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UNIVERSITI PUTRA MALAYSIA
DEVELOPMENT AND IN VITRO BIOEVALUATION OF COCKLE
SHELLCALCIUM CARBONATE (ARAGONITE) NANOPARTICLES
FOR INTRACELLULAR DRUG DELIVERY
TIJANI ISA
IB 2015 3
PM
By
TIJANI ISA
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DEVELOPMENT AND IN VITRO BIOEVALUATION OF COCKLE SHELLCALCIUM CARBONATE (ARAGONITE) NANOPARTICLES FOR
INTRACELLULAR DRUG DELIVERY
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in
Fulfillment of the Requirements for the Degree of Master of Science
October, 2015
COPYRIGHT
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All material contained within the thesis, including without limitation text, logos, icons,
photograph and all other artwork, is a copyright material of the Universiti Putra Malaysia unless
otherwise stated. Use may be made of any material contained within the thesis for noncommercial purposes from the copyright holder. Commercial use of material may only be made
with the express, prior written permission of Universiti Putra Malaysia.
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Copyright © Universiti Putra Malaysia
DEDICATION
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This thesis is sincerely dedicated to the memory of my late parents Sheikh Isa Ladan
Yakub and Hafsat Isa Ladan Yakub and my step-mammy Aishat Isa Ladan Yakub
(May your souls continue to rest in Jannah). You will forever remain in my dear
heart.
Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfillment of the
requirements for the Degree Master of Science.
DEVELOPMENT AND IN VITRO BIOEVALUATION OF COCKLE SHELLCALCIUM CARBONATE (ARAGONITE) NANOPARTICLES FOR
INTRACELLULAR DRUG DELIVERY
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By
TIJANI ISA
Professor Md Zuki Bin Abu Bakar @ Zakaria, PhD
Institute of Bioscience
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Chairman :
Institute
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October, 2015
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The use of safe and efficient delivery systems, capable of delivering therapeutic agents to subcellular levels are an ultimate goal in enhancing therapeutic effect. It is also a promising
strategy in overcoming microbial resistance and the emergence of intracellular bacterial
infections. The challenge, however, is that the interaction of nanoparticles with biological
systems at the cellular level must be established prior to biomedical applications. In this study,
ciprofloxacin conjugated cockle shells-derived calcium carbonate (aragonite) nanoparticle (CCSCCAN) was developed and characterized for its physicochemical properties and
antibacterial activities. Biocompatibilities were evaluated on macrophage cell line (J774.A1)
using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Bromo-2ʹdeoxyuridine (BrdU) assays. The nanoparticles were spherical in shape, with particles sizes
ranging from 11.93 to 22.12 nm as determined through a transmission electron microscope
(TEM). The highest percentage entrapment efficiency (EE) and loading content (LC) were
99.5% and 5.9%, respectively, with an optimum negative zeta potential. X-ray diffraction
(XRD) patterns revealed strong crystallity of the formulations. Fourier transforms infrared
(FT-IR) spectra shows evident of interactions exist between the drug and nanoparticles at the
molecular level. No burst effect, but a sustained drug release was observed from the
formulation. The mean diameter of inhibition zone was 18.6 ± 0.5 mm, which was better than
ciprofloxacin alone (11.7 ± 0.9 mm), while the minimum inhibitory concentration (MIC) and
the minimum bactericidal concentration (MBC) of the formulation were lower than those of
free drugs. Study of biocompatability suggested non-toxic effects of the formulations. In
conclusion, the results indicated that the ciprofloxacin- nanoparticle conjugate (C-CSCCAN)
enhanced susceptibility of Salmonella and antibacterial efficacy of the antibiotic, which could
potentially improve the clinical efficacy of the drug.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk Ijazah Master Sains
