ABSTRACT
Chemotherapy is one of the leading means of treating cancer. However, the side effects and complications arising from the dose-dependent toxicity of anticancer drugs are often as severe as the disease itself. This research was aimed at preparation and characterization of nanocomposites from hydroxyapatite (HA) and sodium alginate (SA), and evaluation of their applications in controlled delivery of doxorubicin (DOX) and methotrexate (MTX). In situ preparation of hydroxyapatite-sodium alginate (HASA) nanocomposite was carried out by the wet precipitation methods. The prepared HA and the nanocomposite were characterized by Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), X-Ray Fluorescence (XRF) and X-Ray Diffraction (XRD) analyses. Drug loading was carried out at neutral pH, while in vitro drug release study was carried out in synthetic body fluid (SBF) at pH 7.4 and 37oC. Four different loading methods were investigated. Method 1 involved adsorbing the drug in already prepared nanocomposites. In method 2, the dried HA was cross-linked with SA in the drug solution using calcium chloride solution; in method 3 the hydroxyapatite was first incubated in the drug solution before the cross-linking stage; while in method 4 the freshly prepared hydroxyapatite was cross-linked with sodium alginate in the drug solution. The effect of pH of the release medium on release profile was studied using pH 3.0, pH 5.0 and pH 7.4; and drug combination study was also carried out. FTIR study showed peaks that confirmed the formation of HA as well as the formation of the composite. Image analysis revealed that the HA and the nanocomposites were of nanometre size (24.67 nm – 997.09 nm) with irregular morphologies as shown by the circularity (0.119 – 0.988) and aspect ratio (0.149 – 1). The particle size decreased with increase in SA composition from 359.46 nm for HASA-1%wt to 109.98 nm for HASA-50%wt. A similar trend was observed for crystallite size (28.39 nm – 9.47 nm) and degree of crystallinity (49.29% – 1.82%), while circularity and aspect ratio did not show any noticeable change with SA addition. XRD analysis showed the apatite phase to be composed of pure HA and carbonate-HA. Both phases were responsible for the major peaks at 2𝜃 = 25.880 (d-value = 3.4394 Å), 31.770 (d-value = 2.8144 Å), and 32.150 (d-value = 2.7818 Å), which were assigned Miller indices of (002), (121), and (112) planes, respectively. These planes were also present in all the HASA samples. There was no notable effect on the peak positions with the addition of SA. The result of drug loading study showed that the nanocomposites have high loading efficiency for DOX, which increased with increase in SA composition reaching a
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maximum value of 92.03% for HASA-50%wt, while for MTX the loading efficiency was relatively low and decreased with increase in SA composition with the highest loading efficiency of 35.24% for pure hydroxyapatite. There was sustained release of DOX by the nanocomposites with SA composition of 5%wt and above for about 57 hours, while MTX showed short release time for all the formulations with maximum release time of 5 hours for HASA-5%wt. Except for HA and HASA-1%wt, the DOX release followed first order release kinetics, with Fickian diffusion as the predominant release mechanism. Nanocomposite prepared in aqueous medium had higher loading efficiency (83.69%) compared with those prepared in organic solvents (52.46%, 47.50%, and 46.50%). The release profiles also showed that nanocomposites from aqueous medium had least burst release effect and more sustained release. The release kinetics and mechanism however, did not depend on the synthetic medium. DOX was loaded well (above 80% loading efficiency) by all the four loading methods studied, while for MTX, method 2 and 4 had better loading efficiency (39.98% and 37.10% respectively) than method 1 and 3 (10.39% and 15.21% respectively). Release study for DOX, indicated that the adsorption method had faster release rate than other methods; while for MTX, only method 4 sustained the release of the drug for about 9 hours, while other methods had high burst release effects. DOX release rate was initially faster at acidic conditions than at physiologic condition, but became slower at later release times. From the drug combination study, the release profiles for all the combination ratios showed high burst release for MTX with total release time not exceeding five hours. However for DOX, there was sustained release throughout the thirty three hours of the study. In conclusion, HASA nanocomposite was successfully prepared and characterised. Its ability to load and release the drugs depended on the nature of the drug as well as the synthetic medium and the loading method employed. The nanocomposite if prepared under optimal conditions is a potential carrier for effective delivery of DOX.
