INVESTIGATION OF IN VITRO LOADING AND RELEASE KINETICS OF HYDROXYAPATITE/CELLULOSE BIOCOMPOSITES By BENJAMIN MAWULI GANE (10479794) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL BIOMEDICAL ENGINEERING DEGREE UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES DEPARTMENT OF BIOMEDICAL ENGINEERING DECEMBER 2021 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION I, Benjamin Mawuli Gane, do hereby declare that the work in this thesis titled “Investigation of in vitro loading and release kinetics of hydroxyapatite/cellulose biocomposites” was carried out and written by me under strict supervision according to the academic rules and regulations of University of Ghana and that this thesis has never been presented either in part or in whole for any degree in this University or elsewhere. …20/12/2021… Benjamin Mawuli Gane (10479794) Date (Student) This thesis has been submitted for examination with our approval as supervisors. ………………………… …20/12/2021… Prof. Elvis K. Tiburu Date (Principal Supervisor) …… …………… …20/12/2021… Dr. Bernard Asimeng Date (Co-Supervisor) University of Ghana http://ugspace.ug.edu.gh iii ABSTRACT Cellulose nanocrystal (CNC) and hydroxyapatite (HAP) offer numerous advantages in drug delivery. A significant challenge encountered is the occurrence of burst release when these materials are used as drug carriers. This renders drugs ineffective when used at sites that require a sustained release. In this work, CNC and HAP composites were synthesized and used as drug carriers to investigate the release behavior of a model drug (curcumin). Two synthesis techniques were used where one material serves as the base material and the other as a modifier in varying concentrations. The formulated composites were characterized by Fourier-transform infrared spectroscopy (FTIR) and X-Ray powder Diffraction (XRD). The drug was loaded onto the composites for 22 hours. Composites with HAP as base material demonstrated successful loading of curcumin whereas composites with CNC as base material did not exhibit drug uptake. There were no significant differences (p > 0.05) in terms of drug uptake between the formulated composites and the control (HAP). The release properties of the composites were evaluated in vitro for 6 hours at acidic (pH ≈ 6.2) and basic (pH ≈ 7.4) conditions. The percentage drug release in acidic medium was found to be higher than basic medium. One-way ANOVA revealed that there was no difference in the means of the release data between the composites and the control group at acidic pH (p > 0.05). However, at basic condition, the release data of the composites and the control group showed significant differences (p < 0.05). Significant differences (p < 0.01) were observed for all composites across groups, indicating that changes in pH had a significant effect on the drug release. All composites and control at both pH (6.2 and 7.4) were best described by the Korsmeyer-Peppas model. Cell viability studies showed that modified composites inhibited cell growth at all time points during the drug release as compared to HAP only. This shows that the combination of HAP and CNC serves as a better drug delivery system than HAP only. University of Ghana http://ugspace.ug.edu.gh iv DEDICATION I dedicate this thesis to the Almighty God, my parents and to everyone who played an important role in my life. University of Ghana http://ugspace.ug.edu.gh v ACKNOWLEDGEMENTS First and foremost, I thank the almighty God for His strength throughout my graduate study. My profound gratitude goes to my supervisors. Prof. Elvis K. Tiburu and Dr. Bernard Asimeng for their invaluable advice, continuous support, encouragement and constructive suggestions on this study. I gratefully recognize the help of Mr. Solomon Kingsley Katu for his assistance in keeping my progress on schedule and offering me the resources available in the laboratory for my research work. Special thanks to Dr. Martin Egblewogbe and Mr. Samuel Owusu-Atuah for their help in the X-Ray powder Diffraction (XRD) and Fourier-Transform Infrared spectroscopy (FTIR) characterizations respectively. I would also like to thank Mr. Edward Amenyaglo for his enormous help. I also thank Mr. Alex Atia, the Assistant Overseer at the Department of Biomedical Engineering. Finally, I wish to thank my parents (Mr. Benjamin Seloame Gane and Mrs. Charlotte Agudu) and Ms. Benedicta Bubune Adatorvor for their prayers, support and encouragement throughout the entire study. University of Ghana http://ugspace.ug.edu.gh vi TABLE OF CONTENTS DECLARATION ............................................................................................................................ ii ABSTRACT ................................................................................................................................... iii DEDICATION ............................................................................................................................... iv ACKNOWLEDGEMENTS ............................................................................................................ v LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi LIST OF ACRONYMS AND ABBREVIATIONS...................................................................... xii CHAPTER ONE ............................................................................................................................. 1 1.0 Introduction ........................................................................................................................... 1 1.1 Background ........................................................................................................................... 1 1.2 Problem statement ................................................................................................................. 6 1.3 Research question and hypothesis ......................................................................................... 6 1.3.1 Research questions ......................................................................................................... 7 1.3.2 Hypothesis ...................................................................................................................... 7 1.4 Aim and objectives ................................................................................................................ 8 1.5 Scope and limitations ............................................................................................................ 8 1.5.1 Scope .............................................................................................................................. 8 1.5.2 Limitations ..................................................................................................................... 9 CHAPTER TWO .......................................................................................................................... 10 2.0 Literature review ................................................................................................................. 10 University of Ghana http://ugspace.ug.edu.gh vii 2.1 Drug delivery ...................................................................................................................... 10 2.1.1 Drug delivery methods ................................................................................................. 12 2.1.2 Drug delivery systems .................................................................................................. 12 2.1.3 Requirements for drug delivery systems ...................................................................... 14 2.2 Cellulose nanocrystals (CNCs) ........................................................................................... 16 2.2.1 Modification of CNC ................................................................................................... 19 2.3 Hydroxyapatite (HAP) ........................................................................................................ 20 2.3.1 Modification of HAP ................................................................................................... 22 CHAPTER THREE ....................................................................................................................... 24 3.0 Materials and Experimental Techniques ............................................................................. 24 3.1 Materials and chemicals ...................................................................................................... 24 3.2 Experimental methods ......................................................................................................... 24 3.2.1 Dissolution of CNC ...................................................................................................... 24 3.2.2 Dissolution of HAP ...................................................................................................... 25 3.2.3 Composite Formation ................................................................................................... 25 3.2.3.1 CNC as base material ................................................................................................ 25 3.2.3.2 HAP as base material. ............................................................................................... 26 3.2.4 Calibration Curve of Model Drug (Curcumin) ................................................................ 26 3.2.5 Drug Loading ................................................................................................................... 26 3.2.6 In vitro drug release ......................................................................................................... 28 3.3 Material Characterization Methods ..................................................................................... 28 University of Ghana http://ugspace.ug.edu.gh viii 3.3.1 Fourier-Transform Infrared spectroscopy (FTIR) ........................................................ 28 3.3.2 X-ray powder diffraction (XRD) ................................................................................. 29 3.3.3 Ultraviolet-visible spectroscopy (UV-VIS) ................................................................. 29 3.4 Cell viability studies ............................................................................................................ 31 3.4.1 Cell culture conditions ..................................................................................................... 31 3.4.2 MTT assay ........................................................................................................................ 31 3.5 Data analysis ....................................................................................................................... 32 CHAPTER FOUR ......................................................................................................................... 34 4.0 Results and Discussion ............................................................................................................ 34 4.1 Fourier-transform infrared spectroscopy (FTIR) ................................................................ 34 4.1.1 Composites with CNC as base material ....................................................................... 34 4.1.2 Composite using HAP as base material. ...................................................................... 36 4.2 X-Ray Diffraction (XRD) ................................................................................................... 38 4.2.1 Composites with CNC as base material ....................................................................... 38 4.2.2 Composites with HAP as base material ....................................................................... 40 4.3 Standard calibration curve of model drug (curcumin) ........................................................ 