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PEMBANGUNAN DAN PENILAIAN BIOLOGI SECARA IN VITRO KE ATAS
KALSIUM KARBONAT (ARAGONITE) NANOPARTIKEL UNTUK
PENGHANTARAN DADAH INTRASEL
Oleh
TIJANI ISA
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Oktober 2015
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Pengerusi : Professor Md Zuki Bin Abu Bakar @ Zakaria, PhD
Institut
: Institut Biosains
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Penggunaan sistem penghantaran selamat dan cekap, mampu menyampaikan agen
terapeutik ke tahap sub-selular merupakan matlamat utama dalam meningkatkan kesan
terapeutik. Ia juga merupakan satu strategi yang boleh dipercayai dalam mengatasi
rintangan mikrob dan kemunculan jangkitan bakteria intrasel. Cabaran itu,
bagaimanapun, adalah bahawa interaksi nanopartikel dengan sistem biologi pada tahap
sel perlu diwujudkan sebelum aplikasi bioperubatan. Dalam kajian ini, ciprofloxacin
terkonjungsi kalsium karbonat (aragonite) nanopartikel dari cengkerang kerang (CCSCCAN) telah dibangunkan dan mempunyai ciri-ciri fizikokimia dan aktiviti antibakteria. Biocompatibiliti telah dinilai pada baris sel makrofaj (J774.A1)
menggunakan 3-(4,5-Dimethylthiazol-2-YL)-2,5-diphenyltetrazolium bromida (MTT)
dan 5-Bromo-2'-deoxyuridine (BrdU) asei. Nanopartikel adalah berbentuk bulat,
dengan zarah saiz antara 11.93 – 22.12 nm seperti yang ditentukan melalui mikroskop
elektron transmisi (TEM). Kecekapan peratusan pemerangkapan (EE) dan kandungan
yang dimuatkan (LC) yang tertinggi adalah masing-masing 99.5% dan 5.9%, dengan
potensi zeta negatif yang optimum. Pola pembelauan sinar-X (XRD) mendedahkan
kristaliti yang kuat daripada formulasi. Spektrum fourier mengubah inframerah (FTIR) menunjukkan bukti wujud interaksi diantara dadah dan nanopartikel pada
peringkat molekul. Tiada kesan pancutan rembesan dadah, tetapi perembesan dadah
yang berterusan diperhatikan dari formulasi. Garis pusat min zon perencatan adalah
18.6 ± 0.5 mm, adalah lebih baik daripada ciprofloxacin sahaja (11.7 ± 0.9 mm),
manakala kepekatan perencatan minimum (MIC) dan kepekatan bakteria minimum
(MBC) formulasi adalah lebih rendah daripada yang dadah sahaja. Kajian
biocompatabiliti mencadangkan tiada kesan toksik daripada formulasi.
Kesimpulannya, keputusan menunjukkan bahawa ciprofloxacin terkonjugsi kalsium
karbonate nanopartikel (C-CSCCAN) meningkatkan kecenderungan Salmonella dan
keberkesanan anti-bakteria antibiotik, yang berpotensi meningkatkan keberkesanan
klinikal dadah.
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ACKNOWLEDGEMENTS
All thanks and praises be to The All-Mighty Allah, the Most Beneficent and the Most Merciful
by whose power I accomplished this challenging task. I would like to extend thanks to the
following people:
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To my supervisor, Professor Dr. Md Zuki Bin Abu Bakar @ Zakaria for providing invaluable
assistance, academic and moral support through all my years of study. This project would not
have come to fruition without his constant encouragement and kindness.
My sincere thanks and regard to my co-supervisors Assoc. Professor Dr. Yaya Rukayadi and
Dr. Mohd Hezmee Bin Mohd Noor for the contributions in their own fields of expertise.
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To Dr. Mustapha Umar Imam and Dr. Aminu Umar Kura for their ready assistance and expert
advice on my research.
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My utmost regard goes to my uncles Dr. Sani Ibn Yakubu and Dr. Haliru Yakubu and the
entire members of El-Yakub family for their concern and prayers.
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I cannot forget The University of Maiduguri, Nigeria for the utmost financial support and
granting me a study leave to pursue my studies in Malaysia.
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Lastly, to my friends and my entire research fellow (Cockle shells R- group) Universiti Putra
Malaysia for your advice, companionship, conversation, kindness and humor never failed to
brighten my day.
