International Journal of Biological Macromolecules 110 (2018) 133–139

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Amphotericin B loaded sulfonated chitosan nanoparticles for targeting

macrophages to treat intracellular Candida glabrata infections
Sandhya Murali a,1 , V. Aparna b,1 , Maneesha K. Suresh b , Raja Biswas b,∗ , R. Jayakumar b,∗ ,
S. Sathianarayanan a,∗
a
School of Pharmacy, Amrita Vishwa Vidyapeetham, Kochi, 682041, Kerala, India
b
Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi, 682041,
India

a r t i c l e i n f o a b s t r a c t

Article history: The current study assesses the potential of functionalised chitosan nanoparticles (CNPs) for proficient
Received 30 November 2017 macrophage delivery of amphotericin B (AmpB) for the management of Candida glabrata fungemia. Chi-
Received in revised form tosan was functionalised by the method of sulfation by using chlorosulfonic acid and the developed
29 December 2017
compound was confirmed by FTIR, 1 H NMR and degree of sulfation and CHNS analysis. Amphotericin B
Accepted 5 January 2018
encapsulated sulfated chitosan (AmpB-SCNPs), when characterized showed a hydrodynamic diameter
Available online 13 January 2018
of 310 ± 14 nm and zeta potential of 41.5 ± 2 mV. The safety of AmpB-SCNPs was established by the ala-
mar cytotoxicity assay in nanoparticle treated macrophages following 24 h incubation. The AmpB-SCNPs
Keywords:
Amphotericin B
showed a significant increase in the reduction of C. glabrata in comparison with the bare AmpB and
Sulfated chitosan AmpB-CNPs (55.2 and 42.7 vs 11.12 cfu/ml) indicating that AmpB-SCNPs could be a promising carrier for
Macrophages specific delivery of AmpB to macrophages for effective treatment of Candida glabrata fungemia.
Nanoparticles © 2018 Published by Elsevier B.V.
Targeted drug delivery
Candida glabrata

1. Introduction dida glabrata. AmpB binds to ergosterol, an essential component of

Intracellular pathogens are responsible for many severe
diseases, like tuberculosis, listeriosis and salmonellosis, etc. Intra-
cellular pathogens are protected from the host defence as well as
from the antimicrobial therapies, presenting the treatment regime
with a number of unusual challenges. 2/3rd of the antimicrobials
are unsuccessful against intracellular pathogens.
Fungemia caused by candida glabrata, a haploid yeast that inhab-
its inside the macrophages, is difficult to treat because ˇ-lactams
and aminoglycosides the most common antimycotics, have limited
cellular penetration due to their high hydrophilicity. The high stress
resistance and efficient iron acquisition capacity of C. glabrata aids
its survival within macrophages [1].
Amphotericin B (AmpB), a polyene antifungal produced by
Streptomyces nodosus is the most common drug used against Can-
∗ Corresponding authors.
E-mail addresses: rajabiswas@aims.amrita.edu (B. Raja),
rjayakumar@aims.amrita.edu (J. R.), sathyanarayanans@aims.amrita.edu (S. S.).
1
These authors contributed equally.
the fungal cell membrane, leading to depolarization of the mem-
brane, alteration of cell membrane permeability and eventually
cell death [2]. AmpB will bind not only to ergosterol but also to
cholesterol in the host cells, which makes it highly toxic. This
toxicity can be reduced by delivering the drugs to specific loca-
tions (where the bacteria resides), thereby ensuring reduced drug
dosage. Nanomedicine has the capacity to make intracellular dis-
ease therapy better by facilitating properties like drug targeting,
sustained drug release and delivery to the pathogen’s intracellu-
lar location [3–5]. Here nanoparticles can be engineered to deliver
the drugs into the macrophages thereby ensuring better antimi-
crobial action [6]. Sulfated polysaccharides can be easily employed
for macrophage targeting the mechanism behind being the effi-
cient binding of the sulfate domain of the polysaccharide with the
cysteine rich domain of mannose receptor [7].
Chitosan, a linear polysaccharide extracted from crustacean
shells has been widely for the development of biocompatible drug
delivery systems [8,9]. Drug activity can be further enhanced by
the application of chitosan nanoparticles instead of chitosan solu-
tion [10–14]. Chemical modification of chitosan with sulfate would
not change the fundamental skeleton or physicochemical and bio-

https://doi.org/10.1016/j.ijbiomac.2018.01.028
0141-8130/© 2018 Published by Elsevier B.V.
134 M. Sandhya et al. / International Journal of Biological Macromolecules 110 (2018) 133–139
chemical properties of chitosan, but would bring new or improved
properties like macrophage targeting [15–17]. Here the possibil-
ity of using the generated Amphotericin B encapsulated sulfated
chitosan nanoparticles (AmpB-SCNPs) to kill the intracellular C.
glabrata was examined.
2. Materials and methods
2.1. Materials used
Chitosan (KOYO, Japan), Amphotericin B (LYKA), Chlorosul-
fonic acid, Tripolyphosphate (TPP) (Sigma-Aldrich), Pyridine, Citric
acid monohydrate, Nitric acid (Nice chemicals), Glacial acetic acid
(Sd Fine Chem), Sodium hydroxide, Potassium chloride, Sodium
chloride, Potassium dihydrogen phosphate, Disodium hydrogen
phosphate and Hydrochloric acid (Merck).
2.2. Preparation of sulfated chitosan
4 g of chitosan was dissolved in 15 ml of 1% acetic acid in a
beaker. To this, pyridine was added and noted as solution A. Chloro-
sulfonic acid was added dropwise to a three necked round bottom
flask containing 80 ml of dry pyridine under nitrogen atmosphere.
This solution is noted as solution B. 8 ml of solution B was added
dropwise to solution A under nitrogen atmosphere. The final solu-
tion is heated to 60 ◦ C for 3 h and rapidly cooled. To this, added
200 ml of water, 75 ml of 2.5 N NaOH and 400 ml of ethanol and
maintain the pH at 7.4. The solution is then filtered and the precip-
itate is washed twice with deionized water and ethanol [15].
2.3. Characterization of prepared sulfated chitosan
2.3.1. Estimation of sulfation
The C H N and S was estimated using Vario EL III elemental
analysis, the degree of sulfation was estimated by barium chloride
method. Briefly, 1 g of the sulfated chitosan was dissolved with 2 ml
of HCl and HNO3 solution and further mixed with 2 ml of barium
chloride to obtain a precipitate. The weight of the precipitate was
calculated and degree of sulfation was estimated by atomic mass
method [18].
2.3.2. Zeta potential, FTIR and NMR
DLS utilizing DLS-ZP/molecule sizer NicompTM 380 ZLS was
used for the determination of zeta potential. Fourier Transform
Infrared Spectrophotometer (FT-IR, SHIMADZU) was used to ver-
ify the sulfation of chitosan. The chemical structure of the product
was characterized by using NMR spectroscopy, where deuteriated
ethanol was used as a solvent and TMS was used as an internal
standard. NMR study was done with Bruker Fourier- NMR spec-
trometer.
2.4. Cell viability studies of sulfated chitosan
The Raw 264.7 macrophage cell line was cultured in
RPMI medium supplemented with 10% FBS, 1% (v/v) antibi-
otic/antimycotic solution at 37 ◦ C in a 5% CO2 containing balanced
air incubator. The cell viability was determined using alamar assay.
103 cells were added in 96 well plated and incubated for 12 h, then
the medium was removed and the medium with different concen-
trations of sulfated chitosan. It is then further incubated for 24 h
and 48 h time point. Instead of sulafted chitosan, PBS was added in
positive control well (blank) and cells incubated with 1% triton X
were treated as negative control. Following incubation the media
was replaced with 10% alamar solution and the reduction of the
alamar compound is measured after 4 h [19].
2.5. Heamocompatibility studies of sulfated chitosan
Blood samples were obtained from healthy individuals in EDTA
containing vacutainers, diluted 1:10 using PBS and aliquoted in
different eppendorfs. Increasing concentrations of sulfated chi-
tosan were added to the diluted blood. 1% Triton X-100 treated
blood samples were used as positive control. Untreated blood solu-
tion served as negative control. The samples were incubated for
24 h in an incubator chamber at 37 ◦ C. The samples were then
centrifuged at 3000 rpm (Eppendorf 5418 R) for 10 min at room
temperature. 200 ␮l of the supernatants was collected and pres-
ence of hemoglobin in the supernatants was measured at 540 nm
using a microtiter plate reader (Biotek Power Wave XS, USA).
2.6. Preparation of sulfated chitosan nanoparticles (AmpB-SCNPs)
Sulfated chitosan nanoparticles were synthesized by the
method of ionic cross linking. 0.25 g of sulfated chitosan was dis-
solved in 10 ml of 1% acetic acid. 8 ml 0.5% (w/v) of tripolyphosphate
(TPP) was added dropwise into the sulfated chitosan solution by
using a micropipette under constant stirring and the appearance
of turbidity was analysed. The solution was continuously stirred
for 30 min at 600 rpm in a magnetic stirrer. The sulfated chitosan
nanoparticles (SCNPs) were then harvested by centrifugation at
12,000 rpm for 15 min and washed with water twice to remove
acetic acid content [20].
2.7. Preparation of Amphotericin B loaded sulfated chitosan
nanoparticles (AmpB-SCNPs)
Amphotericin loaded sulfated chitosan nanoparticle were pre-
pared by ionic gelation method. 10 ml of 0.25% (w/v) of sulfated
chitosan solution was prepared with 1% acetic acid. 1 mg of Ampho-
tericin B was added into this solution and stirred for 30 min at
600 rpm. Add 0.5% (w/v) TPP dropwise by using a micropipette into
prepared solution until it forms a turbidity. This solution was mixed
well for 30 min by stirring at 600 rpm and centrifuged for 9500 rpm
for 15 min to yield AmpB-SCNPs.
2.8. Characterisation of nanoparticles
2.8.1. Size, zeta and FTIR
Dynamic light scattering and Zeta potential (DLS utilizing
DLS-ZP/molecule sizer NicompTM 380 ZLS) were used for the deter-
mination of size, distribution and zeta potential. Functional groups
were also analysed using FTIR.
2.8.2. TEM analysis
Surface morphology and the size were studied by TEM
(Transmisssion Electron Microscopy). Diluted nanoparticles were
dropped on carbon coated copper grid and allowed to dry. TEM
images were taken for the analysis.
2.9. Encapsulation efficiency determination
The encapsulation efficiency, determined as the amount of
AmpB encapsulated into the nanoparticles to the initial amount
of AmpB added was determined by the measurement of free AmpB
obtained in the supernatant after the AmpB-SCNPs were prepared.
2.10. Stability of AmpB-SCNPs
AmpB-SCNPs were suspended in PBS at a concentration of
1 mg/ml. These nanoparticles (NPs) were stored for 1 month at tem-
perature conditions: 5 ± 2 ◦ C, 30 ± 2 ◦ C and 70 ± 2 ◦ C, to determine
their physical and chemical stabilities. Aliquots were withdrawn
 M. Sandhya et al. / International Journal of Biological Macromolecules 110 (2018) 133–139 135

at regular intervals and assessed for their particle size and their
encapsulation efficiencies [21].

2.11. In vitro AmpB release and kinetics of release

The kinetics of the AmpB release form the prepared NPs were
studied by spectrophotometry. In order to determine the release of
AmpB from SCNPs citrate buffer solution (CBS) was used. The 1 ml of
freshly prepared AmpB-SCNPs was redispersed in 1 ml CBS at 20 ◦ C
in. One eppendorf was taken at regular time intervals, centrifuged
and the supernatant was used for determining AmpB concentration
at 406 nm [22]. Data obtained from in vitro release studies were
further evaluated to determine kinetic models drug release (Zero
order, First order, Higuchi model and Korsemeyer-Peppas Kinetic
models).

2.12. Cytotoxicity and hemolysis of AmpB-SCNPs

The cytotoxicity of increasing concentrations of AmpB-SCNPs

was studies by the previously reported alamar assay. Cells incu-
bated in the medium without AmpB-SCNPs served as positive
control (Blank) and cells treated with 1% triton X were treated as
negative control.
The effect of increasing concentrations of AmpB-SCNPs (0.5, 1,
2.5, 5, and 10 ␮g/ml) and 10 ␮g/ml of AmpB-GNPs in blood cells
Fig. 1. Reaction scheme is showing the preparation of sulfated chitosan.
were analysed by hemolysis test. Blood treated with 1% Triton X
served as positive control and PBS acted as negative control.

3.1.2. FTIR and NMR analysis

2.13. Efficacy studies with AmpB loaded nanoparticles
Antibiotic/antimycotic free DMEM medium was used for cul-
turing RAW 264.7cells for intracellular survival assays. Confluent
monolayers of RAW 264.7 cells were infected with C. glabrata at
a multiplicity of infection (MOI) of 1:10. After 3 h the cells were
washed thrice with PBS to remove the nonadherent fungi and sub-
sequently incubated for an additional 90 min with 25 ␮g/ml of
AmpB to kill the extracellular fungi. The C. glabrata internalized
cells were further incubated with 2× MIC of bare AmpB, AmpB-
CNPs and AmpB-SCNPs. After 6 h the cells were lysed using 0.1%
(v/v) triton X-100. The lysates were further diluted and plated on
SD Agar plates. The number of colonies formed on the agar surface
after 24 h incubation was counted [23,24].
All the experiments were carried out in triplicates and the
results were represented as average ± standard deviation. The sig-
nificance between samples was analysed using Student’s t-test.
The chitosan FTIR spectra exhibited peaks of NH3 + (protonated
amino group) vibrations at 1150 cm−1 and C O (carboxyl group)
stretching of the secondary amide group at 1650 cm−1 . The peaks
at 1150 and 1026 cm−1 designated asymmetric C O C stretching
and C O skeletal vibrations of chitosan. The characteristic peaks
of sulfated chitosan spectrum at 1160–1260 and 845 cm−1 were
linked with S O asymmetric stretching and C O S stretching of
the sulfate group [18].
1 H NMR data of chitosan showed signals at 2.7 and 1.9 ppm,
which is due to the presence of two protons in the hydroxyl group.
In sulfated chitosan the peak at 3.07 ppm shifted down field due
to the elecronegativity of S and O present in the SO3 H group.
This explains the mesomeric effect of sulfated chitosan [25,26].
The other peaks of the chitosan remained unaffected in sulfated
chitosan (Fig. 2).
3.2. Cytocompatibility and hemolytic activity of sulfated chitosan

3. Results and discussion
3.1. Preparation and characterisation of sulfated chitosan
Sulfated chitosan was synthesized according to the proto-
col mentioned and was further characterized. Sulfonation was
achieved in alcohol using chlorosulfonic acid (Fig. 1). The hydroxyl
group of the chitosan was reacted with the chlorosulfonic acid and
then the hydrochloric acid was removed in presence of pyridine
to form SO3 H. Chlorosulfonic acid is a mild oxidating agent and
therefore the reaction was carried under nitrogen atmosphere.
The cytotoxicity of the different concentrations of sulfated chi-
tosan tested in vitro on RAW 264.7 cells showed (Fig. 3A) no
significant difference in the viability of the cells in comparison to
the control. Alamar assay showed that the cells remained viable at
all the tested concentrations. The hemolytic activities of the mate-
rial were investigated and the results are shown in Fig. 3B. The
results illustrated that the synthesized compound didn’t cause any
detectable disturbance to the erythrocyte membranes. The material
caused less than 5% disruption of blood cells.
3.3. Preparation and characterisation of sulfated chitosan
nanoparticles

3.1.1. Zeta analysis 3.3.1. Size and zeta analysis

The zeta potential was done to determine the surface charge of Hydrodynamic size of nanoparticles was obtained using
the product and the charge of chitosan and sulfated chitosan was dynamic light scattering method. 1 mg/ml of nanoparticles were
found to be +30.7 ± 3 mV and −32.5 ± 7 mV respectively. The shift dispersed in distilled water and sonicated before particle size
in zeta potential supported the presence of sulfate group. determination. SCNPs had a diameter of 120 ± 7 nm with a poly
136 M. Sandhya et al. / International Journal of Biological Macromolecules 110 (2018) 133–139

Fig. 2. (A) FTIR image showing successful sulfation of chitosan. NMR data of chitosan (B) and sulfated chitosan (C).

Fig. 3. Biocompatibility assays: (A) RAW 264.7 cell line was checked for viability after 24 and 48 h of treatment with increasing concentrations of sulfated chitosan by means
of alamar blue assay. Here, Blank represents cells alone, C1-1 ␮g/ml Chitosan solution, SC1-1 ␮g/ml Sulfated chitosan solution, SC2-3 ␮g/ml Sulfated chitosan solution, SC3-
5 ␮g/ml Sulfated chitosan solution, SC4-7 ␮g/ml Sulfated chitosan solution and SC5-9 ␮g/ml Sulfated chitosan solution (B) Bar diagram showing % hemolysis by increasing
concentrations of sulfated chitosan using 1% TX-100 as positive control. Data represent the average ± SD and were analysed by paired Student’s t-test, **P < 0.01, and *P < 0.05.

dispersity index (PDI) of 0.283 and AmpB-SCNPs had a diameter Morphology of these nanoparticles were confirmed by further
of 310 ± 14 nm with PDI of 0.321. The zeta potential value of plain using TEM. The nanoparticles were spherical in morphology and
and the drug loaded sulfated chitosan nanoparticle was found to be the average diameter of SCNPs and AmpB-SCNPs were obtained as
−30.5 ± 7 and −41.5 ± 2 mV respectively. 120 ± 17 nm and 290 ± 24 nm respectively (Fig. 4A).
 M. Sandhya et al. / International Journal of Biological Macromolecules 110 (2018) 133–139
Fig. 4. (A) Morphology of SCNP and AmpB-SCNP as visualized using Transmission electron microscope, (B) FTIR of sulfated chitosan, AmpB, SCNPs and AmpB-SCNPs provig
successful entrapment of AmpB in SCNPs, Stability studies of AmpB-SCNPs by observing (C) Particle size and (D) entrapment efficiency for 90 days and (E) Drug release profile
of AmpB from SCNPs in citrate buffer (pH 5).
3.3.2. FTIR analysis
The peaks at 1103 cm−1 show the C O stretching, characteristic
peak at 1500 cm−1 shows the C N, peak at 1603 cm−1 shows C O
stretching, peak at 3030 cm−1 shows the aromatic C H stretch-
ing and characteristic c peak at 2600–3200 cm−1 shows the O H
stretching of AmpB. AmpB-SCNPs had the characteristic peaks of
all its individual components (Fig. 4B).
3.4. Stability of the AmpB-SCNPs
EE of Amphotericin B in nanoparticle was assessed by drug
loss through the supernatant and was found to be 89 ± 0.4%.
The purpose of stability study was to provide evidence that
the product remain stable for a specified period of time and
it is carried out at room temperature (30 ± 2 ◦ C) at refrigerated
temperature condition (4 ± 2 ◦ C) and at accelerated temperature
(70 ± 2 ◦ C). The particle size and percentage entrapment efficiency
(EE) were measured to ensure that the product characteristics
and drug content of the product remain unchanged. At accel-
erated temperature, the product shows significant variation in
the particle size and entrapment efficiency due to the degrada-
tion of polymers whereas at refrigerated temperature and the
room temperature, product did not show any significant variation
(Fig. 4C). Therefore, from the results we can say that the storage
of AmpB-SCNPs at room temperature and refrigerated condition is
preferable.
137
3.5. Release of AmpB and its kinetics
The release of Amphotericin B from the nanoparticle was
observed in pH 5 (citrate buffer) and the determined with respect
to time. Figure shows the release profile of Amphotericin B from
SCNPs which measured over a period of 72 h. The sulfated moiety
in the nanoparticle prohibited the initial burst release of Ampho-
tericin B and it shown the sustained release of 96% at 72 h. Fig. 4E
demonstrated the sustained release of Amphotericin B from SCNPs.
Initially the drug release from SCNPs was less and it was reached
to 96% on 3rd day. The kinetic modelling of release followed
korsmeyer peppas model and it confirmed the non-Fickian diffu-
sion with release exponent (n) value of 0.956.
3.6. Cytocompatibility and hemolytic activity of AmpB-SCNPs
The minimum fungicidal concentration of CMC-AmpB-GNPs
against C. glabrata was obtained as 1.25 ␮g/ml by RPMI dilution
method. The cytotoxicity of the NPs tested in vitro on RAW 264.7
cells showed (Fig. 5A) no significant difference in the viability of
the cells in comparison to the control. Alamar assay showed that
the cells remained viable at all the tested concentrations.
The hemolytic activities of the NPs were investigated and the
results are shown in Fig. 5B. The formulation caused less than 5%
disruption of blood cells. The results illustrated that the NPs didn’t
cause any detectable disturbance to the erythrocyte membranes.
138 M. Sandhya et al. / International Journal of Biological Macromolecules 110 (2018) 133–139

Fig. 5. Biocompatibility assays (A) RAW 264.7 cell line were checked for viability after 24 h and 48 h of treatment with increasing concentrations of NPs by means of alamar
blue assay. Here, Blank represents cells alone, C1: 3 ␮g/ml of chitosan nanoparticle, C2: 3 ␮g/ml of Amphotericin B loaded CNPs, S0-1 ␮g/ml of Sulfated chitosan nanoparticles,
S1-AmpB SCNPs 1 ␮g/ml, S2-AmpB SCNPs 3 ␮g/ml, S3-AmpB SCNPs 5 ␮g/ml, S4-AmpB SCNPs 7 ␮g/ml, S5-AmpB SCNPs 9 ␮g/ml (B) Bar diagram showing % hemolysis by
increasing concentrations of NPs using 1% TX-100 as positive control. (C) Decreased survival of intracellular C. glabrata in RAW 264.7 cells when treated with CMC-AmpB-GNPs
compared to bare AmpB and AmpB-GNPs at MOI 1:10. Data represent the average ± SD and were analysed by paired Student’s t-test, **P < 0.01, and *P < 0.05.

3.7. Efficacy studies with AmpB loaded nanoparticles
We then assessed whether there was any difference between
the efficiencies of free drug and nanoparticles during 24 h post
infection treatment. Raw 264.7 cells (105 /well) were infected with
104 IFU/well C. glabrata. Cells were treated for 24 h after infection
with AmpB, AmpB-CNPs and AmpB-SCNPs after the removal of
unphagocytosed yeast. The cells were the washed, lysed and the
numbers of intracellular C. glabrata colonies were counted (Fig. 5C).
The intracellular yeast burden was significantly less in AmpB-
SCNPs treated group (11.12 ± 4.7%) in comparison with bare AmpB
group (55.26 ± 11.7%) and AmpB-CNPs 42.7 ± 12.8%. The formula-
tion was efficient in the removal of intracellular Candida glabrata.
The better antifungal activity of AmpB-SCNPs may be because
AmpB-SCNPs are better phagocytosed by the Raw 264.7 cells than
AmpB-CNPs due to the presence of the sulfate group. We assume
the uptake of AmpB-SCNPs is via the cysteine rich domain of man-
nose receptor whereas AmpB-CNPs is via general endocytic process
[7]. The bare AmpB is diffused into the cells leading to poor pene-
tration and less antifungal activity.
4. Conclusion
In this study a straightforward and simplistic method was devel-
oped to make stable AmpB-SCNPs. The AmpB-SCNPs was prepared
and it was characterized by FTIR and 1 H NMR. The size of the
prepared AmpB-SCNPs is approximately 300–310 nm. The AmpB-
SCNPs was capable to release the AmpB in a sustained manner. The
developed particles were compatible with blood and RAW 264.7
cells. The synthesized nanoparticles showed superior intracellu-
lar killing of Candida glabrata in infected RAW 264.7 macrophage
cell lines. The better antifungal activity of AmpB-SCNPs could
be unambiguously attributed to the presence of sulfate group in
AmpB-SCNPs. This strategy can be used to synthesize new thera-
peutics for treating chronic and stubborn infection associated with
intracellular pathogens.
Acknowledgments
The authors are grateful to Centre for Nanosciences and Molec-
ular Medicine, Amrita University for infrastructure support. The
authors are grateful to Dr. Sabitha M, Principal, Amrita School
of Pharmacy, for continuous support and for providing the facili-
ties for this study. The authors are also grateful to Nanomission,
Department of Science and Technology (DST) Reference No:
SR/NM/PG-01/2015, Government of India, for providing M. Tech.
PG Program research grant to Centre for Nanosciences and Molec-
ular Medicine, Kochi. We thank Mr. Sajin P. Ravi and Ms. Arya Raju
for their help in electron microscopy.
 M. Sandhya et al. / International Journal of Biological Macromolecules 110 (2018) 133–139
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