42 4.4 Drug loading ....................................................................................................................... 43 4.5 Drug release characteristics ................................................................................................ 46 4.6 In vitro drug release kinetics ............................................................................................... 50 4.6.1 Drug release in pH ≈ 6.2 .............................................................................................. 50 4.6.2 Drug release in pH ≈ 7.4 .............................................................................................. 51 University of Ghana http://ugspace.ug.edu.gh ix 4.7 Cell viability ........................................................................................................................ 55 CHAPTER FIVE ........................................................................................................................... 58 5.0 Summary ............................................................................................................................. 58 5.1 Conclusion .......................................................................................................................... 58 5.2 Recommendation ................................................................................................................ 60 6.0 References ............................................................................................................................... 61 7.0 Appendices .............................................................................................................................. 79 University of Ghana http://ugspace.ug.edu.gh x LIST OF TABLES Table 4.1: Crystallinity of samples in the range 20° – 25° 2θ ...................................................... 40 Table 4.2: Summary of crystallinity of samples ........................................................................... 41 Table 4.3: Entrapment efficiency of composites with HAP as base material ............................... 45 Table 4.4: Percentage cumulative release of curcumin from composites ..................................... 46 Table 4.5: R-squared values from fitted models ........................................................................... 54 University of Ghana http://ugspace.ug.edu.gh xi LIST OF FIGURES Figure 2.1: Pharmacokinetic profiles of sustained release vs. conventional release of drugs. ..... 11 Figure 2.2: Different types of carriers used for drug delivery ...................................................... 16 Figure 2.3: Chemical structure of CNC showing the rich hydroxyl groups ................................. 19 Figure 2.4: Chemical structure of hydroxyapatite. ....................................................................... 22 Figure 3.1: CNC powder in solvent system .................................................................................. 24 Figure 3.2: HAP powder in 5% glacial acetic acid ....................................................................... 25 Figure 3.3: Agitation of composites in drug solution to ensure adsorption of drug ..................... 27 Figure 4.1: FTIR spectra of composites with CNC as base material ............................................ 36 Figure 4.2: FTIR spectra of composites with HAP as base material. ........................................... 37 Figure 4.3: XRD plot of composites with CNC as base material. ................................................ 39 Figure 4.4: XRD plot of composites with HAP as base material. ................................................ 41 Figure 4.5: Standard calibration curve of curcumin ..................................................................... 42 Figure 4.6: Drug upload on CNC and composites with CNC as base material ............................ 44 Figure 4.7: Drug upload on HAP and composites with HAP as base material ............................. 45 Figure 4.8: Drug release profiles of samples in acidic medium (pH ≈ 6.2) .................................. 47 Figure 4.9: Drug release profiles of samples in neutral medium (pH ≈ 7.4) ................................ 48 Figure 4.10: Higuchi release profile of all samples ...................................................................... 52 Figure 4.11: Korsmeyer-Peppas release profile of all samples ..................................................... 52 Figure 4.12: Cubic degree polynomial release profile of all samples ........................................... 53 Figure 4.13: Cell viability of all composites and control using MTT assay. ................................ 55 Figure 4.14: Cell viability of 2.0 mg CNC/HAP composite using MTT assay. ........................... 57 Figure 4.15: Cell viability of HAP using MTT assay. .................................................................. 57 University of Ghana http://ugspace.ug.edu.gh xii LIST OF ACRONYMS AND ABBREVIATIONS ANOVA One-way analysis of variance CNC Cellulose nanocrystal CTAB Cetyltrimethylammonium bromide DDS Drug delivery system DMSO Dimethyl sulfoxide FTIR Fourier Transform Infrared Spectroscopy HAP Hydroxyapatite LiOH Lithium hydroxide MTT 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide NaOH Sodium hydroxide NP Nanoparticle PVA Poly (vinyl alcohol) SDS Sodium dodecyl sulfate SEM Scanning Electron Microscopy UV Ultraviolet UV-Vis Ultra-violet Visible spectroscopy XRD X-Ray powder Diffraction University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 Introduction This chapter presents a standard synopsis of the project considering the project’s background, research justification, problem statement, aim and objectives, research question and hypothesis, the scope and limitations. 1.1 Background There has been an unprecedented growth in the field of nanotechnology research pertaining to drug delivery in the treatment of diseases. This is observed as researchers investigate various approaches for diagnosis and treatment for cancer. Nanotechnology is defined by the National Nanotechnology Initiative in the United States as the understanding and control of matter at nanoscale dimensions which is approximately 1 to 100 nanometers (Heath, 2015). Nanoparticles (NPs) are used as drug carriers as they can deliver active ingredients targeted to malignant cells or tissues without causing harm to surrounding healthy cells and tissues while producing a desired pharmacological effect. NPs that act as drug carriers should be biodegradable, should not trigger any immunological response, and be able to release the drug at the target site (Portioli et al., 2017). NPs provide major advantages over conventional delivery methods in terms of high stability, high specificity, high drug loading capacity, capability for controlled release of drug and the ability to transport both hydrophilic and hydrophobic molecules. Despite the numerous advantages of NPs as drug delivery systems, they still pose some challenges which include burst or rapid release at targeted sites that require a sustainable or prolonged release and goes a long way to affect therapeutic efficiency. There is a growing demand for drug carriers that provide long-term drug release. Material composites are considered in order to make the development of these types of drug carriers easier. University of Ghana http://ugspace.ug.edu.gh 2 However, the uniformity of these composite materials has an impact on drug release rates. According to Huang and Brazel's research, drug release is influenced by drug dispersion heterogeneity (Huang & Brazel, 2001). As a result of the heterogeneous dispersion of drugs introduced by the aggregation of composites, caution should be exercised during material processing for composite formation to be used as drug carriers. Combining organic and inorganic materials results in enhancing their individual physical and chemical properties. These enhanced properties possess various characteristics (Sanchez, Julián, Belleville, & Popall, 2005) which are used in many applications particularly in biomedical fields. Biopolymers such as cellulose, chitosan, collagen and alginate among others are usually incorporated into nanocomposites due to their outstanding properties such as biocompatibility, biodegradability and availability. Cellulose has however received much attention with a number of applications ranging from paper, textile, and composite to pharmaceutical excipients for over the past years (Eyley & Thielemans, 2014). Cellulose has been found to be readily available and the most sustainable biopolymer on earth (Moniri et al., 2017). Generally, cellulose has the same molecular formula (C6H10O5)n, irrespective of its source. Various sources of cellulose are wood, plant fibers, bacteria and invertebrates among others. Plant cellulose, unlike bacterial cellulose, contains lignin, hemicelluloses, and pectin which compromises the purity of cellulose from plants. However plant cellulose are highly soluble than cellulose from bacterial source (Zhong, 2020). Chemically, cellulose is a linear polysaccharide made up of glucose monosaccharide units that link each other by β-1-4 glycosidic bond (Kenawy et al., 2020). Intermolecular hydrogen bonds bind hydroxyl groups and oxygen atoms to glucose in a rigid and stable ring structure. Comparatively such rigid intermolecular bonds to the glucose base is in multiples and highly stable in bacterial cellulose making it uneasy to dissolve (Lee, University of Ghana http://ugspace.ug.edu.gh 3 Blaker, & Bismarck, 2009). These cellulose molecules exist as fibers (which are about 5-50 nm in diameter) held together by intramolecular hydrogen bonds and van der Waals interactions. Thus, cellulose is partially soluble in water and some solvents unlike alcohol. This is as a result of the strong intramolecular and intermolecular hydrogen bonding between the chains that make up cellulose (Lee et al., 2009). Such a limitation can be mitigated by doping cellulose with other polymers or inorganic materials such as HAP (hydroxyapatite) to break and limit inter and intramolecular hydrogen bonds. Depending on the type of application, cellulose is purified and separated into its nanocrystal components. To separate into nanocrystals, cellulose is treated with an acid which catalyzes the amorphous regions, thereby leaving the crystalline regions only (Shankaran, 2018). The purification methods include treatment with sodium hydroxide (NaOH) or sodium dodecyl sulfate (SDS) (Banerjee, Saraswatula, Williams, & Brettmann, 2020). It is reported in literature that a synthesized poly(vinyl alcohol) PVA/cellulose composites exhibited a highly proven mechanical and thermal properties and also a high flexibility as compared to the pure cellulose only (Dai, Wang, Samanipour, Koo, & Kim, 2016). In another paper, Poly (ethylene oxide)/cellulose nanofiber biocomposites were formulated by aqueous solution method. The results showed that the modified biocomposites exhibited enhanced mechanical properties. These works show that blended polymer biocomposites showed improved properties which can be used for many biomedical applications (Safdari, Carreau, Heuzey, Kamal, & Sain, 2017). Cellulose’s biocompatibility in vitro and in vivo has been proven by several studies. Research has shown that plant cellulose-derived scaffolds had a promising potential when subcutaneously implanted in wild immunocompetent mice. The scaffolds supported the production of natural extracellular matrix and promoted vascularization (Modulevsky, Cuerrier, & Pelling, 2016). Literature also reported the in vivo and in vitro evaluations of plant-derived cellulose hydrogels University of Ghana http://ugspace.ug.edu.gh 4 enriched with Larrea tridentata