TABLE OF CONTENTS
Cover page i
Title Page ii
Declaration iv
Certification v
Acknowledgement vi
Abstract vii
Table of Contents ix
List of Figures xiv
List of Tables xvii
List of Appendices xviii
Abbreviation xx
CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Preamble 1
1.2 Statement of the Problem 5
1.3 Justification 6
1.4 Aim and Objectives 7
CHAPTER TWO
2.0 LITERATURE REVIEW 8
2.1 Drug Delivery System 8
2.1.1 Sustained drug delivery systems 8
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2.2 Material for Drug Delivery 10
2.2.1 Hydroxyapatite 10
2.2.2 Polymers 11
2.3 nanocomposites 17
2.3.1 Synthetic strategies for polymer-inorganic nanocomposites 18
2.3.2 Methods for characterization of nanocomposites 21
2.4 Drug Loading Methods 30
2.4.1 Incorporation method 31
2.4.2 Adsorption/absorption method 32
2.5 Drug Release Mechanisms and Kinetic Models 34
2.5.1 Drug release mechanisms 34
2.5.2 Drug release models 37
2.6 Cancer Therapeutics 43
2.6.1 Cancers 43
2.6.2 Enhance permeability and retention effect (EPR) 44
2.6.3 Stealth characteristics 46
2.6.4 Multidrug resistance 47
2.6.5 Overview of the anticancer drugs –DOX and MTX 48
CHAPTER THREE
3.0 MATERIALS AND METHODS 50
3.1 Materials 50
3.2 Preparation of Hydroxyapatite 50
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3.2.1 Preparation of reagents 50
3.2.2 Procedure 51
3.3 Preparation of Hydroxyapatite-Sodium Alginate Nanocomposites 51
3.3.1 Preparation of reagents 51
3.3.2 Procedure (first synthetic route) 52
3.3.3 Procedure (second synthetic route) 52
3.4. Material Characterizations 53
3.4.1 Fourier Transform Infrared spectroscopy (FTIR) 53
3.4.2 Scanning electron microscopy (SEM) 53
3.4.3 Image analysis 53
3.4.4 X-Ray Powder Diffraction analysis 54
3.4.5 X-Ray fluorescence analysis 55
3.5 Drug Loading Study 56
3.5.1 Preparation of drug solutions 56
3.5.2 Preparation of drug loaded hydroxyapatite 56
3.5.3 Preparation of drug-loaded hydroxyapatite-sodium alginate 57
3.6 Evaluation of Drug Loading Methods 57
3.6.1 Preparation of reagents 57
3.6.2 Procedures 58
3.7 Drug Release Study 59
3.7.1 Preparation of Synthetic Body Fluid 59
3.7.2 In-Vitro Drug Release Study 60
3.7.3 Comparison of Drug Release Profiles 61
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3.7.4 Drug Release Kinetics and Mechanistic Study 62
3.8 Preparation of Hydroxyapatite-Sodium Alginate Nanocomposites
in Different Solvents 62
3.9 Investigation of the Effect of Release Medium pH on Drug Release Profiles 63
3.10 Drug Combination Study 63
CHAPTER FOUR
4.0 RESULTS 64
4.1 Fourier Transform Infra-Red (FTIR) Spectra Analysis 64
4.2 Scanning Electron Microscope (SEM) Analysis 65
4.3 Image Analysis 76
4.4 X-Ray Powder Diffraction Analysis 85
4.5 Energy Dispersive X-Ray Fluorescence (XRF) Analysis 85
4.6 Comparison of Nanocomposites Prepared by the two Different Methods 85
4.7 Drug Loading and Release Results 93
4.7.1 Drug loading 93
4.7.2 Drug release 94
4.7.3 Comparison of doxorubicin release profiles from HASA and 2HASA 104
4.8 Evaluation of Different Loading Methods 104
4.9 Effect of Synthetic Solvent on Drug Loading and Release 105
4.10 Effect of pH Medium on Release of Doxorubicin 105
4.11 Drug Combination Study 121
CHAPTER FIVE
5.0 DISCUSSION 128
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5.1 Results of FTIR Analysis 128
5.2 Scanning Electron Microscope (SEM) Analysis 130
5.3 X-Ray Diffraction (XRD) Analysis 132
5.4 X-Ray Fluorescence (XRF) 134
5.5 Comparison of the two Synthetic Routes of Hydroxyapatite-Sodium Alginate
Nanocomposites 135
5.6 Drug Loading and Release Results 136
5.6.1 Doxorubicin Loading 136
5.6.2 Methotrexate Loading 136
5.6.3 Comparison of the Encapsulation Efficiencies of the two Synthetic Methods 136
5.6.4 Methotrexate Release Study 137
5.6.5 Doxorubicin Release Study 138
5.6.6 Doxorubicin Release Mechanisms and Kinetics 139
5.6.7 Comparison of the Release Profiles from the two Synthetic Methods 140
5.7 Evaluation of Different Loading Methods 141
5.7.1 Doxorubicin 141
5.7.2 Methotrexate 142
5.8 Effect of Synthetic Medium on Drug Loading and Release 142
5.9 Effect of the pH of the Release Medium on Doxorubicin Release Profile 144
5.10 Drug Combination Study 145
CHAPTER SIX
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS 147
6.1 Summary 147
6.2 Conclusion 148
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6.3 Recommendations 149
REFERENCES 150
APPENDIX 170
CHAPTER ONE
1.0 INTRODUCTION
1.1 Preamble
In recent times, there has been a surge in research interest in the use of nanoparticles in several areas such as medical applications (Khotimchenko, 2001; D‘Ayala et al, 2008; Lee and Mooney, 2012); analytics (De Vols et al., 2006; Antosiak-Iwariska, et al., 2009); cosmetics (Kailasapathy et al., 2002; Sugiura et al., 2005); optical and electronic applications (Cho et al., 2008). This is as a result of the unique properties possessed by nanoparticles such as high surface area to volume ratio which gives them advantages over macro and micro materials. Nanoparticles are particles with size ranging from 1 to 1000 nm (Jung et al., 2000). US National Cancer Institute however defined therapeutic nanoparticles as colloidal particles in the size of 1 – 100 nm (Chen, 2010).
Interestingly, one of the major promising areas of application of nanoparticles is in controlled drug delivery. As a drug delivery system, nanoparticles can entrap drugs or biomolecules onto their exterior surfaces. They could show controlled release properties due to the biodegradability, pH, and/or temperature sensibility of the materials; and they can improve the effectiveness of drugs and reduce side effects (Devanand et al., 2011).
Marques et al. (2014), stated that the most popular drug delivery systems are based on polymers and ceramics and their nanocomposites. Extensive applications of polymers in drug delivery have also been realized because polymers offer unique properties which, so far, have not been attained by any other materials.
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Sodium alginate is a naturally occurring, water soluble linear polysaccharide polymer extracted from brown sea weed and is composed of alternating blocks of 1-4 linked α-L-guluronic acid and β-D-mannuronic acid residues (Kato et al., 2003). It is widely used for drug delivery and tissue engineering applications due to its many unique properties such as biocompatibility, biodegradability, low toxicity, non-immunogenicity, water solubility, relatively low cost, gelling ability and stabilizing properties (Aggarwal et al., 2012). According to Sun and Tan (2013), alginate based microcapsules and scaffolds have shown minimal or negligible cytotoxicity and are histocompatible. The polymer based carriers can protect drugs from degradation and may improve plasma half time to ensure transport and release of drugs.
However, ionically cross-linked alginate hydrogel has limited drug loading efficiency which limits its applications (Pongjanyakul et al., 2010; Ruvinov et al., 2010). Other major disadvantages of alginate beads are their fast disintegration, and their high porosity, which result in burst release. In order to improve drug entrapment efficiency and modulate drug release, water insoluble materials (e.g. hydroxyapatite and clay) can be incorporated to the alginate matrix (Pongjanyakul et al., 2010).
Hydroxyapatite (HA) is a calcium phosphate salt. It is the main component of vertebrate hard tissues such as bone and teeth in the form of nanometer-sized needle-like crystals of approximately 5-20 nm width by 60 nm length (Ferraz et al., 2004). In recent years, synthetic nano-HA has been widely used in biomaterials such as drug delivery, in orthopedic as well as dental applications. This is as a result of its excellent useful properties such as biocompatibility, bioactivity and osteo-conductivity. It is also non-toxic, non-inflammatory, and non-immunogenic (Mateus et al., 2008). HA/polymer nanocomposites have attracted much attention since such
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nanocomposites lead to improved properties (Khaled et al., 2014) such as sustained drug delivery (Raj et al., 2013).
Sustained drug delivery involves releasing the drug into the target site over a prolonged period of time. It is characterized by releasing the drug in a controlled fashion to maintain an appropriate therapeutic plasma concentration for a long period of time, while controlled release involves providing the drug only where and when it is needed (Devanand et al., 2011). Conventional form of drug delivery is not target specific and release of drug to the target site is not sustained (Raj et al., 2013).
Most drug carriers are associated with the problem of burst releases (Hasan et al., 2007). According to Martinho et al. (2011), burst releases lead to a significant and unpredictable toxicity for potent drugs and in treatment of chronic diseases. One of such diseases is cancer. Cancer is mainly treated by chemotherapy. The success of chemotherapy depends on the selection of an optimum carrier system. These carriers include nanoparticles, nanotubes, nanorods, dendrimers, liposomes, microspheres and so forth (Kakde et al., 2011). The aim of an ideal cancer chemotherapy is to deliver the correct amount of drug at controlled rate for sufficiently long time (sustained release) to the site of action (tumour cells) while minimizing contact with normal cells.
In order to endow nanosystems with long circulation properties, new technologies aimed at the surface modification of their physicochemical features have been developed. In particular, stealth nanocarriers can be obtained by polymeric coating (Salmaso et al., 2013). It is now recognised that long circulating nanocarriers, ―stealth‖ systems, can be obtained by surface coating with hydrophilic polymers that prevent the opsonisation process (Moghimi et al., 2001; Yan et al.,
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2005). Stealth nanocarriers are invisible to the biological system involved in clearance of particles from the bloodstream, namely the reticuloendothelial systems (RES) and Kupffer cells.
Conventional chemotherapy with anticancer drugs has no tumour selectivity and is randomly distributed in the body; resulting in severe side effects associated with anticancer drugs with low therapeutic index (Kakde et al., 2011). Researchers have tried to develop therapeutic agent with tumour specific antibody or ligand. However, Matsumure and Maeda, (1986), reported that macromolecular drug delivery system with prolonged blood circulation can accumulate by passive retention mechanism in tumours even in the absence of targeting ligands. This is referred to as the Enhanced Permeability and Retention Effect (EPR effect). Due to the leaky vasculature and low lymphatic drainage, solid tumours present erratic fluid molecular transport dynamics. These features can yield specific accumulation of colloidal anticancer drug delivery systems into the tumour tissues by the EPR effect. The pore sizes in solid tumour vasculature vary from 100 nm to 760 nm, which is much larger than that of the normal tissue where the gaps are usually less than 6 nm (Drummond et al., 1999).
Nanocomposite is a composition having dispersed material that has one or more dimensions, such as length, width, and thickness in the nanometre size range (Roul et al., 2013). The final properties of the nanocomposite are very often not a simple addition of the properties of the independent components, but a unique result from synergistic effect (Hood et al., 2014).
It has been shown that the drug loading efficiency and controlled release behaviour can be enhanced because of the synergistic effect between biopolymer and inorganic materials (Devanand et al., 2011). Venkatesan et al. (2011), carried out a study on chitosan modified HA nanocomposite loaded with celecoxib. The anticancer nanocomposite showed high entrapment efficiency and sustained release profile. HA is a good biocompatible material, but when used as a
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carrier in pure form, the impregnated drug can elute only for a short period (Krisanapiboon et al., 2006). In vitro drug release study by Raj et al., (2013) showed that normal HA showed a burst release in the initial stage. However, coating of HA by polyvinyl alcohol (PVA) showed sustained release of about 70% of the drug in 7 days 12 hours. Addition of cloisite nanoparticles to poly (ethylene-co-vinyl acetate) resulted in slower release of dexamethasone (Cypes et al., 2003), while modification of SA with hydrophobic poly (butyl methacrylate) led to the prolonged release of the model drug as compared with unmodified alginate gels (Yao et al., 2010). By controlling the release kinetics of drugs, one can not only optimize the therapeutic effects of the drugs, but also influence their biological activity (Jana et al., 2013).
1.2 Statement of the Problem
According to Martinho et al. (2011), sustained drug delivery is most useful in treatment of chronic diseases such as cancers. Globally, cancer remains the second most common cause of death (only exceeded by heart diseases) despite the advances in prevention, early detection and protocols of treatment (Marques et al., 2014). In 2012, the International Agency for Research on Cancer (IARC) gave an estimated 14.1 million new cancer cases and 8.2 million cancer related deaths, compared with 12.7 million and 7.6 million respectively in 2008 (Ferlay et al., 2013). A report by World Health Organization (WHO) has shown that cancer related mortality is still on the increase globally including in Nigeria. Between August 2014 and 2016, 3.4 million people have died from cancer globally out of which 36, 000 are from Nigeria alone.
Although cancer treatment is mainly based on surgery, radiotherapy and chemotherapy, chemotherapy is one of the most important treatments currently available among the various approaches. The present status of chemotherapy is far from being satisfactory. Its efficacy is
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limited and patients suffer from serious side effects, some of which are life-threatening (Marques et al., 2014).
Methotrexate (4-amino-10-methylfolic acid) is a hydrophilic anticancer drug widely used for treatment of a number of diseases such as leukaemia, lymphomas, osteosarcoma, and rheumatoid arthritis (Nerves et al, 2009). Despite its importance, its use is limited by dose-dependent toxicity ranging from malaise, asthenia, to gastrointestinal and hepatic toxicity, and pancytopenia which can be fatal (Khan et al., 2010). MTX efficacy is also limited by its short plasma half-life (Yousefi et al., 2010).
Doxorubicin (DOX), on the other hand, is commonly used in the treatment of a number of diverse malignant tumours like acute leukaemia, non-Hodgkin‘s and Hodgkin‘s lymphoma and several solid tumours. However, the side effects of dose – dependent cardiotoxicity, myelosuppression as well as large distribution volume and low life time represent the limitations of its clinical use (Asadishad et al., 2010).
1.3 Justification of the Research
Despite advancement in cancer research, cancer related morbidity and mortality is still increasing. The situation is even worse in developing countries, where access to radiotherapy and some costly chemotherapy is limited; and according to Boyle and Levin (2008), most people in developing countries have no access to cancer screening and early diagnosis. It is also known that the available drugs have dose-limiting toxicity which limits their use. There is, therefore, need to fabricate drug delivery materials that are biodegradable, cheap and readily available and capable of exploiting the EPR effect to reduce wastage and dose-limiting toxicity associated with anticancer drugs.
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Since the discovery of the EPR effect by Maeda et al. (1986), research interest on cancer has focused on exploitation of this phenomenon. For any material to make use of the EPR effect, it must have size in the range of 100 nm – 760 nm (the pore size in solid tumour vasculature, (Drummond et al., 1999), possess stealth characteristics, and provide sustained release profile. To this end, nanocomposite comprising a hydrophilic polymer and inorganic particles like HA is a promising material. It has been shown that the drug loading efficiency and controlled release behaviour can be enhanced because of the synergistic effect between biopolymer and inorganic materials (Devanand et al., 2011). The stealth characteristics can be provided by SA (because of its hydrophilicity); and research has shown that incorporating HA with a biopolymer prolongs the release of encapsulated drug (Raj et al., 2013).
Although numerous researches have been carried out on encapsulation of DOX and MTX for drug delivery application, to the best of our knowledge, the use of HASA for this application has not been investigated.
1.4 Aim and Objectives
1.4.1 Aim of the Study
The aim of this study is to prepare and characterize HASA nanocomposite and to evaluate its application in delivery of two anticancer drugs – DOX and MTX.
1.4.2 Objectives of the study
i. To prepare HA nanoparticles and HASA nanocomposite and characterize them using XRD, XRF, SEM, and FTIR.
ii. Drug loading and in vitro drug release study of the HA and the HASA nanocomposite.
iii. Determination of release kinetics and release mechanism for the drug delivery system.
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iv. To investigate the effects of different solvents used in the synthesis of the nanocomposites on drug loading and release profile.
v. To investigate the effects of drug loading methods on the drug release behavior.
vi. To investigate the effect of pH of the release medium on drug release profiles.
vii. To study co-delivery of the two drugs – DOX and MTX, by the nanocomposite in order to evaluate its suitability for combination therapy.
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