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Md Zuki Bin Abu Bakar @ Zakaria, PhD
Professor
Institute of Bioscience
Universiti Putra Malaysia
(Chairman)
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Mohd Hezmee Bin Mohd Noor, PhD
Senior Lecturer
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
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Yaya Rukayadi, PhD
Associate Professor
Institute of Bioscience/Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as
fulfillment of the requirement for the degree of Master of Science. The members of the
Supervisory Committee were as follows:
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BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
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Date:
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Declaration by graduate student
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I hereby declare that:
the thesis is my original;
quotations, illustrations and citations have been duly referenced;
this thesis has not been previously or concurrently for any other degree at any other
institutions;
intellectual property of the thesis and copyright of thesis are fully-owned by Universiti
Putra Malaysia, as according to the Universiti Putra Malaysia (Research) Rules 2012;
written permission must be obtained from supervisor and the office of Deputy ViceChancellor (Research and Innovation) before thesis is published (in the form of
written, printed or in electronic form) including books, journals, modules,
proceedings, popular writings, seminar papers, manuscripts, posters, reports, lecture
notes, learning modules or any other materials as stated in the Universiti Putra
Malaysia (Research) Rules 2012;
there is no plagiarism or data falsification/fabrication in the thesis, and scholarly
integrity is upheld as according to the Universiti Putra Malaysia (Graduate Studies)
Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia (Research) Rules
2012. The Thesis has undergone plagiarism detection software.
Signature: _____________________________ Date: _____________________
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Name and Matric No.: Tijani Isa (GS39975)
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Declaration by Members of Supervisory Committee
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Signature:
Name of
Member of
Supervisory
Md Zuki Bin Abu Bakar @ Committee: Yaya Rukayadi, PhD
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Signature:
Name of
Chairman of
Supervisory
Committee:
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This is to confirm that:
the research conducted and the writing of the thesis was under our supervision;
supervision responsibilities as stated in the Unversiti Putra Malaysia (Graduate
Studies) Rules 2003 (revision 2012-2013) are adhered to.
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Zakaria, PhD
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Signature:
Name of
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Member of
Supervisory
Mohd Hezmee Bin Mohd Noor, PhD
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Committee:
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TABLE OF CONTENTS
Page
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ABSTRACT
ABSTRAK
AKNOWLEDGEMENT
APPROVAL
DECLARATION
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
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LITERATURE REVIEW
2.1
Intracellular Bacteria
2.1.1 Bacteria intracellular survival
2.1.2 Passive immunity in phagocytes
2.2
Fluoroquinolones Efflux and Decrease Permeation in Eukaryotes
2.3
Accumulation and Subcellular Distribution of Flouroquinolones
2.4
Efflux Mechanisms and Decrease Permeation in prokaryotes
2.5
Antibiotics Therapy
2.6
Nanotechnology in Medicine and Biotechnology
2.7
Delivery System in Nanotechnology: Advantages and Disadvantages
2.7.1 Recombinant proteins
2.7.2 Viral carriers
2.7.3 Cationic carriers
2.7.4 Nanoparticles carrier system
2.7.4.1 Inorganic nanoparticles and nanobiocomposite
2.7.4.2 Liposomal nanoparticles
2.7.4.3 Polymeric nanoparticles
2.7.4.4 Solid lipid nanoparticles
2.7.4.5 Chitosan nanoparticles
2.7.4.6
Dendrimeric nanostructures
2.7.4.7
Nanoemulsions
2.7.5
Nanoparticles drug loading
2.7.6
Nanoparticles sustained release mechanisms
2.7.7
Physicochemical properties of nano-particulate system
2.8
Inorganic Calcium Carbonate
2.8.1 Synthesis of calcium carbonate nanoparticles
2.8.2 Microemulsion-based
synthesis
of
calcium
carbonate
nanoparticles
2.8.3 Calcium carbonate nanoparticles cellular delivery system
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2.
INTRODUCTION
1.1
Background
1.2
Problem Statement
1.3
Research Objective
1.3.1 General objective
1.3.2 Specific objectives
1.4
Hypothesis
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CHAPTER
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2.9
2.10
2.11
2.12
2.13
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MATERIALS AND METHODS
3.1
Materials
3.1.1 Laboratory equipment
3.1.2 Reagents, chemicals and media
3.1.3 Bacterial strain
3.2
Development of Ciprofloxacin-loaded Calcium Carbonate Nanoparticles
3.2.1 Preparation of micron-size cockle shells calcium carbonate
powder
3.2.1.1 Top down synthesis of cockle shells calcium
carbonate aragonite nanoparticles
3.2.2 Drug loading
3.2.3 Determination of drug loading content and encapsulation
efficiency
3.3
Characterization of Cockle Shells Calcium carbonate Aragonite
Nanoparticles and Ciprofloxacin- Loaded Cockle Shells Calcium
carbonate Aragonite Nanoparticles
3.3.1 Transmission electron microscopy
3.3.2 Field-emission scanning electron microscope
3.3.3 Zeta potential determination
3.3.4 X-ray powder diffraction
3.3.5 Fourier transform infrared spectroscopy
3.4
In Vitro Drug Release Study
3.5
In Vitro Biocompatibility Assays
3.5.1 Cell culture
3.5.2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) viability assay
3.5.3 5-Bromo-2ʹ-deoxyuridine (BrdU-ELISA) proliferation assay
3.6
In Vitro Antibacterial Assays
3.6.1 Preparation of drug stock solutions
3.6.2 Disk diffusion susceptibility assay
3.6.3 Determination of minimum inhibitory concentration and minimum
bactericidal concentration
3.6.4 Statistical analysis
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3.
Ciprofloxacin Conjugated Nanoparticles
Nano-Antimicrobial Therapy
Targeted Intracellular Therapy
Molecular Biocompatibility of Nanoparticles
Microbiology in Nanotechnology
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4.
RESULTS AND DISCUSSION
4.1
Development of Ciprofloxacin Nanoparticles
4.1.1 Synthesis of cockle shells derived calcium carbonate nanoparticles
4.1.2 Ciprofloxacin loading and encapsulation efficiency
4.1.3 Transmission electron microscopy of ciprofloxacin-nanoparticles
formulation
4.1.4 Field emission scanning electron microscopy of ciprofloxacinnanoparticles formulation
4.1.5 Zeta potential analysis of ciprofloxacin-nanoparticles formulation
4.1.6 X-Ray powder diffraction patterns of ciprofloxacin-nanoparticles
formulation
4.1.7 Fourier transforms infrared spectroscopy of ciprofloxacinix
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4.4
SUMMARY, CONCLUSION AND RECOMMENDATIONS
5.1
Summary
5.2
Conclusion
5.3
Recommendation for Future Research
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REFERENCES
APPENDICES
BIODATA OF STUDENT
PUBLICATION
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4.2
4.3
nanoparticles formulation
Assessment of In Vitro Ciprofloxacin Release
Biocompatability Evaluation
4.3.1 Effect of nanoparticles formulation on cell viability
4.3.2 Effect of nanoparticles formulation on cell proliferation
Antibacterial Activity of Ciprofloxacin Nanoparticles Suspensions
4.4.1 Measurement of diameter of inhibition zone
4.4.2 Minimum inhibitory concentrations and minimum bactericidal
concentrations
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LIST OF TABLES
Table
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Loading content and encapsulation efficiency of ciprofloxacin- conjugated
cockle shells calcium carbonate (aragonite) nanoparticles
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Mean zone of inhibition (mm) of free ciprofloxacin, ciprofloxacinconjugated cockle shells calcium carbonate (aragonite) nanoparticles and
cockle shells calcium carbonate (aragonite) nanoparticles suspension (10
µL)
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3
Minimum inhibitory concentration and minimum bactericidal
concentration of ciprofloxacin- conjugated cockle shells calcium
carbonate (aragonite) nanoparticles and free ciprofloxacin
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LIST OF FIGURES
Figure
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Molecular structure of ciprofloxacin showing it’s zwitterionic nature
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2.
Structure of ciprofloxacin depicting it’s piperazine and pyridone moieties
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3.
Diagram of active and passive targeting drug delivery
4.
TEM micrographs showing nanoscale spherical-shaped cockle shells
calcium carbonate (aragonite) nanoparticles (a), and FESEM micrograph
showing the pore structured micron-size cockle shells calcium carbonate
aragonite powder (b)
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5.
TEM micrograph showing homogenized, spherical shaped ciprofloxacinconjugated cockle shells calcium carbonate (aragonite) nanoparticles
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6.
FESEM micrographs showing dispersed and homogenized spherical
shaped ciprofloxacin-conjugated cockle shells calcium carbonate
(aragonite) nanoparticles
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7.
XRD spectra for micron-size cockle shells calcium carbonate aragonite
powder (a), cockle shells calcium carbonate (aragonite) nanoparticles (b),
ciprofloxacin-conjugated cockle shells calcium carbonate (aragonite)
nanoparticles (c), and free ciprofloxacin (d) showing crystalline phases and
purity
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8.
FT-IR spectra for micron-size cockle shells calcium carbonate aragonite
powder (a), cockle shells calcium carbonate (aragonite) nanoparticles (b),
ciprofloxacin-conjugated cockle shells calcium carbonate (aragonite)
nanoparticles (c), and free ciprofloxacin (d) depicting the samples
absorption or molecular interaction
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9.
In vitro ciprofloxacin release profile of cockle shell calcium carbonate
aragonite nanoparticles. Each bar represents mean ± standard deviation
(n=3) of three independent experiments
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The MTT percentage viability of proliferating cells. The values represent
mean ± standard deviation (=3); P ˂ 0.05 compared with ciprofloxacin
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11.
The percentage of BrdU incorporation into the DNA of proliferating cells.
The values represent mean ± standard deviation (n=3); P ˂ 0.05 compared
with ciprofloxacin
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LIST OF ABBREVIATIONS
American Type Culture Collection
BrdU
5-bromo-2ʹ- deoxyuridine
CaCO3
Calcium Carbonate
Ca2+
Calcium ion
C-CSCCAN
Ciprofloxacin-cockle shells calcium carbonate aragonite nanoparticles
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ATCC
Colony-forming unit
Clinical and Laboratory Standards Institute
CLSI
Centimeter
Cm
Carbon dioxide
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CFU
CO2
Cockle shells calcium carbonate aragonite nanoparticles powder
DMEM
Dulbecco’s Modified Eagle’s Medium
DMSO
Dimethyl sulfoxide
EE
Encapsulation efficiency
FBS
Foetal Bovine Serum
FESEM
Field Emission Scanning Electron Microscopy
HPH
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Fourier Transform Infrared Spectroscopy
High Pressure Homogenizer
Water
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FTIR
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CSCCAP
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LC
Loading Capacity
Minimum Bactericidal Concentration
mg/mL
Milligram per milliter
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MBC
MHA
Mueller–Hinton Agar
MHB
Mueller–Hinton Broth
MIC
Minimum Inhibitory Concentration
Min
Minute(s)
mL
Milliliter
Μm
Mircometer
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Millimete
MTT
3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide
Mv
Millivolts
Nm
Nanometer
PBS
Phosphate Buffer Saline
Rpm
Revolutions per minute
R2
Regression square
SD
Standard deviation
TEM
Transmission Electron Microscopy
µg/mL
Microgram per millilitre
Wf
Weight of free drug
WnP
Weight of nanoparticles
Wt
Total weight of drug fed
W/O
Water in Oil microemulsions
XRD
X-ray Diffractometry
ºC
Degree Celsius
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Mm
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CHAPTER 1
INTRODUCTION
1.1
Background
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Facultative intracellular bacterial pathogens are notorious causative agents for a
number of severe diseases world-wide. These pathogenic agents have developed a
number of inventive mechanisms to replicate and spread inside numerous multicellular eukaryotes including the antagonistic phagocytic cells, resulting in persistent,
latent and life threatening infections (Pinto-Alphandar et al., 2000; Carryn et al., 2003;
Imbuluzqueta et al., 2010; Xie et al., 2014). The host organisms are pose with greater
challenge as the body defense mechanism were not just infected but as well act as
reservoir for the pathogenic organisms, while reaching and spreading the infection to
the adjacent uninfected tissues (Pinto-Alphandary et al., 2000). However, owing to
their ability to escape the mammalian host phagocytic protection mechanism and
establish a specialized intracellular milieu outside the host immune system,
intracellular bacterial infection has remain a rising trend among humans and animals
(Monack et al., 2004; Steinberg and Grinstein, 2008). Several human infectious
diseases such as leishmaniasis, brucellosis, tuberculosis, salmonellosis and
histoplasmosis, are caused by intracellular microorganisms. They also caused
opportunistic infections in immunosuppressed patients with AIDS or other conditions,
where Mycobacterial infections are involved to cause more complications (Briones et
al., 2008; Monack et al., 2004; Steinberg and Grinstein, 2008; Mehta, 1996; Peters et
al., 2000). Due to their opportunistic nature, no detailed explanations have been
highlighted on the physiological adaptation mechanism to intracellular survival and
replication strategies. Though, the continuous intracellular survival of these pathogens
is not an important virulence factor in their life cycle (Van Bambeke et al., 2006).
Chronic infections are generally characterized by diverse changes in the intracellular
microenvironment. Thus successful pathophysiological adaptation led to dormancy of
specialized lymphoid tissues and prolonged or persistent invasion of the body by the
pathogens (Ranjan et al., 2012). In addition, chronic inflammation and autoimmune
disorders are commonly associated with intracellular pathogens which usually
participate in malignant processes (Kaufmann, 2011). Besides the famous facultative
and obligate intracellular bacteria, several other common pathogenic bacteria are now
recognized for intracellular survival under definite conditions (Andrade et al., 2013).
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Intracellular bacterial infections presents considerable challenges to antibiotic
treatment, this was due to limited cellular penetration, and poor intracellular retention
of the antibiotic in the phygocytes, thus imposing insignificant inhibitory or microcidal
effects on the pathogens (Akbari et al., 2013), (Drulis-Kawa and Dorotkiewicz-Jach,
2010). Consequently, the required intracellular drug levels for bacterial elimination are
not met (Ranjan et al., 2012). Such kind of infections have been connected with
deterioration in health and treatment failure (Akbari et al., 2013). Life-threatening
infections are often caused by resistant intracellular bacteria, making them more
difficult to treat. Treating patients with resistant intracellular strains, requires high
doses of drugs which presents adverse effects to healthy organs and tissues (Andrade
et al., 2013).
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Microbial resistance have become more complicated over time, greatly limiting
antimicrobials success, and is an increasingly emergent crisis (Hajipour et al., 2012).
These include, among others impediments, reduces drug permeability, increased drug
extrusion from cells, mutation at key antimicrobial-genes-binding sites and drug
inactivation or modification by enzymes (Jayaraman, 2009; Deurenberg et al., 2009;
Périchon and Courvalin, 2009). Resistance to conventional antimicrobials is also
ascribed to the alteration in microbial growth cycle, as well as decreased in bacterial
susceptibility to antibiotics (Pinto-Alphandary et al., 2000; Sandhiya et al., 2009).
Furthermore, bacteria express higher resistance to antibiotics by forming biofilms
which provides protection for them. Thus, the reduced membrane permeability of
bacteria is recognized as the main reason for antibiotic resistance (Jayaraman, 2009;
Blecher et al., 2011; Huh and Kwon, 2011; Davin-Regli et al., 2008). With the
emergence of multi-drug resistant bacteria, antibiotic resistance remain a top challenge
for drug delivery against bacterial cells (Davin-Regli et al., 2008).
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Antibiotic delivery systems represent a promising solution for the challenges of
intracellular bacterial infections and are alternative to conventional antibacterial
therapy for efficient eradication of pathogens. In this regard, the use of antibiotics
carrier systems may represent an incredible approach towards the treatment of
intracellular bacteria. Antibiotic carrier systems can be endocytose and/or phagocytose
in a similar manner with bacteria and then release into phagocytic cells carrying
intracellular pathogens the drug payload (Briones et al., 2008; Abed and Couvreur,
2014). In another development, foreign materials immediately after injection are
subjected to opsonization. The same process by which bacteria surface molecules are
tainted by opsonins and become more readily engulf by phagocytes. In this manner,
the carriers system are recognized by the reticuloendothelial system (Pinto-Alphandary
et al., 2000).
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The increased global assembly of engineered nanoparticles as impending drug carrier
system necessitates a comprehensive understanding of their potential toxicity (Kroll et
al., 2012). However, the clinical application of nanoparticles for diagnostic procedure
and therapeutic purposes, imaging or as a delivery vehicles represent deliberate
exposure to considerable dosage of the particles (Oostingh et al., 2011). Many
European Communities responsible for implementing laws have laid down a number
of legislations concerning the use and exposure to nanoamterials (Commission of the
European Communities, 2005; Commission, 2004). This has become indispensable as
the general awareness of nanotechnology can be threaten by events such as ―Germany
2006 nano scare‖, concerning the spray glass protective Magic-Nanoing (Ross, 2006),
and the controversy of nanometer-sized sunscreen in the United States (Long et al.,
2006). Despite many reports on the toxicity of nanomaterials, the precise association
between the engineered nanoparticles and the immune system have not been
tentatively studied (Kroll et al., 2012; Samberg et al., 2010; McNeil et al., 2007).
1.2
Problem Statement
Since the 1980s, flouroquinolones have been used in clinical practice, and they have
contributed to major advances in the medical treatment of gram negative bacterial
infections as frontline drugs (Pestova et al., 2000; Pinto-Alphandary et al., 2000).
Active efflux from prokaryotes as well as eukaryotic cells strongly modulates the
activity of this class of antibiotics (Van Bambeke et al., 2000). Thus, the intracellular
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flouroquinolones act hastily in a concentration-dependent manner but in an inadequate
fashion and a sub-optimal/therapeutic way (Carryn et al., 2003). This contributes to
failure of conventional fluoroquinolones therapies as a result of decreased
accumulation and poor retention of the antibiotics inside the cells (Jacoby, 2005;
Briones et al., 2008). Flouroquinolones resistance is often due to increased efflux
(Singh et al., 2011; Ahmed et al., 2013). Over recent decades, the increasing
emergence of antibiotic resistance in pathogenic bacteria has been worsening and
resulted in severe and often lethal infections that cannot be treated by conventional
therapy (Freire-Moran et al., 2011).
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Ciprofloxacin is one of the frequently used antimicrobial of the fluoroquinolones
group available all over the world (Ambulkar et al., 2009). It is required in several
systemic diseases, having strong bactericidal effect against a broad range of clinically
relevant gram-negative and gram-positive bacteria (Jain and Banerjee, 2008; Jeong et
al., 2008). Ciprofloxacin have been available only as conventional, immediate- release
tablets, and has a biological half-life of about 3-5 hours for a single or repeated
administration (Henry et al., 2002; Bhalerao and Rote, 2012). Other factors limiting
the success and clinical usage of ciprofloxacin includes poor solubility at physiological
pH, which prevent it from diffusing into the lung fluid to instigate a therapeutic
response at key affected site, bitter taste in solution, and rapid renal clearance, in
which minimum of 70% of the oral dose is excreted unchanged in the urine (El-gendy
et al., 2010; Bhalerao and Rote, 2012). However, the frequent administration of
ciprofloxacin is most associated with side effects such as central nervous system
disorder and kidney problems (Spratto, 2012).
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Whilst antimicrobial treatments usually involve a prolonged period of therapy,
adequate antibiotic exposure is desirable to guarantee eradication of the microbial
pathogens. Nevertheless, prolonged therapy is often associated with patient
noncompliance, and incomplete treatment may result in the development of resistance.
Poor compliance is exclusively a problem for drugs with short half-lives, since these
drugs have short dosing intervals, and the number of doses require for microbial
eradication is high (Gao et al., 2011). Therefore, in order to attained a successful
treatment, antibiotic must fulfill a series of criteria, including the ability to penetrate
and be retained by the cell, the capacity to reach the intracellular target, and the
display of activity against bacteria residing in the peculiar environment (Imbuluzqueta
et al., 2010). On the other hand, due to the deficient in new antibacterial agents, there
is considerable interest in restoring the activity of older and conventional
antimicrobials (Piddock et al., 2010).
©
The use of safe and efficient delivery systems, capable of delivering therapeutic agents
to sub-cellular levels is an ultimate goal in enhancing therapeutic effect. It is also a
promising strategy in overcoming microbial resistance (Pelgrift and Friedman, 2013).
It has recognized that extended-release drug formulation is beneficial in improving
patient compliance, as regular administration can be avoided by maintaining stable and
continuous plasma drug concentration over a prolonged period, and maximize the
therapeutic effect of antibiotics while minimizing resistance (Blasi et al., 2007; Gao et
al., 2011). Modifications in drug delivery to redirect the antibiotic from the circulation
and target it to cells, tissues, or organs where infection occurs may lessen the chance
for the flouroquinolones travels to bone and cartilage (Lee et al., 2011). The
encapsulation of antibiotics in carriers could avoid antibiotic efflux and enhance the
drugs‘ intracellular retention, since delivery systems like nanoparticles are not
3
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substrates of the efflux pump proteins (Plapied et al., 2011). Moreover, encapsulation
of antibiotic improve their pharmacokinetic by increasing serum half-life and
decreasing apparent volume of distribution which can increases the maximum
tolerated dose (Pinto-Alphandary et al., 2000). Nanoparticles can be phagocytosed by
host phagocytes containing intracellular microbes. Once inside host phagocytes, the
antibiotic-nanoparticles delivery system could release high dose of the antibiotic to
eliminate the intracellular microbes before developing resistance (Blecher et al., 2011;
Huh and Kwon, 2011; Huang et al., 2011).
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Many studies have reported the increased antimicrobial activity of ciprofloxacinconjugated nanoparticles (Akbari et al., 2013; Chono et al., 2008; Hono et al., 2007;
Ong et al., 2012). Likewise decreased antibiotic resistance was reported in the
presence of Zinc Oxide nanoparticles (Banoee et al., 2010). It is anticipated that the
use of nanoparticles-based drug delivery systems will continue to improve treatment of
bacterial infections and multidrug-resistant microbes (Huh and Kwon, 2011).
However, no studies have been conducted on the potential of ciprofloxacinconjugated biobased-cockle shells-derived calcium carbonate (aragonite) nanoparticles
(CSCCAN), to enhance the efficacy of the drug. Moreover, calcium carbonate has
been used for controlled delivery of biomolecules due to it biodegradability,
biocompatibility, and porous nature with huge promises (Rodríguez-Ruiz et al., 2013).
CSCCAN has good physicochemical properties and a simple technique of preparation
in a bulk-scale (Kamba et al., 2013). The cockle shells (Anadara granosa), which is
available in abundance, is often considered a waste (Mohamed et al., 2012). A, porous
aragonite calcium carbonate nanoparticles loaded with gentamicin sulphate with
controlled released have been used successfully in ostoemyelitis treatment (LucasGirot et al., 2005). Thus, calcium carbonate nanoparticles are expected to also enhance
the efficacy of ciprofloxacin.
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Human exposure to nanoparticles is inevitable as the particles become more widely
used; hence nanotoxicology research is gaining attention. The challenge, however, is
that the interaction of nanoparticles physicochemical properties with biological
systems at the cellular level must be established prior to biomedical applications
(Lewinski et al., 2008; Shukla et al., 2005; Kroll et al., 2012; Oostingh et al., 2011).
Study conducted on the biocompatibility of cockle shells-derived calcium carbonate
(aragonite) nanoparticles (CSCCAN) revealed their non-toxic effects and therefore
considered potential agent for drug delivery (Kamba et al., 2014). However,
understanding the biological response of the nanoparticles at sub cellular and
molecular level is crucial and can certainly present another line of information to
appraise the interactions between the nanomaterials and cells.
©
1.3 Research Objectives
1.3.1 General objective
The main objective of the present study was to develop ciprofloxacin-cockle shellsderived Calcium carbonate (aragonite) nanoparticles (C-CSCCAN) as advanced
vehicle for intracellular drug delivery and controlled released with good
cytocompatibility, insignificant genotoxicity and high antibacterial efficacy.
4
1.3.2 Specific objectives
This research was carried out specifically:
ii.
iii.
To develop C-CSCCAN hybrid molecules and study its physicochemical
properties, and delivery system
To evaluate in vitro biological toxicity and immunogenic potential of CCSCCAN and CSCCAN using macrophage (J7741.A) cell line.
To evaluate the in vitro antibacterial activity of C-CSCCAN by identifying
the diameter of inhibition zone and minimum inhibitory concentration and
minimum bactericidal concentration against Salmonella Typhi.
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1.4 Hypothesis
It is hypothesized that;
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ii.
iii.
CSCCAN is an effective carrier for ciprofloxacin, improves the chemical
and physical stability of the drug substance and could sustained release of
the drug in the surrounding intracellular milieu.
C-CSCCAN is biocompatible and nontoxic.
The physicochemical properties of C-CSCCAN enhance susceptibility of
intracellular S. Typhi, and augment the antibacterial performance of
ciprofloxacin.
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i.
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