in a rat model. It showed no negative effect on their biocompatibility properties (Tovar-Carrillo et al., 2020). A study reported a fabrication method of plant-derived scaffolds that support cell attachment and invasion both in vitro and in vivo. Their fabrication method is reported to improve cell attachment and the scaffold remains highly biocompatible and maintains in vivo vascularization properties (Hickey, Modulevsky, Cuerrier, & Pelling, 2018). These studies affirm the biocompatibility of plant cellulose and the reason why it is useful in diverse range of applications especially biological or medical applications. The use of cellulose nanocrystal (CNC) as a drug carrier is becoming more popular. Most drugs released from this polymer, however, are released rapidly, which could be due to ion exchange between ions on the CNC surface and ions present in the release medium (Jackson et al., 2011). This affects the therapeutic efficiency of the drug at sites that require sustained released of drug. CNC is also being studied as a composite material due to its large surface area, high aspect ratio, and tremendous stiffness and strength (Jackson et al., 2011). Combining CNC and a polymer or inorganic material as a composite material may result in a prolonged or sustained release. The use of inorganic nanoparticles has aroused interest among scientists. The high surface energy of inorganic nanoparticles makes them thermodynamically unstable (Huang, Li, Liu, He, & Tan, 2015). However, from experiments, during the production and treatment of these inorganic nanoparticles, agglomeration, which is a challenge, occurs leading to the aggregation of the individual materials involved. This comes about as a result of the attraction of the particles through chemical bonding as the nanoparticles are being formed in surfactant-free chemical reactions. This affects their properties and applications (Li & Kaner, 2006). The study further reported that this limitation is solvable by causing the nanoparticles that are formed to be absorbed onto a supporting material, examples being an inorganic material or a polymer. Research works are geared towards University of Ghana http://ugspace.ug.edu.gh 5 using these inorganic nanoparticles in the biomedical field. A typical example is the use of hydroxyapatite (HAP) as drug carriers because it possesses no, or minimum toxicity and it is one of the excellent biocompatible materials since it is found in the human teeth and bone. Despite the numerous advantages of HAP, it has also been proven that drugs released from HAP is very fast due to weak interaction between the drugs and HAP. This weak interaction is due to electrostatic interactions between the ions on HAP and the drug (Mizushima et al., 2006). This challenge can be reduced by combining HAP nanoparticles with a polymer which will result in a prolonged release of the drug. From literature, polymer/HAP composites are used to achieve sustained release of drug molecules, growth factors and as scaffolds for cell growth (Wei & Ma, 2004). Typically, in the synthesis of inorganic nanoparticles, large aggregates are formed instead of well dispersed nanoparticles due to the presence of high surface energy. This aggregation is caused by hydrogen bonding and van der Waal’s forces (Laaksonen, Ahonen, Johans, & Kontturi, 2006). To mitigate this problem, the surfaces are mostly protected. Stabilizers are added during the aqueous synthesis of inorganic nanoparticles to help control the nucleation, growth rate, and particle shape of the nanoparticles. The stabilizers with metal affinity groups bind to the inorganic surface and thus induce repulsive forces which help to stabilize the inorganic particles. When the stabilizers degrade or undergo oxidation, the stabilization of the inorganic nanoparticles is compromised. Also, conditions such as pH change and the charge present on the inorganic nanoparticle surface is affected resulting in the initiation of nanoparticle aggregation (Sperling & Parak, 2010). Another approach in resolving the problem of aggregation of the particles is by depositing inorganic nanoparticles onto a material with high surface area. Literature has reported that carboxylic or sulphonic acid groups offer longer stability for inorganic nanoparticles (Sperling & Parak, 2010). The above approaches will prevent aggregation and make the formulated composites University of Ghana http://ugspace.ug.edu.gh 6 suitable to be used as a drug carrier for sustained release of drugs. Research scientists have gained interest in the use of biomaterials from plants for various biomedical applications, for example the use of CNC as a substrate to promote the growth of HAP crystals. In this work, CNC and HAP were functionalized, a model drug (curcumin) was loaded onto the resulting composites and released at pre-determined times. The functionalized composites were characterized by Fourier-Transform Infrared spectroscopy (FTIR) and X-Ray powder Diffraction (XRD), and the release studies were done by Ultra-violet Visible spectroscopy (UV-Vis). The release study and kinetics of the composites were examined by using existing kinetic models. 1.2 Problem statement Cellulose nanocrystal and hydroxyapatite when used as drug carriers, exhibit burst release of the drug (Mizushima et al., 2006; Jackson et al., 2011). This affects therapeutic efficiency when used at targeted sites that require a sustained release of the drug. This, therefore, increases the ineffectiveness of the drug. Despite the numerous advantages of polymers and inorganic materials as drug carriers, they still pose some challenges which include sustained and safe delivery of drug to cells for improved efficacy. Most of these materials have not been able to completely deliver sustainably on the promise of targeted diseased sites (Pasut, 2019). 1.3 Research question and hypothesis The purpose of this research is to provide answers to questions raised by literature and to lay the framework for adopting or rejecting the hypothesis indicated below. University of Ghana http://ugspace.ug.edu.gh 7 1.3.1 Research questions 1. Can CNC (as a base material) be modified with HAP? 2. Can HAP (as a base material) be modified with CNC? 3. Can the modified composites be loaded with the model drug (curcumin)? 4. Does the interaction between the composites enhance the release rate of the drug? 5. What is the effect of pH on the rate of release of the drug (curcumin)? 6. How do the modified composites affect the delivery of curcumin to MDA-MB-231 cells? 1.3.2 Hypothesis Hydroxyapatite (HAP) and cellulose nanocrystals (CNC) possess complementary properties and are compatible which will ensure effective interfacial bonding between them and result in composites with improved properties and functionalities. The composites formed from HAP and CNC will be capable of loading the model drug curcumin and exhibit sustained release properties resulting in improved drug delivery characteristics. The pH of the surrounding environment influences the release rate of drugs from modified composites leading to different rates of drug release depending on variations in pH. It is therefore predicted that the drug release profile will exhibit a pH-dependent behavior with a more pronounced effect observed at extreme pH levels. It is also anticipated that the modified composites will exhibit controlled drug release, thereby effectively reducing cell viability. University of Ghana http://ugspace.ug.edu.gh 8 1.4 Aim and objectives The aim of this work is to synthesize hydroxyapatite/cellulose biocomposites for sustained release of drug to improve therapeutic efficiency. To achieve the overall aim of this work, the following objectives will be followed: 1. Synthesize and characterize HAP/CNC composites using HAP and CNC as base materials. 2. Assess drug upload and release kinetics in the HAP/CNC composites. 3. Examine the drug release kinetics of the HAP/CNC composites in different pH. 4. Assess the impact of loaded composites on drug delivery to MDA-MB-231 cells. 1.5 Scope and limitations 1.5.1 Scope This work centers primarily on using HAP to modify CNC and to use CNC which is a polymer as a dopant during the synthesis of HAP as a base material. The model drug will be loaded on the resulting composites and study the release kinetics at different pH (6.2 and 7.4). The thesis will consist of 5 chapters. Chapter one will be the introduction, which gives a background of this research, the objectives and the relevance of the work. Chapter two will talk about literature review, which surveys scholarly articles, books and other sources relevant to this research. Chapter three will outline the materials and techniques used. Chapter 4 will cover the results and discussions of the findings and chapter five, which is the last chapter, will be the conclusion of the work with some recommendations. University of Ghana http://ugspace.ug.edu.gh 9 1.5.2 Limitations The following studies were not covered in this work: i. Zeta potential of synthesized composites. ii. Scanning Electron Microscopy (SEM) of composites. University of Ghana http://ugspace.ug.edu.gh 10 CHAPTER TWO 2.0 Literature review This chapter reviews the underlining theoretical concepts used in this work. It focuses on drug delivery systems, synthesis of cellulose nanocrystals and hydroxyapatite, drug release studies from synthesized composites in literature. 2.1 Drug delivery Drug Delivery is a fast-evolving multidisciplinary field within the health sciences. Drug delivery is a broad field which describes the development of novel carrier systems for safe and effective delivery of therapeutic agents to their site of action to achieve therapeutic effect (Tiwari et al., 2012). Pharmacodynamics and pharmacokinetics are essential for drug delivery. Pharmacodynamics describes the physiologic and biochemical effects of the drug on the body (Rimmington, 2020). Pharmacokinetics refers to the time course of the concentration of a drug in the body, from absorption, bioavailability, distribution, metabolism through to excretion (Thürmann, 2020). For a disease to be properly managed, pharmacokinetic profiles of therapeutic drugs must be consistent. This is contingent on the patient's continued compliance in taking the medication as prescribed. Patients on prolonged release therapies have been shown in the literature to have lower rates of relapse due to constant dosing of the medicine (Senior, Radomsky, & Group, 2000). This enables the achievement of a consistent and stable pharmacodynamics profile. Conventional method of drug delivery is effective when a quick response of the drug is required. However, drugs with narrow therapeutic window and low solubility are ineffective when delivered by the conventional method. University of Ghana http://ugspace.ug.edu.gh 11 Figure 2.1: Pharmacokinetic profiles of sustained release vs. conventional release of drugs. Adapted from “Modeling of controlled-release drug delivery from autocatalytically degrading polymer microspheres” by A. F. Versypt (2015) In figure 2.1, sustained release is achieved when a drug concentration increases steadily and plateaus in the therapeutic range in the blood plasma of a specific target and releases the drug for an extended period and that is where maximum therapeutic efficiency is reached. When the drug concentration goes above the therapeutic range, it is considered toxic and when the concentration goes below the therapeutic range, it is rendered ineffective. The first controlled release formulation was approved in the 1950s (Lee & Li, 2010). Since then, there has been a steady increase in the development of new carriers for drug delivery as compared to development of new drugs (Park, 2014). One of the major factors for this shift is due to the high cost and time involved in developing new drugs. A review in 2013 found that the cost involved in developing a drug carrier was 10% of the cost of developing a new drug (He et al., 2013). As such research scientists prefer to develop drug carriers to increase the effectiveness of drug targeting and delivery other than go through the University of Ghana http://ugspace.ug.edu.gh 12 long process of drug discovery, clinical testing, and drug development through to regulatory approval. 2.1.1 Drug delivery methods Drugs are administered by swallowing, absorption, inhalation, intravenous injection, or through the skin to create a systemic pharmacological effect. The oral route which is the common route experiences the following problems: low bioavailability, poor absorption of protein and this results in low permeability of drugs (Gopinath & Wilson, 2020). For pulmonary delivery as a route for systemic administration, inhalation is used. Intravenous infusion is often used to deliver sedatives, analgesics, chemotherapy agents, anesthetics and other fluid to patients (Ferrari, Sponchioni, Morbidelli, & Moscatelli, 2018). For transdermal route of drug delivery, the skin acts as a barrier allowing molecules with appropriate physiochemical properties to penetrate the skin (Alkilani, McCrudden, & Donnelly, 2015). Some advantages of delivery of drugs through the skin are less frequent dosing, large area for absorption and noninvasiveness among others (Saravanakumar, Swapna, Nagaveni, Vani, & Pujitha, 2015). 2.1.2 Drug delivery systems Drug delivery systems (DDSs) are pharmaceutical formulations or engineered devices that aid in the targeted delivery and/or controlled release of pharmaceutical compounds in the body and improves its efficacy and safety by regulating the rate, timing, and location of medication release in the body (Jain, 2020). DDSs release active pharmaceutical compounds when administered. When these compounds are encapsulated within a protective structure, potential disruptions of the active compounds are reduced (Wang et al., 2021). Aside the increment in bioavailability of the pharmaceutical compound, undesirable side effects resulting from systemic distribution and frequent dosing are reduced (Kok-Yong & Lawrence, 2015). The bioactive molecules reach the University of Ghana http://ugspace.ug.edu.gh 13 site of action as they pass through many biological barriers. Research has revealed that these barriers play different roles in the transport of drugs in the circulatory systems and through cells and tissues (Yang et al., 2017). Research scientists have capitalized on the roles of these biological barriers to develop new modes of drug delivery. Current novel DDSs trigger the release of the drug only at the target site with sustained or controlled release. Others deliver based on stimuli such as light, and temperature among others. One important advantages offered by delivery systems is the controlled drug release in a time dependent manner through passive and active targeting (Kumar & Gupta, 2012). Passive targeting works based on the pathophysiological features of tumor vessels which allow DDS to accumulate in the tumor at much higher concentrations than in normal tissues since tumor vessels become more permeable than healthy state and the compromise of the lymphatic drainage in tumors also enhances the retention of theses delivery systems in tumors (Attia, Anton, Wallyn, Omran, & Vandamme, 2019). This type of targeting depends on the size and circulation time of the delivery system. Active targeting relies on the ligand-receptor binding, which improves the accumulation of DDS to target sites (Yoo, Park, Yi, Lee, & Koo, 2019). This facilitates the uptake of DDS by the tumor cells. DDS could be activated by a trigger that is peculiar to the target site, such as a pH responsive material. Example, most parts of the body have a consistent neutral pH. Some other parts may become more acidic than others, especially tumor environments. DDSs can however use the change in pH to trigger the release of the drug. Another mechanism that can be used to trigger drug release is the redox potential (Mollazadeh, Mackiewicz, & Yazdimamaghani, 2021). Tumors change the redox potential in their environment. Based on this, a payload of drug can be released based on a change in redox potential in the tumors. University of Ghana http://ugspace.ug.edu.gh 14 2.1.3 Requirements for drug delivery systems Ideal Drug Delivery Systems ensure that drug is released at the targeted site of action in accordance with the need of patient for an intended duration. The concentration of the drug at the targeted site should be between minimal effective concentration and minimal toxic concentration (Hedaya, 2012). The DDSs must also regulate the rate of release of the drug in a controlled manner. Increase in patient compliance and reduction in frequency of drug dosing can be achieved when the drug fluctuation is reduced in the plasma level while extending its duration of action. The materials used for DDSs should not trigger any adverse response, the matrix material and its degraded products should be biocompatible, the mechanical properties of the material should be able to provide prolonged protection of the therapeutic cargo thus ensuring chemical stability of the drug before it gets to the target site (Parhiz et al., 2018). The surface of the DDS should be functionally modified for cell-specific moieties. There should also be a triggerable or controlled drug release. The stability of the product and delivery should be maintained under various physiological variables. They should be easy to administer to patients and ultimately, they should be safe and reliable. Figure 2.2 illustrates various examples of DDSs that have been developed over the years, such as liposomes, micelles, polymeric particles, hydrogels, dendrimers, inorganic particles, quantum dots, and nanospheres. Liposomes are made up of one or more concentric phospholipid bilayers (Haisheng He et al., 2019). Polar drugs are loaded in its core because of its polar nature. They can entrap both amphiphilic and lipophilic compounds depending on the compounds’ affinity towards the phospholipids. In literature, Ketoprofen drug was encapsulated in pharmacosomes and showed an improved dissolution of 93.3% as compared to free ketoprofen which showed 49.77% dissolution (Amandeep Kaur, 2013). University of Ghana http://ugspace.ug.edu.gh 15 Dendrimers are highly branched and monodispersed with symmetrical structure. They have a central core, branching units and terminal functional groups (Dias et al., 2020). Ligands are attached to the terminal ends of dendrimers to enhance drug targeting. The dendrimers are functionalized by polyethylene glycol chains to keep them stable and protect them from Mononuclear Phagocyte System. It was observed from research that the solubility of aceclofenac, a nonsteroidal anti-inflammatory drug, was improved by using G0 PAMAM dendrimers. The study showed that the enhancement of aceclofenac was dependent on the concentration of the dendrimer (Patel, Basu, Dharamsi, Garala, & Raval, 2011). Polymers used for drug delivery can be natural or synthetic. Polymeric system formulations can deliver drugs sustainably for a longer time duration based on the properties of the polymer used in the formulation. Polymers can be tuned to change its properties depending on its application (Sharma, Aiswarya, Mirza, & Saha, 2020). This makes research scientists gain interest in the use of polymers for drug delivery applications. Drug release from polymers are affected by the environment of the release media, diffusion from the polymer and the chemical nature of the delivery system. Wilson et al., (2010) developed tacrine loaded chitosan nanoparticles and coated with polysorbate 80 for anti-Alzheimer therapy. The synthesized particles showed improved drug- loading capacity and sustained release of drug. Inorganic materials such as silica, graphene oxide, and hydroxyapatite and gold nanoparticles have been exploited for use in delivery of drugs. The nano-carriers which serve as skeletons in these systems are able to load and release drugs without compromising its framework in the blood (Zhaoqing Shi et al., 2020). They are biocompatible and possess good pharmacological properties. Lei et al., (2019) developed a drug delivery system by coating polydopamine on the surface of University of Ghana http://ugspace.ug.edu.gh 16 mesoporous silica nanoparticles. The resulting carrier could respond to high glutathione or low pH to release drug. The diagram below shows some types of DDSs commonly used. Figure 2.2: Different types of carriers used for drug delivery. From “Designing dendrimer and miktoarm polymer based multi-tasking nanocarriers for efficient medical therapy” by A. Sharma, & A. Kakkar. (2015). Molecules, Vol. 20, pp. 16987–17015. 2.2 Cellulose nanocrystals (CNCs) Cellulose consists of crystalline and amorphous regions. Acid treatment of cellulose, irrespective of the source, hydrolyses the glycosidic bonds. This is in conjunction with the hydrolysis of the amorphous regions of cellulose. This is because the acid can permeate and catalyze the amorphous regions. The resulting crystalline units are referred to as CNCs. The size of the resulting nanocrystals depends on the source of the cellulose. Example CNCs from cotton are 70 – 300 nm (Elazzouzi-Hafraoui et al., 2008), from bacteria is 100 – 1700 nm (George & Siddaramaiah, 2012), University of Ghana http://ugspace.ug.edu.gh 17 from wood; 100 – 300 nm (Beck-Candanedo, Roman, & Gray, 2005) in length. Hydrolysis using sulfuric acid results in the formation of sulfate esters on the surface of the nanocrystal. The hydroxyl groups of CNC as shown in figure 2.3, react partially with the acid to the formation of charged sulfate ester groups on its surface. This provides good dispersibility in aqueous suspension. The concentration of sulfuric acid and the duration of hydrolysis affect the number of sulfate groups present on the nanocrystal (Wang, Zhao, & Zhu, 2014). However, if CNC with no sulfate groups on the surface are required, then hydrobromic or hydrochloric acid is used in the hydrolysis (Montanari, Roumani, Heux, & Vignon, 2005). CNCs are rod-like in shape and have high aspect ratio (Habibi, Lucia, & Rojas, 2010), large surface area (Lam, Male, Chong, Leung, & Luong, 2012), high mechanical strength (Moon, Martini, Nairn, Simonsen, & Youngblood, 2011), high stiffness (Lam et al., 2012) and hydrophilic in nature (Farnia, Fan, Dory, & Zhao, 2019). The negatively charged surface of the CNC, along with its large surface area, enables the binding of high levels of drugs to its surface and the release of drugs by ion exchange processes in a regulated manner (Jain, Sahu, Mohan, & Jain, 2016). CNC can be distributed in polymer matrices of the same charge and bind with others of opposite charge. This results in a drug delivery system that releases drugs at a controllable rate. Research has shown the excellent sustained release of pilocarpine hydrochloride from poloxamer modified CNC up to more than twenty hours (Åhlén, Tummala, & Mihranyan, 2018). Abo-Elseoud et al., (2018) developed a contact lens platform for ocular drug delivery by incorporating in situ gelled nanoparticles and CNC in PVA lenses. The drug release was prolonged over 28 hours and the developed system exhibited a greater potential for extended drug release. A study reported a burst drug release, followed by a slow and sustained release at 37°C when poly (N-isopropylacrylamide) reinforced with CNC was loaded with drug. The results showed that the University of Ghana http://ugspace.ug.edu.gh 18 modified CNC has a good potential for being used as in ophthalmic drug delivery system and injectable hydrogels for wound dressing (Zubik, Singhsa, Wang, Manuspiya, & Narain, 2017). There is the tendency of CNC aggregating when used in the preparation of nanocomposites though CNCs are well-dispersed in aqueous media. This arises due to lack of compatibility with the polymer which comes from the numerous surface hydroxyl groups as a result of the presence of strong hydrogen bonding (Wohlhauser et al., 2018). This challenge can be overcome by the introduction of an interaction between the polymer and CNCs. Preparation of CNC-polymer nanocomposites with covalent bonding between the polymer and CNC is one way to overcome the problem of aggregation (Meesorn, Shirole, Vanhecke, De Espinosa, & Weder, 2017). The direct attachment of polymer chains onto the surface of CNC creates the covalent bond. The binding of polymer chains to the CNC surface can be facilitated by the reactive functionality of the polymer chain (Kang, Liu, & Huang, 2015). The reacting polymer chains have larger groups and so have a significant steric hindrance to access the reaction site on CNC because of the presence of attached polymer chains (Chakrabarty & Teramoto, 2018). Another way aggregation can be prevented is by growing the polymer chains on CNC’s surface. This is done by attaching species that initiate polymerization on the surface of CNC. Polymerization on the surface is highly probable in the presence of monomers and this will produce a high grafting density (Wu et al., 2015). The grafting process of other polymers through reactive hydroxyl groups on the surface of CNC yields low graft ratios. Researchers have gained interest in the functionalization of CNC with inorganic nanoparticles since the high surface area of CNC enhances nanocomposite formation between CNC and inorganic particles. Functionalization of CNC promotes the sustained release of drug since there will be a good dispersion of the drug in the absence of aggregation. University of Ghana http://ugspace.ug.edu.gh 19 Figure 2.3: Chemical structure of CNC showing the rich hydroxyl groups. From “In situ nano- assembly of bacterial cellulose-polyaniline composites” by Z. Shi et al., (2012). RSC Advances, 2(3), 1040–1046. 2.2.1 Modification of CNC Features such as biocompatibility, biodegradability and surface charge, among others have an effect on how drugs are released and delivered (Jackson et al., 2011). CNC is a good drug carrier for hydrophilic drugs due to its negative surface charge (Hare et al., 2017). The surface of CNC, which is rich in hydroxyl groups, may be modified with various chemical substances to other functional groups. Even though CNC does not carry hydrophobic drugs, the surface modification can enhance the loading and release of hydrophobic drugs like curcumin (Roman, Dong, Hirani, & Lee, 2009). Surface modification of CNC can also be employed to introduce new electrostatic properties (Kargarzadeh et al., 2018). This may also aid in drug delivery purposes and other applications. Some surface modification methods include cationization, sulfonation, oxidation, and silylation (Jorfi & Foster, 2015; Lam et al., 2012). Cationization has to do with the treatment of surface of a material with cations. This reduces the hydroxyl groups present on the surface of CNC. Oxidation introduces carboxylate groups onto the CNC surface which leads to better dispersion in aqueous phase. The reaction changes the C6 primary hydroxyl groups to carboxylate groups. Silylation refers to the introduction of a substituted silyl group to CNC typically through reactions with organosilane compounds. Sulfonation is the process of introducing sulfonic acid in University of Ghana http://ugspace.ug.edu.gh 20 an organic compound. This replaces the hydrogen atoms present on CNC surface with sulfonic acid making the resulting compound more water-soluble. Another approach to modifying CNC involves the use of surfactant to coat their surface (Brinchi, Cotana, Fortunati, & Kenny, 2013). In a related study, the negative charge of CNC was successfully modified using a cationic surfactant called Cetyltrimethylammonium bromide (CTAB), resulting in CNC acquiring hydrophobic properties allowing for efficient loading of hydrophobic drugs within the CTAB domains. Notably, it was observed that the release of hydrophobic drugs from the modified CNC exhibited a slower rate compared to unmodified CNC (Brinchi, Cotana, Fortunati, & Kenny, 2013). These modifications enhance the surface hydrophobicity of CNC and facilitates the binding of hydrophobic drugs, leading to a sustained release profile for non-steroidal anti-inflammatory drugs (Gupta & Raghav, 2020) and curcumin (Raghav et al., 2020) among others. Similarly, another study reported the effect of natural surfactant saponin, on the modification of cellulose and starch nanoparticles through hydrophobic drug loading and release (Putro, Ismadji, Gunarto, Soetaredjo, & Ju, 2019). 2.3 Hydroxyapatite (HAP) HAP with chemical formula Ca10(PO4)6(OH)2 is a bioceramic that is similar to the constituent of bones and teeth. It belongs to the calcium phosphates family. HAP has gained interest in science because of its exceptional characteristics; bioactivity and excellent biocompatibility (Mohd Pu’ad, Koshy, Abdullah, Idris, & Lee, 2019), absence of immune response (Ghiasi, Sefidbakht, & Rezaei, 2019), affinity to biopolymers (Setiawan, Widiyastuti, Winardi, & Nugroho, 2017) osteoinductivity and osteoconductivity (Yokota et al., 2018). HAP has good adsorptive properties due to the presence of its ion composition (Asimeng et al., 2019). HAP’s thermodynamic stability in biological environment and adsorptive properties make it one of the best options for drug University of Ghana http://ugspace.ug.edu.gh 21 delivery to cancer cells. HAP has a hexagonal structure (Luque et al., 2017) with high amount of hydroxyl on the surface and polar charge. This property makes it able to attract peptides and DNA (Sadat-Shojai, Khorasani, Dinpanah-Khoshdargi, & Jamshidi, 2013) and as such a good candidate for drug delivery systems for the controlled release of drugs (Andrés et al., 2018). However, the surface of pure HAP makes it lack biofunctionality which is a problem in drug delivery and this effect could be combated by adding a polymer to HAP to reduce leakage hence enhancing its biofunctionality. Research has reported that drugs loaded onto HAP are released very fast at the initial stage due to the weak force of interaction between drugs loaded and HAP particles (Zhu et al., 2015). Amidst the performance advantages of HAP, it has low strength and toughness, low fatigue resistance in biological environment. These disadvantages make it difficult for HAP to be used for medical and biological application. This problem can be alleviated by changing preparation methods of HAP and adding other materials like polymers to enhance its properties. Studies have reported the use of natural polymers such as chitosan, collagen, alginate, gelatin and starch to modify HAP which resulted in improved properties (Chen, Shi, Luo, & Ma, 2019). Literature reported that HAP/chitosan composite showed an improved mechanical strength and biocompatibility. HAP nanoparticles were synthesized with polymers (polyvinyl alcohol and sodium alginate) and loaded with amoxicillin. Amoxicillin was observed to be released at a sustained rate for 30 days. The sustained release was achieved by coating HAP with polymers in a layer-by-later method (Prasanna & Venkatasubbu, 2018). Literature has reported enhancement in the antibacterial activity against Staphylococcus aureus through the introduction of silver-doped hydroxyapatite (Riaz et al., 2018). Due to the chemical makeup and orientation of HAP crystals, they have two binding sites: C-sites (Ca2+) and P-sites (PO4 -) as shown in figure 2.4. In aqueous media, the C-sites are positively University of Ghana http://ugspace.ug.edu.gh 22 charged and release OH- ions from the surface of the crystal. Ca2+ ions interact with the negatively charged proteins after release of OH- ions. The P-sites are negatively charged and thus interact with positively charged proteins (Haider, Haider, Han, & Kang, 2017). Literature confirmed that –COO- ions from leucine-rich amelogenin protein interacted with the surface of the HAP (Chen, Wang, Shen, Pan, & Wu, 2007). This confirms the presence of the C-sites on the surface of HAP. There are no side effects recorded for using HAP as drug carrier, there is enhanced bioavailability and efficacy. However, drawbacks still exist such as low mechanical strength and rapid release of the loaded drug. Figure 2.4: Chemical structure of hydroxyapatite. From “Direct crystal formation from micronized bone and lactic acid: The writing on the wall for calcium-containing crystal pathogenesis in osteoarthritis” by A.A. Bulysheva, N. Sori, & M. P. Francis, (2018). PLoS ONE, 13(11). 2.3.1 Modification of HAP The surface of HAP is modified through ions adsorption, reprecipitation, encapsulation and surface reactions. The majority of surface ion exchange occurs due to hydroxyapatite's high specific surface area and the presence of a metastable hydrated layer on the surface containing loosely bound ions (Cazalbou et al., 2005). This is evident in the literature because apatites have metal ion sorption and exchange behavior to remove heavy metal ions from water or soils (Smičiklas, Onjia, Marković, & Raičević, 2005; Sugiyama et al., 2003). Reprecipitation is the process of dissolving a precipitate and precipitating it again. This ensures that metal ions are continuously distributed to the surface layer (Skwarek & Janusz, 2019). University of Ghana http://ugspace.ug.edu.gh 23 Surface reaction of hydroxyapatite involves the reaction between ions present at the surface. An example of such a reaction is the adsorption of fluoride ions onto hydroxyapatite in aqueous solution. Calcium and phosphate ions together with fluoride ions were in solution and reacted. Fluoride was adsorbed to H2PO− 4 ions and favored the formation of brushite. As the amount of calcium was increased in the solution, calcium reacted with fluoride to form CaF2 and was precipitated. Phosphate ions present favored the formation of fluorinated apatite (Gasser, Haikel, Voegel, & Gramain, 1994). University of Ghana http://ugspace.ug.edu.gh 24 CHAPTER THREE 3.0 Materials and Experimental Techniques This chapter explains the experimental approach used in this research work. The materials and chemicals used, synthesis of CNC/HAP composites, drug loading, release studies, characterization techniques have been described in this chapter. 3.1 Materials and chemicals All reagents used for this work needed no further purification and were of analytical grade. Curcumin (99% purity), Cellulose Nanocrystals, Hydroxyapatite (99.5% purity), Dimethyl sulfoxide (DMSO) (99.0 % purity), Lithium hydroxide (LiOH), Urea (CH4N2O), Sulphuric acid (98.9% purity) H2SO4, Acetic acid (CH3COOH). 3.2 Experimental methods 3.2.1 Dissolution of CNC CNC was dissolved in a solvent system consisting of 6% LiOH, 4% Urea and 90% water as shown in figure 3.1. The CNC powder (60 mg) was weighed and added to the solvent system of 6 ml and vortexed for 20 minutes. The resulting concentration of the CNC solution was 10 mg/ml. The CNC solution was then kept in the refrigerator for 48 hours to obtain a clear solution. Figure 3.1: CNC powder in solvent system University of Ghana http://ugspace.ug.edu.gh 25 3.2.2 Dissolution of HAP The HAP powder (0.6 g) was dissolved in 60 ml of 5 % glacial acetic acid as shown in figure 3.2. The resulting mixture was stirred for 20 minutes to attain complete dissolution. The concentration of HAP obtained was 10 mg/ml. Figure 3.2: HAP powder in 5% glacial acetic acid 3.2.3 Composite Formation 3.2.3.1 CNC as base material Dissolved CNC solution is thawed to form a transparent solution and divided into five equal parts: 5 ml each. To each 5 ml CNC solution, varied masses of HAP (0.5 mg, 1.0 mg, 1.5 mg, and 2.0 mg) were added. The CNC solution without HAP served as a control. The resulting suspensions were vortex at a speed of 1400 rpm for 15 minutes. The HAP/CNC composite solutions were placed in a vacuum oven at 40 °C for 5 minutes. The solutions were allowed to cool and stored for 72 hours. The solution is then centrifuged at 3600 rpm for 10 minutes to obtain the HAP/CNC composites. The products are washed with distilled water and dried in vacuum to obtain dry powders of the composites. University of Ghana http://ugspace.ug.edu.gh 26 3.2.3.2 HAP as base material. HAP solution was divided into 5 parts (5 ml each). To each of the parts, 20 µl, 40 µl, 60 µl, and 80 µl of CNC were added. The added volumes of CNC solution represent 0.5 mg, 1.0 mg, 1.5 mg and 2.0 mg of CNC added. HAP only was used as a control in this study. The resulting mixtures were agitated for 15 minutes with the help of the vortex at a speed of 1400 rpm to obtain a uniform mixture. The mixture is then heated at 45°C for 5 minutes. The mixture is allowed to cool and stored for 72 hours. The mixture is centrifuged at 3600 rpm for 10 minutes to obtain the CNC/HAP composites. The product is washed with distilled water several times. Dry powders of the composites were obtained by drying the product in vacuum. 3.2.4 Calibration Curve of Model Drug (Curcumin) The calibration curve developed in this study was utilized to assess the amounts of curcumin in the release medium. 8 mg of curcumin powder was dissolved in 8 ml of DMSO-water (v/v 1:1) to obtain a concentration of 1 mg/ml of the resulting solution. The solution was vortexed for 30 minutes in order to obtain a uniform solution. Subsequent concentrations were obtained through serial dilution. A calibration curve (R2 = 0.979) was plotted by measuring the absorbance of the diluted solutions at a wavelength of 428 nm. 3.2.5 Drug Loading Curcumin (30 mg) was dissolved in 14 ml of DMSO-water (v/v 1:1) to obtain a concentration of 2.14 mg/ml. Composites (15 mg) were weighed and transferred into Eppendorf tubes. Curcumin solution (1.5 ml) was pipetted and added to the composites (and control). The mixture was then agitated gently using the rotating equipment at 18 rpm to ensure the adsorption of the drug onto the composites as shown in figure 3.3. At predetermined times the mixture was centrifuged at 3000 rpm for 1 minute and 50 µl of the supernatant was pipetted and diluted with 950µl of 50% DMSO University of Ghana http://ugspace.ug.edu.gh 27 solution. The absorbance of the resulting solution was measured at wavelength of 428 nm using the JENWAY 6705 UV-Vis Spectrophotometer (Antylia Scientific, USA). 50 µl of fresh 50% DMSO solution was replaced in the solution to maintain sink conditions. The drug loading was done for 22 hours. Equation (3.1) was used to determine the amount of curcumin entrapped onto the formulated composites. Entrapment Efficiency (% EE) = x−y x × 100% (3.1) Where, x = total concentration of drug added y = concentration of drug in the supernatant. Entrapment efficiency estimates the percentage of the active ingredient that is adsorbed on the composites compared to the total amount of drug added. Figure 3.3: Agitation of composites in drug solution to ensure adsorption of drug onto composites. University of Ghana http://ugspace.ug.edu.gh 28 3.2.6 In vitro drug release The curcumin solution is discarded, leaving the curcumin-loaded composites in the Eppendorf tubes. 1.5 ml release medium made up of DMSO-water (v/v 1:1) at pH (≈ 6.2 and ≈ 7.4) was added to each composite and rocked on the rotating equipment at 18 rpm. At fixed time intervals, the mixture was centrifuged at 6000 rpm for 1 minute and 50 µl of the supernatant was carefully pipetted and diluted with 950 µl of 50% DMSO solution. Absorbance of the resulting solution was measured at wavelength of 428 nm using the UV-Vis Spectrophotometer. 50 µl of fresh 50% DMSO solution was replaced in the solution to maintain sink conditions. The drug release was done for 6 hours at 37 °C. The concentration of the released curcumin was determined using a standard curve of curcumin in 50% DMSO. Technical replicates were incorporated into the experimental design to ensure reliability and reproducibility of the data. The same experimental approach was conducted in triplicates under same experimental conditions. This made it possible to evaluate measurement precision and find any potential variations or errors in the experimental design. 3.3 Material Characterization Methods 3.3.1 Fourier-Transform Infrared spectroscopy (FTIR) FTIR is a well-established technique for chemical compound identification and structural analysis. Peaks in an infrared spectrum represent the excitation of vibrational modes of molecules in the sample and are thus associated with the various chemical bonds and functional groups present in the molecules (Nandiyanto, Oktiani, & Ragadhita, 2019). The infrared beam is focused on the sample thereby allowing various interactions with the sample. A slit selects the various wavelengths that pass through the sample at any given time. Absorption of light by the sample depends on the sample’s chemical properties. A detector then collects the radiation that passes University of Ghana http://ugspace.ug.edu.gh 29 through the sample and compares its energy to the reference. The analysis was conducted on Hydroxyapatite (HAP), Cellulose Nanocrystals (CNC) and the formulated HAP/CNC composites. The FTIR was recorded with a Nicolet MAGNA-IR 750 Spectrometer (Nicolet Instrument Co., Madison, WI, USA). The analysis was done from 450 to 4000 cm-1 and the data was analyzed. 3.3.2 X-ray powder diffraction (XRD) X-ray powder diffraction is a non-destructive technique that reveals information about the crystallographic structure, physical properties, and chemical composition of materials (Jurásek, Bartůněk, Huber, & Kuchař, 2019). Constructive interference of a monochromatic beam of X-rays at different angles from each set of lattice planes in a material produces XRD peaks (Bunaciu, Udriştioiu, & Aboul-Enein, 2015). These peaks are determined by the positions of atoms within the lattice planes. This technique uses Bragg’s law of diffraction. Which is defined as, nλ = 2dSinθ, where n is the reflection order, λ is the wavelength, d is the interplanary distance and θ is the angle of reflection. XRD analysis was conducted on Hydroxyapatite (HAP), Cellulose Nanocrystals (CNC) and the formulated HAP/CNC composites. The XRD spectra of samples were obtained by an Analytical diffractometer with CuKα radiation operated at 40 mA and 45 kV. Samples were scanned from 5° to 100° with a scan step size of 0.05° and a scan step time of 38.5 seconds. 3.3.3 Ultraviolet-visible spectroscopy (UV-VIS) UV-vis spectroscopy is used to determine the absorbance spectra of a compound in solution. It works based on Beer-Lambert law as seen in equation (3.2) which states that there is a linear relationship between the concentration and the absorbance of the solution. A = ε x b x c (3.2) University of Ghana http://ugspace.ug.edu.gh 30 where, A is the absorbance, ε is the molar absorptivity of the solution, b is the path length of the cuvette which is usually 1 cm, and c is the concentration of the solution. A source of light is needed to emit light over a wide range of wavelengths. Since the UV-vis spectrophotometer measures absorbance in the ultraviolet and visible region, a source of light that measures both UV and visible light ranges is appropriate. An example of a source of light that exhibits such a characteristic is the xenon lamp. However, xenon lamps are less stable and very expensive. Therefore, tungsten or halogen lamps and deuterium lamps are used. Tungsten or halogen lamp is used for measurement in the visible light range, while deuterium lamp is used for the UV range. Since two sources of light are used, there is a switchover when measurements are across the UV and visible ranges. The incoming light is split into a narrow band of wavelengths by a monochromator. The selected wavelength then passes through the sample. Measuring a reference sample is essential for all analyses. In this work, the upload and release rates were studied using the UV-vis spectrophotometer. It helps to determine the amount of drugs that was adsorbed onto the formulated composites and the amount of drugs released from the loaded composites by measuring the absorbance within a specific time interval. The JENWAY 6705 UV-vis spectrophotometer was used for UV-vis characterization. The samples were measured at a wavelength of 428 nm. OriginPro 8.5 (OriginLab Corp., Northampton, MA, USA) was used to analyze the data obtained from the absorbance measurements. University of Ghana http://ugspace.ug.edu.gh 31 3.4 Cell viability studies Biocompatibility is one of the key properties of bio-ceramic and human biological system and this is assessed by performing in vitro cell viability assays (Williams, 2014). In this study, commercial HAP and formulated composites were assessed by utilizing MTT assay to evaluate the viability of MDA-MB-231 cells on various formulated composites. A percentage of cell viability greater than 90% indicates that the sample is biocompatible to MDA-MB-231 cells. 3.4.1 Cell culture conditions Prior to experiments, MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Corporation, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; R&D Systems, Minneapolis MN, USA), 1% penicillin and streptomycin (Life Technologies Corporation, Grand Island, NY, USA) in a humidified 5% CO2 environment at 37 °C (cell culture incubator). Confluent cell monolayers were detached from the culture flask with 2ml of 1x trypsin- EDTA solution (Gibco, Waltham, MA, USA), suspended in culture medium and incubated for 5 minutes for subculturing. The trypan blue exclusion experiment was used to measure cell viability and density. 3.4.2 MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay MTT assay was used to test curcumin's anticancer activity. The MTT assay is based on the ability of cells to convert the yellow soluble salt of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) to an insoluble formazan (purple-blue) precipitate. MDA-MB-231 (10,000 cells/100 µL) cells were seeded overnight in 96-well plates. Prior to testing loaded composites on cells, 2.5 µL of raw composites (composites without curcumin) and HAP in 5% DMSO were tested on cells to assess compatibility and viability of the samples with the cell culture system. This is done to determine if the samples are toxic to cells and to assess baseline effects. University of Ghana http://ugspace.ug.edu.gh 32 MDA-MB-231 cells were then treated with 2.5 µL of loaded composites in 5% DMSO and incubated for 48 hours. After the incubation period, 20 µL of MTT was added to each well and incubated for further 4 hours after which 100 µL of acidified isopropanol was added to dissolve the MTT. A microplate reader spectrophotometer was used to record the optical densities at 570 nm after 30 minutes of incubation. 3.5 Data analysis Data were obtained from the drug loading and release experiments. The XRD and FTIR test generated data in the form of spectra. Origin Pro software was employed for the analysis of the XRD and FTIR spectra and cell viability studies. The data obtained from the drug release were tested for normality using Kolmogorov-Smirnov test. Data showing p > 0.05 were accepted to be normally distributed, hence parametric test was performed on the data. One-way analysis of variance (ANOVA) was used to find out whether there were statistically significant differences in the release data with respect to the synthesis of composites and cumulative release in different pH release media. Tukey test was used to find out the means that were significantly different from each other at p < 0.05. The drug release data was fitted using the Higuchi and Korsmeyer-Peppas models and cubic degree polynomial. A higher regression coefficient (R2) indicated a strong correlation and improved linearity. Origin Pro software was used to determine the Coefficients of Determination when fitting the data to the different models. These kinetic models provide insights into the underlying mechanism of drug release. The Higuchi model is based on diffusion. It describes drug release from insoluble matrix dependent on the square root of time. This model hypothesizes that the initial concentration of drug in the matrix is higher than the solubility of the drug. Korsmeyer-Peppas model is used to ascertain the type of diffusion taking place. This is University of Ghana http://ugspace.ug.edu.gh 33 characterized by the diffusion coefficient factor, (n) in the Korsmeyer-Peppas equation as shown in table 4.5. University of Ghana http://ugspace.ug.edu.gh 34 CHAPTER FOUR 4.0 Results and Discussion This chapter presents and discusses the experimental and characterization outcomes of the modified HAP/CNC composites. 4.1 Fourier-transform infrared spectroscopy (FTIR) 4.1.1 Composites with CNC as base material The FTIR spectra of the HAP, CNC and the HAP/CNC composites are presented in figure 4.1. The FTIR spectral data are used to study the changes that occurred in the functional groups of CNC when reacted with HAP. The distinct transmission peaks of CNC were observed at the wavenumbers of 3566, 3354, 1625, 1009 cm-1. For HAP, the distinct transmission peaks were at 1029, 601 and 565 cm-1. 3566 and 3354 cm-1 bands are attributed to -OH intramolecular hydrogen bonding (Fan, Dai, & Huang, 2012) present in CNC. 1625 cm-1 indicates C-H symmetrical stretching (Terpáková, Kidalová, Eštoková, Čigášová, & Števulová, 2012) and 1009 cm-1 is attributed to C–O Stretching vibration at the crystalline region of cellulose (Abidi et al., 2008). 1029 and 601 cm-1 of HAP indicate PO4 3- Stretching (Varma & Babu, 2005) and PO4 3- Bending (Yusufoglu & Akinc, 2008) respectively. 565 cm-1 is attributed to P-O symmetric stretching of PO4 group (Varma & Babu, 2005). The weak peak present at 875 cm-1 is ascribed to bending in the CO3 2- ions (Berzina-Cimdina & Borodajenko, 2012). It can also be attributed to the presence of HPO4 2- (Gozalian, Behnamghader, Daliri, & Moshkforoush, 2011). After the modification of CNC with varied masses of HAP, two shoulders 3443 and 3215 cm-1 corresponding to OH Stretching (Kallel et al., 2016) and -OH due to intermolecular hydrogen bonding, reduced. The peak at 3696 cm-1 which is attributed to –OH University of Ghana http://ugspace.ug.edu.gh 35 intramolecular hydrogen bonding (Fan et al., 2012) was enhanced in all composites except 2 mg HAP/CNC composite. The peak at 3566 cm-1 reacted when HAP was added and moved to a higher wavenumber of 3622 cm-1. However, the peak at 3622 cm-1 was not distinct in the 2 mg HAP/CNC composite. The broad region at 2911 – 2871 cm-1 which indicates the presence of CH Stretching (Schwanninger, Rodrigues, Pereira, & Hinterstoisser, 2004) was absent in the 1.5 mg HAP/CNC composite (it was weakened and disappeared), and was reduced in the 1 mg HAP/CNC composite and enhanced in the rest of the composites. After modification, the peak at 1420 cm-1 which represents the amount of crystalline structure of cellulose (Åkerholm, Hinterstoisser, & Salmén, 2004) increased with the amount of HAP added to the composite. However, the 2 mg HAP/CNC composite increased but did not follow that trend. The increase in the peak may be due to the mass of HAP added to CNC which might have affected the crystallinity of the resulting composite. There was also the emergence of two peaks at 911 and 863 cm-1 in all the composites. All composites show an increase in the peak intensity of 911 cm-1 which is ascribed to the interactions between glycosidic linkages and glucose units of cellulose. The peak at 1029 cm-1 was present in all composites. However, it was broadened and moved to a lower wavenumber for all the composites except that of the 2 mg HAP/CNC composite. This could be due to the presence of hydrogen bonding during the modification of CNC with HAP. University of Ghana http://ugspace.ug.edu.gh 36 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm -1 ) 3566 1029 601 565 (a) 1625 100932153443 3622 3700 (b) 3354 T ra n s m it ta n c e ( % ) (c) 911 (d) 1420 (e) (f) 863 Figure 4.1: FTIR spectra of (a) HAP Only (b) CNC Only (c) 0.5 mg HAP/CNC (d) 1.0 mg HAP/CNC (e) 1.5 mg HAP/CNC (f) 2.0 mg HAP/CNC composites where CNC is the base material. 4.1.2 Composite using HAP as base material. The distinct transmission peaks of HAP only were observed at wavenumbers of 1740 cm-1, 1366 cm-1, 1028 cm-1, 601 cm-1 and 565 cm-1 as shown in figure 4.2. The peak position at 1740 cm-1 is C=O stretching vibrations (Predoi et al., 2018), 1366 cm-1 represents C–O stretching vibrations (Predoi et al., 2018). Peaks at wavenumbers 1028 cm-1 and 601 cm-1 of HAP represent PO4 3- stretching (Varma & Babu, 2005) and PO4 3- Bending (Yusufoglu & Akinc, 2008) respectively. The peak at 565 cm-1 indicates P-O symmetric stretching of PO4 group (Varma & Babu, 2005). FTIR spectra labelled ‘c’, ‘d’, ‘e’ and ‘f’ represent modified HAP with varied masses of CNC (0.5 University of Ghana http://ugspace.ug.edu.gh 37 mg, 1.0 mg, 1.5 mg and 2.0 mg). After modification, there was appearance of the peak at 3566 cm- 1 on all the modified samples which indicates OH intramolecular hydrogen bonding in CNC (Fan et al., 2012). The peak positions 1740 cm-1, 1366 cm-1 and 1217 cm-1 disappeared in all modified samples. The peak at 962 cm-1 which is attributed to the (PO4) 3- symmetric stretching (El Khouri, Zegzouti, Elaatmani, & Capitelli, 2019) present in HAP became relatively intense in all the composites. The appearance of 3566 cm-1, disappearance of various peaks and increase in intensity of 962 cm-1 confirms the modification of HAP. 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm -1 ) (a) 1740 1366 1217 565 3566 T ra n s m it a n c e ( % ) (b) 1028 962 (c) 1088 (d) 6013566 (e) (f) Figure 4.2: FTIR spectra of (a) HAP Only (b) CNC Only (c) 0.5 mg CNC/HAP (d) 1.0 mg CNC/HAP (e) 1.5 mg CNC/HAP (f) 2.0 mg CNC/HAP composites where HAP is the base material. University of Ghana http://ugspace.ug.edu.gh 38 4.2 X-Ray Diffraction (XRD) 4.2.1 Composites with CNC as base material Figure 4.3 shows the XRD pattern of HAP, CNC and HAP/CNC composites. HAP shows intense peaks at 2θ = 25.79, 31.67, 32.0, 32.79 corresponding to (002), (211), (300) and (002) which clearly indicates the presence of phosphate groups. CNC shows intense peaks at 2θ = 21.23, 22.13, 29.34, 30.46, 31.67, 64.85, 80.0 which correspond to (110), (110), (111), (202), (002), (-114) and (041). The spectra labelled ‘c’, ‘d’, ‘e’ and ‘f’ represent the XRD patterns of the CNC/HAP composites. Similar XRD patterns were observed for the composites. However, there was disappearance of the peak at 22.13° 2θ position in 0.5 mg HAP/CNC and 1.0 mg HAP/CNC composites but it reduced in the 2.0 mg HAP/CNC composite. There was a preferred orientation in 1.5 mg HAP/CNC composite of peaks at 21.23° 2θ and 22.13° 2θ. The peaks at 22.13° 2θ disappeared on addition of 0.5 mg and 1.0 mg of HAP. However, when 1.5 mg HAP was added, the peak at 22.13° 2θ increased as compared to that of CNC only and when 2.0 mg HAP was added, it decreased. The peaks at 29.34° 2θ, decreased for all composites. These changes confirm the formation of composites. The crystallinity of CNC and the composites where major changes occurred are presented in table 4.1. University of Ghana http://ugspace.ug.edu.gh 39 20 30 40 50 60 70 80 1 3 0 2 1 3 2 2 23 0 02 1 1 0 0 2 2 Theta (degree) (a) 1 1 2 1 0 1 -1 1 22 0 2 1 1 1 -1 1 4 (b) 0 0 2 0 4 1 1 1 0 (c) 1 1 0 C o u n t (d) -3 1 1 (e) (f) Figure 4.3: XRD plot of (a) HAP Only (b) CNC Only (c) 0.5 mg HAP/CNC (d) 1.0 mg HAP/CNC (e) 1.5 mg HAP/CNC (f) 2.0 mg HAP/CNC composites where CNC is the base material. University of Ghana http://ugspace.ug.edu.gh 40 Table 4.1: Crystallinity of samples in the range 20° – 25° 2θ Samples Crystallinity (%) CNC Only 49 0.5 mg HAP/CNC 43.6 1.0 mg HAP/CNC 39.6 1.5 mg HAP/CNC 41.4 2.0 mg HAP/CNC 41.7 4.2.2 Composites with HAP as base material Figure 4.4 shows the XRD pattern of HAP, CNC and CNC/HAP composites. HAP shows peaks at 2θ= 25.79 °, 31.67 °, 32.0 °, 32.79 °, 33.95 ° corresponding to (002), (211), (112), (300) and (202) clearly indicate the presence of phosphate groups. Similar XRD patterns were observed for all composites. 0.5 mg CNC/HAP composite showed a decrease in major peaks of HAP including peaks at 46.72° 2θ and 49.48° 2θ corresponding to (222) and (213) however there was emergence of a peak at 49.48° 2θ and this is due to the interaction of HAP with CNC. The peak at 64.82 ° 2θ position increased more in 0.5 mg CNC/HAP than the rest of the composites. The intensity of peak at 78 ° 2θ which indicates the presence of CNC in the composites increased in 0.5 mg CNC/HAP composite but decreased in the other composites. The XRD plot clearly indicates the formation of composites. The crystallinity of HAP and the composites are presented in table 4.2. University of Ghana http://ugspace.ug.edu.gh 41 20 30 40 50 60 70 80 2 Theta (degree) (a) 2 1 1 3 0 0 2 0 2 0 0 2 1 3 0 2 2 2 2 1 3 2 1 0 -1 1 42 0 2 0 0 2 1 1 1 0 4 1 (b) C o u n t (c) (d) (e) 1 1 2 (f) Figure 4.4: XRD plot of (a) HAP Only (b) CNC Only (c) 0.5 mg CNC/HAP (d) 1.0 mg CNC/HAP (e) 1.5 mg CNC/HAP (f) 2.0 mg CNC/HAP composites where HAP is the base material. Table 4.2: Summary of crystallinity of samples Samples Crystallinity (%) HAP Only 38.5 0.5 mg HAP/CNC 30 1.0 mg HAP/CNC 32.6 1.5 mg HAP/CNC 27.1 2.0 mg HAP/CNC 33 University of Ghana http://ugspace.ug.edu.gh 42 4.3 Standard calibration curve of model drug (curcumin) 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 A b s o rb a n c e Conc (mg/ml) R 2 = 0.9785 Y = 128.29x + 0.09 Figure 4.5: A standard calibration curve of curcumin Figure 4.5 depicts the standard calibration curve used to determine the concentration of the drug during UV-Vis characterization. Calibration curve is plotted using known concentrations, enabling the estimation of drug concentration based on absorbance values during both drug loading and release. University of Ghana http://ugspace.ug.edu.gh 43 4.4 Drug loading Drug loading efficiency onto the HAP/CNC composites were determined using equation (3.1). Figure 4.6 shows absorbance against time of composites with CNC as base material and CNC only. From the graph it is observed that composites with CNC as base material and CNC (control) did not upload the model drug. From literature, CNC cannot upload hydrophobic drugs, however when CNC is modified with polymers or inorganic materials, it tends to load hydrophobic drugs. Even though in this study CNC was modified with varying masses of HAP, the resulting composites did not upload drug. This could be due to the ratio of HAP to CNC used in the composite formation. Figure 4.7 shows absorbance against time of composites with HAP as base material and HAP only. It can be observed from the graph that composites with HAP as base material and modified with varying masses of CNC uploaded the drug. The loading values of the composites (with HAP as base material) are summarized in table 4.3 below. From the table, it can be inferred that after drug loading, all composites together with the control loaded almost the same amount of drug. There was no significant difference between the samples loaded (p > 0.05). This could be due to the ratio of dissolved CNC and HAP used for the composite formation. University of Ghana http://ugspace.ug.edu.gh 44 Figure 4.6: Drug upload on CNC and composites with CNC as base material University of Ghana http://ugspace.ug.edu.gh 45 Figure 4.7: Drug upload on HAP and composites with HAP as base material Table 4.3: Entrapment efficiency of composites with HAP as base material Composites Entrapment Efficiency (%) HAP Only 84 0.5 mg CNC/HAP 85 1.0 mg CNC/HAP 86 1.5 mg CNC/HAP 86 2.0 mg CNC/HAP 85 University of Ghana http://ugspace.ug.edu.gh 46 4.5 Drug release characteristics The drug release profiles of curcumin from composites with HAP as base material in 50 % DMSO at different pH values are shown in figures 4.8 and 4.9 below. Loaded composites were suspended in 50 % DMSO, centrifuged and the drug (curcumin) content present in the supernatant was quantified by UV-Vis spectrophotometer. The drug release profiles showed that the release of curcumin from all the formulated composites in pH ≈ 6.2 was higher than pH ≈ 7.4. The cumulative release of curcumin from the composites at different pH values after 6 hours is shown in table 4.4. Table 4.4: Percentage cumulative release of curcumin from composites % Cumulative release of drug in different pH Composites pH 6.2 pH 7.4 HAP Only 35.36±0.52 27.95±2.38 0.5 mg CNC/HAP 32.87±1.20 18.99±0.23 1.0 mg CNC/HAP 29.89±4.87 16.52±0.83 1.5 mg CNC/HAP 33.78±2.73 23.32±0.38 2.0 mg CNC/HAP 32.14±5.14 19.60±0.16 University of Ghana http://ugspace.ug.edu.gh 47 0 1 2 3 4 5 6 0 10 20 30 40 % D ru g R e le a s e Time (hrs) HAP ONLY 0.5 mg CNC/HAP 1.0 mg CNC/HAP 1.5 mg CNC/HAP 2.0 mg CNC/HAP Figure 4.8: Drug release profiles for samples in acidic medium (pH ≈ 6.2) University of Ghana http://ugspace.ug.edu.gh 48 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 % D ru g R e le a s e Time (hrs) HAP ONLY 0.5 mg CNC/HAP 1.0 mg CNC/HAP 1.5 mg CNC/HAP 2.0 mg CNC/HAP Figure 4.9: Drug release profiles for samples in neutral medium (pH ≈ 7.4) There was a higher amount of drug released acidic medium (pH ≈ 6.2) than in neutral medium (pH ≈ 7.4). This could be because excess hydrogen ions present in the acidic release medium readily broke the hydrogen bonds that hold the drug onto the composites. From the release profiles, the release of the drug (curcumin) from the control (HAP Only) after 6 hours was found to be the highest in both release media (pH ≈6.2 and pH ≈7.4), 35.36±0.52 % in acidic pH and 27.95± 2.38% in neutral pH. The percentage cumulative drug release was higher in pH 6.2 than in pH 7.4 for all corresponding samples. There was a sustained release of all samples in pH 7.4. (0.5 mg, 1.0 mg, 1.5 mg and 2.0 mg) CNC/HAP composites released 18.99± 0.23%, 16.52± University of Ghana http://ugspace.ug.edu.gh 49 0.83%, 23.32± 0.38% and 19.60± 0.16% of the drug respectively. It is observed that there is a rapid release of curcumin from all samples in pH 6.2 release media. This could be because, in acidic medium, the introduction of H+ ions readily break the bonds between the curcumin and the composites, hence, facilitated the initial high release of curcumin during the 30th minute. The effect of ionic interaction was more in the acidic release medium where carboxylic group is dissociated than in neutral pH. The amount of drug released for 6 hours in pH 7.4 release medium is in the order HAP Only > 1.5 mg CNC/HAP > 2.0 mg CNC/HAP > 0.5 mg CNC/HAP > 1.0 mg CNC/HAP. Whereas the amount of drug released in pH ≈ 6.2 is in the order HAP Only > 1.5 mg CNC/HAP > 0.5 mg CNC/HAP > 2.0 mg CNC/HAP > 1.0 mg CNC/HAP One-way independent analysis of variance (ANOVA) showed that there was significant difference between the means of the rate of drug release of the composites in pH ≈ 7.4 (p = 0.004). Tukey post hoc test showed that the drug release rate was significant between all the composites and the control, HAP only. When 0.5 mg, 1.0 mg, 1.5 mg and 2.0 mg CNC were added to HAP, it reduced the percentage release rate significantly (18.99± 0.23%, p = 0.020; 16.52± 0.83%, p = 0.023; 23.32± 0.38%, p = 0.024; 19.60± 0.16%, p = 0.004). These results show that the addition of CNC affected the drug release in pH 7.4. However, there was no significant difference between the composites (p > 0.05). One-way ANOVA showed that there was a significant difference between the means (three replicates) of the drug release rate across groups (samples in pH ≈ 6.2 and pH ≈ 7.4) (p < 0.001). Tukey test showed that the drug release rate was significant between all the composites (p < 0.05) across group except the control, HAP where there was no significant difference (p = 0.7581). This University of Ghana http://ugspace.ug.edu.gh 50 reveals that the change in pH affected the drug release in the composites. However, this is not the case in control, HAP. 4.6 In vitro drug release kinetics In order to predict the release behavior of a solute from a nanocomposite or material, the release data must fit a mathematical model. Regression coefficient (R2) values shows the relationship between the drug release and the time of release. The kinetic models explain the mechanism of drug release from the samples. Figures 4.10, 4.11 and 4.12 show how the release data were fitted to the models and cubic degree polynomial. Table 4.5 shows a summary of the R-squared and constants from the fitted models. From the table, it can be observed that the 2.0 mg CNC/HAP composite in pH ≈7.4 release media showed the highest correlation with Higuchi model (R2 = 0.9831). The correlation coefficient of HAP only in pH ≈ 6.2 release media was the highest with Korsmeyer-Peppas model (R2 = 0.9898). The cubic degree polynomial is not a function of time, so it was not used to show correlation and linearity. It only gives an idea as to how many factors are involved to achieve the release rate of the drug. 4.6.1 Drug release in pH ≈ 6.2 There was a higher correlation with the Korsmeyer-Peppas model than the Higuchi model for all the samples. HAP showed the highest correlation in both models (R2 = 0.9898 for Korsmeyer- Peppas) and (R2 = 0.8138 for Higuchi). Amongst the composites, 0.5 mg CNC/HAP composite showed the highest correlation (R2 = 0.7571) as far as Higuchi model is concerned. Between the composites, 1.0 mg CNC/HAP composite exhibited the highest correlation (R2 = 0.9828) for Korsmeyer-Peppas model. There was not much of a difference between the correlation coefficient for Korsmeyer-Peppas model for all samples. Table 4.5 gives a summary of the R-squared of the University of Ghana http://ugspace.ug.edu.gh 51 various data. One-way independent analysis of variance (ANOVA) revealed that the various masses of CNC did not have any significant influence on the rate of drug release (p = 0.894). 4.6.2 Drug release in pH ≈ 7.4 There was also a higher correlation with the Korsmeyer-Peppas model than the Higuchi model for all the samples in neutral pH. 2.0 mg CNC/HAP composite showed the highest correlation (R2 = 0.9831) with Higuchi model. HAP exhibited the highest correlation (R2 = 0.9838) with Korsmeyer- Peppas model, followed closely by 2.0 mg CNC/HAP composite (R2 = 0.9835) and 1.5 mg HAP/CNC composite (R2 = 0.9753). Between the composites, 2.0 mg CNC/HAP composite (R2 = 0.9835) exhibited the highest correlation for Korsmeyer-Peppas model followed closely by 1.5 mg CNC/HAP composite (R2 = 0.9753). Table 4.5 gives a summary of the R-squared of all samples. Korsmeyer-Peppas model characterizes its release mechanism by the diffusion coefficient factor (n). The values of the diffusion coefficient factor (n) from table 4.5 is less than 0.45 for all samples, which indicate that the drug release followed Fickian diffusion. Fickian diffusion refers to the diffusion of a substance due to concentration gradient. Hence the rate of drug release from the samples is proportional to the concentration gradient of the drug. 1.0 mg, 1.5 mg and 2.0 mg CNC/HAP composites in acidic medium (pH ≈ 6.2) exhibited the same release mechanism. However, the percentage drug release for these composites was slightly lower than the control (HAP only). This could be as a result of the presence of excess H+ ions present in the acidic release medium. All synthesized composites followed the Korsmeyer-Peppas and Higuchi models. The cubic degree polynomial suggests that the drug release was based on three factors. The three factors could be the concentration gradient of the drug, diffusion distance of the drug from the composite to the release environment and the surface area of the composite. University of Ghana http://ugspace.ug.edu.gh 52 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 HAP ONLY 0.5 CNC/HAP 1.0 CNC/HAP 1.5 CNC/HAP 2.0 CNC/HAP % D ru g R e le a s e Time (hrs) R 2 =0.8138 R 2 =0.7571 R 2 =0.7305 R 2 =0.7453 R 2 =0.6763 (a) 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 HAP ONLY 0.5 CNC/HAP 1.0 CNC/HAP 1.5 CNC/HAP 2.0 CNC/HAP % D ru g R e le a s e Time (hrs) R 2 = 0.7817 R 2 = 0.8484 R 2 = 0.7817 R 2 = 0.9718 R 2 = 0.9831 (b) Figure 4.10: Higuchi release profile for all samples in (a) pH ≈ 6.2 and (b) pH ≈ 7.4 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 HAP ONLY 0.5 CNC/HAP 1.0 CNC/HAP 1.5 CNC/HAP 2.0 CNC/HAP % D ru g R e le a s e Time (hrs) R 2 = 0.9900 R 2 = 0.9485 R 2 = 0.9828 R 2 = 0.9786 R 2 = 0.9729 (a) 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 HAP ONLY 0.5 CNC/HAP 1.0 CNC/HAP 1.5 CNC/HAP 2.0 CNC/HAP % D ru g R e le a s e Time (hrs) R 2 =0.9838 R 2 =0.9206 R 2 =0.9375 R 2 =0.9753 R 2 =0.9835 (b) Figure 4.11: Korsmeyer-Peppas release profile for all samples in (a) pH ≈ 6.2 and (b) pH ≈ 7.4 University of Ghana http://ugspace.ug.edu.gh 53 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 HAP ONLY 0.5 CNC/HAP 1.0 CNC/HAP 1.5 CNC/HAP 2.0 CNC/HAP % D ru g R e le a s e Time (hrs) (a) 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 40 HAP ONLY 0.5 CNC/HAP 1.0 CNC/HAP 1.5 CNC/HAP 2.0 CNC/HAP % D ru g R e le a s e Time (hrs) (b) Figure 4.12: Cubic degree polynomial release profile for all samples in (a) pH ≈ 6.2 and (b) pH ≈ 7.4 University of Ghana http://ugspace.ug.edu.gh 54 Table 4.5: R-squared values from fitted models. Kinetic Model Equation Parameter HAP Only 0.5 mg CNC/HAP 1.0 mg CNC/HAP 1.5 mg CNC/HAP 2.0 mg CNC/HAP 6.2 7.4 6.2 7.4 6.2 7.4 6.2 7.4 6.2 7.4 Higuchi Log Q = log k + 1/2 log t R2 0.8138 0.7817 0.7571 0.8484 0.7305 0.7817 0.7453 0.9718 0.6764 0.9831 k 16.1842 12.9652 14.8901 8.6990 14.6178 8.6615 16.2654 8.9471 15.9976 8.1214 Korsmeyer -Peppas Log Q = Log k + n Log t R2 0.9898 0.9838 0.9485 0.9206 0.9828 0.9375 0.9786 0.9753 0.9729 0.9835 k 22.4101 18.0574 20.7692 10.9891 21.0280 11.7755 23.1658 9.6433 23.6014 8.4751 n 0.2507 0.2462 0.2453 0.3226 0.2208 0.2656 0.2287 0.4436 0.2009 0.4380 University of Ghana http://ugspace.ug.edu.gh 55 4.7 Cell viability According to figure 4.13, the MTT assay results showed percentages greater than 90%, indicating that all composites and commercial HAP had a positive interaction with MDA-MB-231 cells in terms of cell proliferation, demonstrating their non-toxic nature. Curcumin was then uploaded on the CNC/HAP composites with HAP as control and released for 9 hours to see the effect of the loaded composites on the breast cancer cells (MDA-MB-231 cells). CNC/HAP composite (2.0 mg) was chosen for the experiment because it is comparable to HAP only, both of which had 667.5% and 666.1% percentage increase in cell growth respectively. 0 100 200 300 400 500 600 700 2.0 mg CNC/HAP 1.5 mg CNC/HAP 1.0 mg CNC/HAP C el l V ia b il it y ( % ) Samples HAP 0.5 mg CNC/HAP Figure 4.13: Cell viability of all composites and control using MTT assay. University of Ghana http://ugspace.ug.edu.gh 56 From figure 4.14, it can be observed that from the first to the ninth hours, the cell viability of the 2.0 mg CNC/HAP composites is lower than it was at time zero. This indicates that the drug released from the composite during the time interval (9 hours) inhibited the growth of MDA-MB-231 cells. