International Journal of Medicine and

Pharmaceutical Science (IJMPS)

ISSN(P): 2250-0049; ISSN(E): 2321-0095
Vol. 6, Issue 5, Oct 2016, 1-8
TJPRC Pvt. Ltd.

PREPARATION, CHARACTERIZATION AND IN VITRO RELEASE PROFILE OF

KETOCONAZOLE LOADED CHITOSAN NANOPARTICLE
KUSHAGRI SINGH, ABHA MISHRA2 & AMIT SINGH3
1
2, 3

School of Biochemical Engineering, Indian Institute of Technology, BHU, India

Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, India

ABSTRACT
Ketoconazole loaded chitosan nanoparticles were prepared for the treatment of fungal infections. Nan
formulation of ketoconazole was prepared using 85% deactivated chitosan as a biodegradable polymer and
tripolyphosphate as a crosslinking agent by ionotropic gelation method. It was further evaluated and characterized on the
basis of morphology, drug loading efficiency, zetapotentialvalue, FTIR study and also In-Vitro release behavior of
ketoconazole. The FTIR spectral studies indicated that there was no interaction between the drug and chitosan.
The formulation CS1 was found stable with good drug entrapement efficiency, fair zeta potential value and its size ranged
between 50-70nm. The in-vitro study showed sustained release with steady rise in cumulative drug release (> 90%) upto
about 8h and thereafter no significant release. The characterized formulations were also used to study antifungal activity.

vehicle for the fungal infection treatment.

KEYWORDS: Chitosan, Nanoparticles, Ketoconazole, Ionotropic Gelation, Cumulative Drug Release

Received: Jul 22, 2016; Accepted: Aug 19, 2016; Published:Aug 23, 2016; Paper Id.:IJMPSOCT20161

Original Article

All the study results showed that ketoconazole loaded chitosan nanoparticle can be used as an effective drug delivery

INTRODUCTION
Chitosan is a polysaccharide derived from deacetylation of naturally occurring chitin. Its unique properties
make it attractive for many industrial and biomedical applications (including controlled drugrelease, wound healing,
nutrition supplements, water purification, removal of toxins and semi-permeable membranes)(1-4).Chitosan matrix
for nanoparticle preparation has advantage over traditional and intravenous methods of administration in terms of
efficiency & effectiveness. It can deliver a higher concentration of pharmaceutical agent to a desired location in a
controlled manner. Chitosannanoparticles are easy to synthesize and are biocompatible, biodegradable, non-toxic,
water soluble and requires very less use of solvents for preparation. The major aim of synthesizing chitosan
nanoparticles is to control size of particle, their surface properties and release of pharmacologically active agents in
order to achieve efficient and controlled release of drugs (5,6).
Chemistry of Chitosan
Chitosan is prepared by the deacetylation of chitin, a linear polymer of (1-4) linked
N-acetyl-D-glucosamine units composed of mucopolysaccharides and amino groups. Chitosan is a linear randomly
distributed heteropolysaccharide composed of (1-4) linked 2-acetamido-2-deoxy--D-glucopyranose and
2-amino-2-deoxy--D-glucopyranose units as shown in Figure 1(7-10).

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Figure 1: Chitosans Structure

Ketoconazoleis an azol-based broad spectrum antifungal drug, which inhibits the biosynthesis of ergosterol in
fungal cell wall, as well as the absorption of DNA and RNA precursors to increase the cell permeability of mycetes and
therefore lead to further damage of mycetes (11). Ketoconazole is also classified in the Bio-pharmaceutics classification
system (BCS) as a class II drug. Although the compound has enough good permeability, their solubility in GI fluids is
insufficient to dissolve the administered dose under normal conditions (12,13). Ketoconazole is widely prescribed to treat
fungal infections. However, due to its hydrophobic nature and poor solubility in GI fluids, variations in bioavailability have
been documented. Among other particulate vehicles, polymeric nanoparticles such as chitosan nano formulations have
presented their great potential in solubilization of poorly water soluble drugs such as Ketoconazole. In the present study,
chitosan nanoparticles containing ketoconazole were prepared using ionotropic gelation method given by Calvo et al in
1997(14,15). The developed formulations were further characterized for size, shape, zeta-potential, FTIR study, entrapment
efficiency and in-vitro release. They were also assessed for anti-fungal activity against Corioulussp., a fungus.

MATERIALS & METHODS

Chitosan with molecular weight of 200kD &TPP of Sodium salt purchased from Sigma Aldrich, Mumbai, India.
The minimum degree of deacetylation of chitosan was 85% (the data provided by the company). Ketoconazole was
obtained as a gift from Hi-Media laboratory, Mumbai, India. All other reagents used were of analytical grade.
Chitosan Nanoparticle Preparation
Chitosan nanoparticlewas prepared using ionotropicgelation method by Calvo et al, 1997. 1% acetic acid solution
was prepared in distilled water. Different concentrations of chitosan solution were prepared by dissolving chitosan in
concentrations suchas 1mg/ml, 2mg/ml&3mg/mlin 1% acetic acid solution.1mg/ml concentration of ketoconazole added to
each chitosan solution.100ml of TPP solution was prepared by dissolving 0.85gms of TPP in 100ml distilled water.TPP
solution was added drop-wise using syringe to chitosan solution in the ratio 1:2 under constant magnetic stirring for 1 hour.
The resulting suspension was subsequently centrifuged at 15,000RPM for 20mins. The pellets obtained were freeze dried
using lyophilizer. The dried pellets were collected for characterization and in-vitro release study.
Measurementof Particle Size & Morphology Study
The ketoconazole loaded nanoparticle size & morphology was determined by Scanning electron microscope study
(Department of Metallurgical Engineering, IIT (BHU), Varanasi) under low vacuum condition.
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Preparation, Characterization and in Vitro Release Profile of Ketoconazole Loaded Chitosan Nanoparticle

Determination of ZetaPotential
The zetapotential of drug loaded chitosan nanoparticles was measured on a Zetasizer (Malvern Instruments) by
determining the electrophoretic mobility in micro electrophoresis flow cell. All the samples were measured in water at
25C in triplicates.
Determination of Entrapment Efficiency
The encapsulation efficiency of nanoparticles was determined by first separating the nanoparticles formed from
the aqueous medium by ultracentrifugation at 15,000RPM for 30mins. The amount of free ketoconazole in the supernatant
was measured by UV spectrophotometer at 257nms. The ketoconazole entrapped in the nanoparticles was calculated as:
Entrapment Efficiency (%) = (Tp-Tf) 100/Tp
Where Tp is the total ketoconazole used to prepare the nanoparticles and Tf is the free ketoconazole in the
supernatant.
Practical Yield Calculation & in-Vitro Release Profile
Freeze dried nanoparticles were collected and weighed to determine practical yield (PY) from following equation:
PY% = Nan particle weight / Theoretical mass (polymer+drug+TPP) 100
For the FTIR study a specified quantity of potassium bromide and samples were blended uniformly. The resultant
blend was then compressed to prepare the pellet as desired. The pellet was subjected for the analysis.
Assessment of Antimicrobial Activity
PDA media was prepared and autoclaved. The strain selected for the study, was Coriolus sp. Thefungal strain was
cultured on the PDA medium. PDA plates were prepared by pouring 20ml of the media into a sterile petridish. fungus was
spread over the media and incubated at 30C for 2 h for stabilization. Further 40l of free drug and ketoconazole loaded
chitosan nanoparticles were dropped over the plate on the marked points. Plates were then kept for incubation at 30C for
48h. Zone of inhibition were determined for both samples after 24 and 48hrs to evaluate extent of fungal inhibition by the
preparation.

RESULTS & DISCUSSIONS

The most satisfactory nanoparticles of chitosan were obtained at a chitosan concentration of 1 mg/ml in 1% acetic
acid and TPP of 0.85%(w/v) in d/w.
Measurement of Particle size &Morphology Study
It is evident from the SEM study of nanoparticles sample that size ranges between 50-70 nm for the sample with
1mg/ml chitosan concentration and with the increase in chitosan concentration size of nanoparticles also increases. Further
clear and uniform morphology was observed (Figure 1).

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Figure 2: SEM Images of Ketoconazole Loaded Chitosan Nanoparticles

Entrapment Efficiency
The results of entrapment efficiency revealed that drug entrapment efficiency is dependent on the polymer
concentration Figure 2. The entrapment efficiency found to be maximum for the CS I formulation and it decreases with
increasing polymer concentration at physiological pH and temperature.
Table 1: Entrapment Efficiency of different Concentrations of Polymer and Drug
Serial No.
1. CS I
2. CS II
3. CS III

Chitosan
Concentration(Mg/Ml)
1
2
3

Drug Concentration
(Mg/Ml)
1.0
1.0
1.0

Entrapment
Efficiency (%)
69.70
50.20
46.60

Figure 3: Bar Diagram Showing Effect of Chitosan Concentration on Efficiency of

Drug Entrapment by Nanoformulationat Physiological pH and Temperature
Practical Yield and In-Vitro Release Profile
Practical yield for Ketoconazole loaded CS 1 nanoparticle was found to be 39.07%. The in-vitro release profile of
ketoconazole loaded chitosan nanoparticles formulation indicates that the formulations exhibit sustained release with a
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Preparation, Characterization and in Vitro Release Profile of Ketoconazole Loaded Chitosan Nanoparticle

steady rise in cumulative drug release (>90%) upto 8 hours. Thereafter, there was no further significant release observed
(Table 2, 3 &Figure 3).

Figure 4: Cumulative Drug Release Profile from Nanoparticle Formulations Upto 8h

Fourier Transform Infrared study
The ability of the ionotropic gelation process to form ketoconazole-loaded chitosan nanoparticles was assessed by
employing FTIR to determine ketoconazole-chitosan interactions. The FTIR spectra of chitosan matrix (a) (16), and
ketoconazole loaded chitosan nanoparticles (b) are shown in Figure 3. In chitosan spectra, the peak for asymmetric stretch
of C-O-C found at around 1023 cm and in drug loaded chitosan nanoparticles at 1085 cm. The tip of the peak in drug
loaded chitosan nanoparticles had been shifted from 3420 cm to 3398 cm and becomes wider with the increase in
relative intensity. The cross-linked chitosan in spectra (b) showing P=O peak at 1224 cm. It may be suggested that the
tripolyphosphoric groups of TPP are linked with ammonium groups of chitosan in spectra (b). The inter - and
intra-molecular interactions are enhanced in chitosan nanoparticles but there was not much difference in the peaks found in
drug loaded nanoparticles. Therefore, it was observed that ketoconazole entrapped in chitosan nanoparticles matrix without
any interaction.

Figure 5: FTIR Spectra of chitosan Matrix (a), & Ketoconazole Loaded

chitosanNanoparticle (b)

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Zetapotential Determination
Zetapotential value for the chitosan nanoparticle was found to be-0.69mv (Figure 4). Zetapotential values provide
an important criterion for the stability of colloidal system. This is the electric potential that exists at the shear plane of a
particle, which is related to both the surface charge and local environment of the nanoparticle. Value of zetapotential
between +10mv to -10mv indicates that the particles are neutral and stable in nature. Therefore the CS1 formulation is
neutral.

Figure 6: Zeta Potential Distribution Plot for the Drug Loaded Chitosan Nanoparticle
Antifungal Activity
Table 4 indicates that all the test preparations containing the ketoconazole containing chitosan nanoparticles,
including the free drug demonstrated that zone of clearance was seen in both the cases for upto 72 hours of incubation.
The commercial preparations of ketoconazole in comparison to the ketoconazole loaded chitosan nanoparticles exhibited
similar antifungal activity within 48h but thereafter, showed very little high activity in 72h.
Table 4: Evaluation of Antimicrobial Activity of Drug
Alone and Drug Loaded Chitosan Nanoparticle
Serial
Number
1.
2.

Impact Factor (JCC): 5.6329

Test Sample

48h

72h

Plain drug(ketoconazole)
Ketoconazole
loaded
chitosan nanoparticle

1.5cm

2.8cm

1.5cm

2.9cm

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Preparation, Characterization and in Vitro Release Profile off Ketoconazole Loaded Chitosan Nanoparticle

Figure 7: Zone of Clearance in the Lawn of CorioulusSp. by Free Drug and

Drug Loaded Chitosan Nanoparticle after 72h of Incubation

CONCLUSIONS
This work confirms
firms that the stable ketoconazole loaded chitosan nanoparticles formulation is useful and efficient
enough to treat fungal infections. They are prepared to maintain a controlled and effective
fective release of drug and thereby
reduces toxicity and patient compliance. With this work, three different formulations of chitosan nanoparticles were
developed using differentconcentrations of polymer and they showed different entrapeement efficiencies as well as
cumulative drug release profile. Three formulations were evaluated where CS1 was found to be most suitable for the
delivery of ketoconazole because of its small particle size with more surface area,, fair zetapotential value and good
entrapment efficiency. All the formulations were observed to have stability even after 60days upto storage temperature
of40C.

ACKNOWLEDGEMENTS
Author would like to acknowledge for the financial support from the Department of Science &
Technology-INSPIRE fellowship,, New Delhi, India.
India Prof. R.K. Mandal (Department of Metallurgical Engineering, IIT,
BHU, Varanasi)) for providing SEM facility&
facility Dr.S.K.Singh(Department of Pharmaceutics, IIT, BHU, Varanasi) for
providing Zetapotential and FTIR facility.

REFERENCES
1.

Aspden TJ, Mason JD, Jones NS.,, 1997. Chitosan as a nasal delivery system: The effect of chitosan solutions on in vitro and in
vivo mucociliary transport rates in human turbinates and volunteers. J Pharm Sci:86: 509-13.
13.

2.

Cooney, M. J., Petermann, J., Lau, C., &Minteer, S. D., 2009.

2009. Characterization and evaluation of hydrophobically modified
modi
chitosan scaffolds: towards design of enzyme immobilized flow-through
through electrodes. Carbohydrate Polymers, 75, 428435.
428

3.

Fan, M., Hu, Q. L.,, &Shen, K.,2009.

K.,2009. Preparation and structure of chitosan soluble in wide Ph range. Carbohydrate Polymers,
78, 6671.

4.

Nwe N, Furuike T, Tamura H.,, 2009. Themechanical and biological properties of chitosan scaffolds for tissue regeneration
templates are significantly enhanced by chitosan from Gongronellabutleri.
Gong
Materials., 2(2):374-98.
2(2):374

www.tjprc.org

editor@tjprc.org

Kushagri Singh, Abha Mishra & Amit Singh

5.

Williams DF., 2005. To engineer is to create. Presentation. The 8th annual meeting of Tissue Engineering Society International,
Shanghai.

6.

Mohanraj VJ, Chen Y., 2006. Nanoparticles- A Review Trop J pharm Res 5(1):561-573.

7.

Pandaya, J.S., D. Harinarayana, D.Jain, J.S.Bidkar, S.Kuthe and M.N.Kadam., 2007. An attractive Biocompatible Polymer for
pharmaceutical application in various dosage form-chitosan. Pharmainfo.Net., 15(3).

8.

Hon, D.N.S., 1996. In: S. Dumitriu, (Ed). Polysaccharides in Medicinal Applications, Marcel Dekker, New york, pp:631-649.

9.

Kurita, K., 1986. Chemical Modifications of chitin and chitosan. In: Muzzarelli R.A.A., Jeuniax C. and Gooday G.W, eds.
Chitin in Nature and Technology. Plenum: New York, NH, pp: 287:293.

10. Dutta, P.K., J. Dutta and V.S. Tripathi., 2004. Chitin and Chitosan: Chemistry, properties and Applications. J.Scientific and
Industrial Res., 63:20-31.
11. Ying YJ, Lu NX, Mei TQ , Yan ZS, Yan De, Z., 2013. Ketoconazole associated hepatotoxicity: A Systemic review and metaanalysis. Biomed Environ Sci 26(7):606-610.
12. Balimane PV, Han YH, Chong S., 2006. Current industrial practices of assessin permeability and P- glycoprotein interaction.
American Association .pharm Sci 8: 1-13.
13. Galia E, Nicolaides E, Horter D, Lobenberg R, Rappas C, Dressman JB., 1998. Evaluation ofvarious dissolution media for
predicting in vivo performance of class I and class II drugs. Pharmacol Res 15; 698-705.
14. P. Calvo, C. Remuan Lopez, J.Vila-Jato, M.Alonso, 1997. J Applpolymsci 63: 125132.
15. P.Calvo, C.Remuan-Lopez, J.Vila-Jato, 1997. Pharm Res 14: 1431-1436.
16. Subhapradha N, Suman S, Ramasamy P, Saravanam R,Shanmugam V, Srinivasan A, Shanmugam A, 2013. Anticoagulant and
antioxidant activity of sulfated chitosan from the shell of domain clam Domaxscrotum(Linnaeus, 1758). Int J
NutrpharmacolNeurol Dis3:39-45.

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