhancing the drug oral bioavail-
ability. Being water soluble and having complex and nonspecific che-
mical structure, GT cannot be directly coated on the surfaces of parti-
cular drug delivery systems. GT coating on metal NPs and then
adsorbing these metal NPs on the surfaces of nano-carriers is best al-
ternative to its chemical conjugation with drug delivery vehicles.
Among metal NPs, gold NPs (Au NPs) have been more attractive for
designing nano-carrier based drug delivery systems. It has been highly
biocompatible, safe and inert, thus providing an ideal base for the
construction of a carrier system (Rao et al., 2017). Thus, Au NPs were
selected for coating of GT in designing the current novel muco-adhesive
nano-carrier system for enhancing AmpB oral bioavailability.
Nano-carriers are versatile tools for increasing solubility and oral
bioavailability of poor aqueous soluble drugs. Designing of nano-carrier
system surface engineered with muco-adhesive biopolymer like GT
would be an effective and practical approach for enhancing AmpB oral
bioavailability. This manuscript reports on fabrication of AmpB loaded
lecithin NPs and their coating with GT stabilized green Au NPs. The
novel hybrid nano-carrier system demonstrated to enhance the oral
bioavailability of the loaded drug efficiently in animal model
2. Materials and methods
2.1. Materials
All the solvents in this study were of analytical grade and were
purchased from Sigma Aldrich (Germany). Soya lecithin, Amphotericin
B, AuCl4·3H2O and Sodium dodecyl sulphate (SDS) were all purchased
from Sigma Aldrich (Germany). Gum Tragacanth was purchased from a
local vendor.
2.2. Synthesis of Amphotericin B loaded lecithin nanoparticles (Lec-AmpB
NPs)
Solvent diffusion method was used for the synthesis of Lec-AmpB
NPs (Javed et al., 2016). Briefly, AmpB (5 mg) and soya lecithin
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Carbohydrate Polymers 194 (2018) 89–96
(20 mg) were dissolved in mixed solvent system of methanol and
chloroform (30 mL, 5:1, v/v). The solution was sonicated (Model
1225E0273, VWR Symphony, USA) for 2 min to ensure complete dis-
solution and mixing of all the ingredients. Double distilled water
(20 mL) was taken in a beaker and maintained at 40 °C with constant
stirring (100 rpm). The organic solvent system containing drug and
lecithin was added drop wise to the aqueous phase. After complete
evaporation of organic solvent, the aqueous phase containing Lec-
AmpB NPs was freeze dried and rehydrated with deionized water
containing 0.02% w/v SDS. The formulation was probe sonicated (VC
505/VC 750, Sonics, Newtown, USA) for 5 min at 30 °C and stored at 4
°C till further use.
2.3. Synthesis of GT stabilized gold NPs
GT (1 g) was dissolved in deionized water (1 L), stirred over night,
and filtered in order to remove the insoluble materials if any and then
freeze dried. Its stock solution (3 mg/mL) was prepared in deionized
water. For synthesis of GT-Au NPs, GT solution (3 mg/mL, 1 mL) was
added to gold solution (5 mM, 0.1 mL) and stirred for 2 h at 65 °C (Rao
et al., 2017). The change in color from yellowish to deep purple con-
firmed the synthesis of GT-Au NPs. NPs synthesis was further confirmed
through UV–vis spectrophotometer (UV-240, Shimadzu, Kyoto, Japan)
for their characteristic surface plasmon resonance (SPR) peak. FT-IR
(IR-470, Shimadzu, Kyoto, Japan) analysis of GT and GT-Au NPs was
used to investigate the involvement of GT functional groups in stabili-
zation of GT-Au NPs.
2.4. Stability of GT-Au NPs
The synthesized GT-Au NPs were subjected to salt, acid/base and
plasma stability studies. Briefly, GT-Au NPs solution was diluted with
NaCl solutions of various concentrations (0.2–1 M) in 1:1 v/v ratios.
They were incubated for 2 h and then absorbance was read spectro-
photometrically for any change in the characteristic SPR. NPs diluted
with only deionized water in the same volume ratio was taken as re-
ference. Similarly, the pH of NPs solution was adjusted in a pH range of
2.5-12.5 with concentrated NaOH or HCl. NPs solutions were read
spectrophotometrically after 2 h incubation and change in character-
istic SPR was observed. For plasma stability study, plasma was diluted
properly; GT-Au NPs solution was added to it in 1:1 v/v ratio and mixed
well. After 2 h incubation, NPs were read spectrophotometrically and
change in characteristic SPR was observed.
2.5. Coating of Lec-AmpB NPs with GT-Au NPs
To remove the unwanted/free gum materials, GT-Au NPs were
centrifuged at 10,000 rpm for 30 min and NPs were collected. GT-Au
NPs were added to Lec-AmpB NPs solution and kept on stirring for 24 h
at room temperature. Surface coated NPs (here we call them as GT-Lec-
AmpB NPs) were collected by centrifugation at 10,000 rpm for 30 min.
GT-Lec-AmpB NPs were kept in light resistant vials and stored at 4 °C
till further use.
2.6. Characterization
2.6.1. Shape, size, PDI and zeta potential determination
Shape of GT-Au NPs, Lec-AmpB NPs and GT-Lec-AmpB NPs was
investigated using AFM (5500, Agilent, Santa Clara, USA). NPs solu-
tions were properly diluted, a drop was placed on mica slide, air dried
and mounted on microscope. The images were recorded in non-contact
mode. Zetasizer (ZS-90 Malvern instruments, Malvern, UK) was used for
determination of size, PDI and zeta potential. Properly diluted samples
solutions were taken and read in triplicate at scattering angle of 90° at
25 °C immediately after samples dilution (Ullah et al., 2017).
T. Jabri et al.
2.6.2. Drug entrapment efficiency
Drug entrapment efficiency of Lec-AmpB and GT-Lec-AmpB NPs was
determined using UV–vis spectrophotometer. Lec-AmpB NPs and GT-
Lec-AmpB NPs (each 1 mL) containing specific amount of the drug were
centrifuged at 10,000 rpm for 30 min. Supernatants containing free
drug were discarded and drug loaded NPs were collected in the form of
pallets. The drug loaded NPs were re-dispersed in methanol. AmpB was
detected spectrophotometrically at 406 nm and then quantified ac-
cording to the following formula:
Amount of AmpB loaded
%EE = × 100
Total amount of AmpB used
2.6.3. FT-IR analysis
To investigate the possible interactions of AmpB with formulation
ingredients, pure drug, drug loaded Lec-AmpB and GT-Lec-AmpB NPs
were subjected to FT-IR analysis. The samples were diluted with KBr
powder and then pressed for preparing their self-supporting disks.
Spectra of all the samples were recorded in a range of 400–4000 cm−1.
2.7. HPLC method development
Mobile phase consisting Acetonitrile (ACN) and NaCH3CO2 (0.01 M)
buffer (pH = 3.7) in 40:60 v/v ratio was prepared. For separation and
detection of analyte, HPLC system (LC20A, Shimadzu, Kyoto, Japan)
with reverse phase C18 column (Merck, Germany, 5 μ, 125 × 4 mm)
was used. The column oven temperature was set at 50 °C while the flow
rate for mobile phase was optimized as 0.8 mL/min. An injection vo-
lume of 50 μL was found appropriate while drug was detected at
406 nm. For extraction of drug from plasma, ACN (200 μL) was added
to plasma (200 μL). The mixture was vortex mixed for 1 min and then
centrifuged it for 12 min at 12,000 rpm. Supernatant (100 μL) was taken
and diluted with 0.01 M NaCH3CO2 buffer (100 μL). It was vortex mixed
for 30 s and 50 μL were injected into HPLC.
2.8. Animals used
In-vivo bioavailability study was carried out in rabbit’s local species
“Oryctolagus Cuniculus”. All the animals were purchased from Dow
University of Health sciences, Karachi, Pakistan. Protocol (2016-0003)
for the study was approved by “Institutional committee for use of ani-
mals in research”, International Center for Chemical and Biological
Sciences (ICCBS), University of Karachi, Pakistan. National Institutes of
Health (NIH) guidelines for the care and use of Laboratory animals were
followed for the in-vivo study involving animals.
2.9. In-vivo oral bioavailability study
All the rabbits (1.7 kg average body weight) were divided into three
groups (n = 4) and housed under standard laboratory conditions with
free access to water and food. Prior to conduct the study, all the animals
were deprived of food and had access to water only. Animals in first and
second groups were given AmpB encapsulated in Lec-AmpB and GT-
Lec-AmpB NPs respectively at 5 mg/mL per kg body weight oral dose.
Animals in third group were given AmpB solution (in 5% DMSO) at the
same oral dose as NPs of the drug. Blood samples of 1 mL were collected
from animals in heparinized tubes form ear marginal vein at pre-de-
termined time intervals (0–24 h). Blood samples were immediately
centrifuged for 5 min at 4000 rpm for separation of plasma. All the
plasma samples were stored at −80 °C till further use. Different in-vivo
pharmacokinetic parameters were determined from plasma drug con-
centration-time curves through linear trapezoidal rule using non-com-
partmental model.
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Carbohydrate Polymers 194 (2018) 89–96
2.10. Statistical analysis
All the experiments were repeated three times and data was pre-
sented as mean ± SEM. Two-way ANOVA followed by the Bonferroni
test was applied for the comparison of multiple groups, and Student’s
independent sample t-test was used for comparison between two
groups. P Values less than or equal to 0.05 were considered statistically
significant
3. Results and discussion
3.1. Synthesis of Lec-AmpB, GT-Au NPs and GT-Lec-AmpB
Lecithin has been safe and is used in food for controlling over lipid
profile in a proper manner. It does not show any toxicity in human at a
daily dose of 20 g. Being an amphiphilic molecule, lecithin has the in-
herent property of self-assembling in aqueous environment (Angelico
et al., 2004; Javed et al., 2015). Its biocompatibility and amphiphilicity
has made it an attractive nano-carrier for entrapment and delivery of
various drug molecules. In the current study, AmpB, a highly hydro-
phobic drug, was entrapped in lecithin NPs (Lec-AmpB NPs), using
solvent diffusion method followed by freeze drying. In order to enhance
their colloidal stability, the freeze dried Lec-AmpB NPs were recon-
stituted in water containing 0.02% w/v SDS. To obtain Lec-AmpB NPs
with increased muco-adhesive properties, their surfaces were coated
with GT gold NPs through stirring.
3.2. Synthesis of GT-Au NPs
Metal NPs intended for drug delivery purposes are required to be
synthesized with biologically safe processes. Synthesis of Au NPs with
biopolymers has got recent attraction of scientists as they eliminate the
need of toxic reducing agents and can also work as modifying agents.
Furthermore, Au NPs synthesized and stabilized with biopolymers de-
monstrate greater stability against changes in pH and salt concentra-
tions (Dhar, Reddy, Shiras, Pokharkar & Prasad, 2008; Padil & Černík,
2013). Au NPs synthesis is based on the reduction of its atoms from
charged state to neutral state. Here, GT was exploited both as reducing
and stabilizing agent for green synthesis of NPs. UV–vis spectral ana-
lysis is the primary and most reliable tool for confirming the synthesis
of Au NPs. GT-Au NPs exhibited a single characteristic SPR peak at
554 nm for GT (3 mg/mL, 1 mL) with Au (5 mM, 0.1 mL). The char-
acteristic peak for GT-Au NPs was narrow and sharp having an absor-
bance of 1.45, as shown in Fig. 1(A). Thus, UV spectral analysis con-
firms the synthesis of maximum GT-Au NPs with almost homogenous
size distribution.
The synthesis of GT-Au NPs was further confirmed through FT-IR
analysis. The FT-IR spectrum of GT showed its characteristic peaks for
eOH and eCH at 3464.7 cm−1 and 2931.5 cm−1 respectively.
Similarly, peaks for eC]O and eCeO groups were evident at
1641.3 cm−1 and 1109.0 cm−1 respectively as shown in Fig. 1(B). The
FT-IR spectrum of GT-Au NPs revealed a characteristic peak at
3450.8 cm−1 that corresponds to stretching vibration of eOH of GT.
The peaks at 2933.0 cm−1 and 1643.0 cm−1 in GT-Au NPs FT-IR
spectrum are at their own places and correspond to eCH and eC]O of
GT. Similarly, the characteristic peak at 1083.8 cm−1 confirms the
stretching vibration of eCeO functional groups of GT. The changes in
GT characteristic peaks from 3464.7 cm−1 to 3450.8 cm−1 and
1109.0 cm−1 to 1083.8 cm−1 confirm that eOH and eCeO of GT are
actively involved in the reduction and stabilization of GT-Au NPs as
shown in Fig. 1(B).
3.3. Stability of GT-Au NPs
Metal nano-particles used in designing drug delivery systems are
desired to be enough stable and should not be denatured by the
T. Jabri et al.
Fig. 1. UV–vis spectral analysis of GT-Au NPs, GT (3 mg/mL, 1 mL): Au (5 mM,
0.1 mL) (A) and FT-IR spectra of GT and GT-Au NPs (B).
destabilizing effects of plasma proteins, saturated salts concentration
and harsh in-vivo acidic or basic environment. Therefore, such systems
are required to be screened for plasma, salt and pH stabilities prior to
their inclusion in drug cargo systems (Khandekar, Kulkarni, &
Devarajan, 2014). GT-Au NPs were screened for their pH stability by
changing the pH in a range of 2.5–12.5. The effects of acidity or basicity
on characteristic SPR of GT-Au NPs were noted. NPs were highly stable
in acidic pH range i.e. 2.5–4, with no obvious decrease in the intensity
Fig. 2. Stability of GT-Au NPs at (A) different pH, (B) salt concentrations and (C) plasma for 2 h incubation.
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Carbohydrate Polymers 194 (2018) 89–96
of SPR. As the pH was increased, i.e. 6.5-12.5, the SPR intensity got
slightly decreased but with no visible shift as shown in Fig. 2(A). Si-
milarly, the effects of various concentrations of NaCl on GT-Au NPs
were also studied. At lower salt concentrations, there was no effect on
the intensity of SPR, but higher concentration of 1 mM caused a mild
decrease in the intensity of SPR with no shifting effect as shown in
Fig. 2(B). This confirms that presence of Cl−1 does not cause any ag-
gregation effect in GT-Au NPs solution and thus they remain highly
stable in presence of higher salt concentration (Link & El-Sayed, 1999).
Upon incubation with plasma for 2 h, GT-Au NPs remained stable,
causing a slight decrease in SPR without any shift as shown in Fig. 2(C).
The stability of GT-Au NPs can be safely assumed form the results of
these investigations.
3.4. Characterization
3.4.1. Shape, size, PDI and zeta potential determination
Size is an important parameter for nano-carriers based drug delivery
systems as it determines the in-vivo performance as well colloidal sta-
bility of particles. Particles with smaller size also demonstrate lower
toxicity as compared to larger particles of the same carrier. Moreover,
particles of smaller size lead to better drug targeting and enhanced
phagocytic uptake (Imran, Shah, Ullah, Ullah, Sadiq et al., 2016; Shi,
Fang, & Pei, 2006). The size of the synthesized NPs was investigated
through zetasizer. GT-Au, Lec-AmpB and GT-Lec-AmpB NPs revealed an
average size of 177.5 ± 1.05, 243.7 ± 0.63 and 358.3 ± 1.78 nm
respectively as shown in Table 1 and Fig. 3(A–C). GT-Lec-AmpB NPs
showed comparatively larger size as compared to Lec-AmpB NPs. This
can be attributed to the adsorption of GT-Au NPs on their surfaces, thus
confirming their successful surface coating with muco-adhesive GT. The
PDI values of all the formulations are shown in Table 1. GT-Au, Lec-
AmpB and GT-Lec-AmpB NPs exhibited 0.280 ± 0.02, 0.32 ± 0.01
and 0.35 ± 0.02 PDI respectively. This indicates that GT-Au NPs are
distributed in almost same size particles population while Lec-AmpB
NPs are slightly varying in their size. GT-Lec-AmpB NPs are not mono-
dispersed and greatly vary in their size as shown in Fig. 3. This greater
T. Jabri et al. Carbohydrate Polymers 194 (2018) 89–96

Table 1
Characterization of GT-Au, Lec-AmpB and GT-Lec-AmpB NPs for size, polydispersity, zeta potential and drug entrapment efficiency.
Formulation Particle Size (nm) Polydispersity Index Zeta Potential (mV) Drug Entrapment Efficiency (%)

Lec-AmpB 243.7 ± 0.63 0.32 ± 0.01 −21.6 ± 0.21 86.54 ± 1.32

GT-Lec-AmpB 358.3 ± 1.78 0.35 ± 0.02 −19.9 ± 0.51 78.91 ± 2.44
GT-Au 177.5 ± 1.05 0.280 ± 0.02 −26.4 ± 0.35 –

size distribution can be attributed to the uneven adsorption of GT-Au
NPs on their surfaces. This further confirms the coating of these NPs
with muco-adhesive GT.
Zeta potential is another important parameter for nano-drug de-
livery systems. Its measurement predicts the colloidal formulations
stability upon their storage. Higher zeta potential is always advanta-
geous as it prevents the particles aggregation efficiently. Similarly, zeta
potential decreases the drug carriers uptake through reticulo-en-
dothelial system, thus decreases the drug clearance for the body and
drug remains in the biological system for a longer period of time with
ultimate increase in its therapeutic efficacy (Müller, 1991). Zetasizer
study revealed −26.4 ± 0.35, −21.6 ± 0.21 and −19.9 ± 0.51 mV
zeta potential values for GT-Au, Lec-AmpB and GT-Lec-AmpB NPs re-
spectively as shown in Fig. 3(D–F) and Table 1. Among all, GT-Au NPs
showed the highest negative zeta potential. This can be due to the
anionic nature of GT. The increased negative surface charge of all the
NPs predicts higher stability and better in-vivo performance for them.
The surface morphologies of the all the NPs were studied through AFM.
All the synthesized NPs revealed to be spherical in shapes as shown in
Fig. 4(A–C). The surfaces of GT-Lec-AmpB NPs are observed compara-
tively deep dark and spotted that confirms the coating of GT-Au NPs on
their surfaces.
3.4.2. FT-IR analysis
The components of drug delivery systems should not change the
chemical nature of their entrapped drug molecules. FT-IR analysis of
AmpB, Lec-AmpB and GT-Lec-AmpB was performed in order to in-
vestigate the possible interactions of the drug with the NPs building
blocks. The FTIR spectrum of AmpB reveals its characteristic peaks at
1597.8 cm−1 and 1104.1 cm−1 for NH3+ and CeOeC of β- glycosidic
linkage respectively. Similarly, characteristic peaks for its NH2 and
trans polyene chromophore appear at 1027.1 cm−1 and 985.0 cm−1
respectively. These characteristic peaks of the entrapped drug appeared
in the spectra of both Lec-AmpB and GT-Lec-AmpB as shown in Fig. 5.
Characteristic peak for NH3+ group of drug AmpB appeared at
1601.0 cm−1 and 1599.4 cm−1 in the FT-IR spectra of Lec-AmpB and

Fig. 3. Size and size distribution of (A) GT-Au NPs, (B) Lec-AmpB NPs and (C) GT-Lec-AmpB NPs and zeta potential of (D) GT-Au NPs, (E) Lec-AmpB NPs and (F) GT-
Lec-AmpB NPs.

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T. Jabri et al. Carbohydrate Polymers 194 (2018) 89–96

Fig. 4. AFM images of (A) GT-au NPs, (B) Lec-AmpB NPs and (C) GT-Lec-AmpB NPs.

solubilization in the lipophilic portion of lecithin. GT-Lec-AmpB NPs

entrapped relatively less amount of the drug as compared to Lec-AmpB
NPs. This slight decrease in drug entrapment of GT-Lec-AmpB NPs can
be due to their surface coating with GT that forces out the loosely
surface found drug. Results show that these NPs are able to entrap in-
creased concentration of AmpB, confirming the enhanced in-vivo per-
formance of formulations.

3.5. In-vivo oral bioavailability study

The oral bioavailability of AmpB was investigated in rabbits after its

Fig. 5. FT-IR spectra of (A) AmpB, (B) Lec-AmpB and (C) GT-Lec-AmpB.
GT-Lec-AmpB respectively. Similarly, peak for the drug CeOeC of β-
glycosidic linkage appeared 1107.5 cm−1 and 1101.9 cm−1 in the FT-IR
spectra of Lec-AmpB and GT-Lec-AmpB respectively. Peaks for the drug
NH2 and trans polyene chromophore also appear at the their respective
positions in the FT-IR spectra of Lec-AmpB and GT-Lec-AmpB. This
shows that the NPs ingredients do not interact chemically with AmpB
and the drug remains well intact within the NPs.
oral administration in the form of GT-Lec-AmpB, Lec-AmpB or its free
solution at 5 mg/ kg body oral dose. Fig. 6 shows the mean plasma
concentration-time curves of AmpB after its oral administration in the
form of aforementioned formulations. Various pharmacokinetic para-
meters were determined and are given in Table 2. AmpB given in the
form of GT-Lec-AmpB revealed elevated “maximum plasma con-
centration” (Cmax, 271.47 ± 12.54 ng/mL) as compared to those of
Lec-AmpB and drug free solution, showing 180.63 ± 17.69 and

3.4.3. Drug entrapment efficiency

Drug entrapment efficiency has been one of the most important
characteristics of NPs based drug delivery systems and defines the
overall success of the therapy. Increased drug entrapment efficiency is
required for delivering therapeutic substances to the target tissues in
higher concentrations, thus results in their better therapeutic efficacy.
Furthermore, drug delivery systems entrapping higher concentrations
of drugs are able to release them over an extended period of time. This
maintains the therapeutic levels of entrapped drugs for longer time,
thus eliminating the need for frequent dosing and their associated
toxicities (Imran, Shah, Ullah, Ullah, Elhissi et al., 2016; Singh & Fig. 6. Plasma drug concentration of AmpB loaded in GT-Lec-AmpB, Lec-AmpB
Lillard, 2009). Lec-AmpB and GT-Lec-AmpB NPs were able to entrap and as drug solution at different time intervals after oral administration at
86.54 ± 1.32% and 78.91 ± 2.44% of the drug respectively as shown 5 mg/kg body weight dose (n = 4, mean ± SEM). P value < 0.05 was con-
in Table 1. Higher drug entrapment efficiency of both the NPs can be sidered statistically significant (ns indicates “non significant” while “*, ** and
attributed to increased lipophilicity of AmpB that gets maximum ***” indicate “levels of significance from ‘significant’ to “highly significant”).

94
T. Jabri et al.
Table 2
Different pharmacokinetic parameters of AmpB after its oral administration in GT-Lec-AmpB, Lec-AmpB and solution at 5 mg/kg body weight dose. P value < 0.05
was considered statistically significant.
Pharmacokinetic parameters GT-Lec-AmpB
Dose (mg/kg body weight) 5 5
Cmax (ng/mL) 271.47 ± 12.54***
Tmax (h) 4 4
AUC0–24 (ng h/mL) 3,329.70 ± 22.19***
AUMC0–24 (ng.h2/mL) 32,948.12 ± 15.68***
MRT (h) 9.89 ± 0.43ns
Cl (L/h.kg) 1.50 ± 0.31ns
ns indicates "non significant" while "*" "88" and "***" indicate "levels of significance from "significant" to "highly significant".
114.87 ± 10.43 ng/mL Cmax respectively. This maximum absorption of
the drug through GT-Lec-AmpB can be due to its increased muco-ad-
hesive nature. The muco-adhesiveness of GT is mainly responsible for
increasing the contact of GT-Lec-AmpB with gastrointestinal mucosa as
described elsewhere for other drug delivery systems prepared with
muco-adhesive polymers (Takeuchi, Matsui, Yamamoto, & Kawashima,
2003; Wang et al., 2000). The size in nano-range can also be a con-
tributing factor for the enhanced oral bioavailability of such systems
(O'hagan, 1996; Ullah et al., 2017). The muco-adhesive nano-hybrid
carrier was also capable of keeping the drug plasma concentration
elevated throughout the study as compared to drug simple lecithin NPs
or solution. This predicts the muco-adhesive nano-hybrid carrier po-
tentials to keep the drug minimum therapeutic level in the blood for a
longer period resulting in the drug improved clinical efficacy.
Interestingly, the time to reach maximum plasma concentration
(Tmax) was significantly increased (4 h) for both GT-Lec-AmpB and Lec-
AmpB NPs as compared to simple drug solution (2 h). This shows that
both the NPs released the entrapped drug in sustained manner over a
longer period of time. This is also evident from the increased mean
residence time (MRT) values of both GT-Lec-AmpB and Lec-AmpB, re-
taining the drug in the biological system for 9.89 ± 0.43 h and
9.15 ± 1.08 h respectively. Furthermore, the AmpB clearance (Cl)
from the body was decreased when administered in the form of GT-Lec-
AmpB NPs (1.50 ± 0.31 L/h kg) as compared to Lec-AmpB NPs and
drug solution showing 2.42 ± 0.54 and 4.96 ± 0.40 L/h kg Cl re-
spectively. This validates the slow release of entrapped drug from GT-
Lec-AmpB NPs for an extended period of time. The comparatively
slower release of drug from GT-Lec-AmpB NPs can be due to its surface
coating with muco-adhesive GT (Yamamoto, Takeuchi, Hino, &
Kawashima, 2000). The drug oral administration in muco-adhesive
nano-carrier also increased its area under the curve (AUC0–24,
3,329.70 ± 22.19 ng h/mL) as compared to its simple lecithin NPs and
solution showing 180.63 ± 17.69 and 1,008.88 ± 18.36 ng h/mL
values of AUC0–24 respectively. Results of the current study confirm that
enhanced oral bioavailability was achieved for AmpB in a controlled
and sustained pattern through its delivery in surface coated muco-ad-
hesive hybrid lecithin NPs.
3.6. Conclusion
Improving the oral bioavailability of poor aqueous soluble drugs has
been a challenging task for formulation scientists. Nano-carriers based
drug delivery systems have recently offered promising solutions to such
problems. Lecithin NPs are increasingly preferred due to their higher
biocompatibility and chances for their surface modulation. Current
study was designed for fabricating muco-adhesive hybrid lecithin NPs
for enhancing AmpB oral bioavailability. Lecithin NPs were coated with
Au NPs of muco-adhesive biopolymer GT. NPs entrapped higher con-
centration of drug without changing its chemical nature and were found
in polydispersed nano-size range with spherical surface morphology.
Results of the current study confirm the effectiveness of novel hybrid
muco-adhesive lecithin NPs for enhancing oral bioavailability of AmpB
95
Carbohydrate Polymers 194 (2018) 89–96
Lec-AmpB AmpB Solution
5
180.63 ± 17.69 114.87 ± 10.43
2
2,060.95 ± 14.77 1,008.88 ± 18.36
18,852.00 ± 27.03 7,897.05 ± 15.92
9.15 ± 1.08 7.83 ± 0.95
2.42 ± 0.54 4.96 ± 0.40
in a fair controlled manner.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
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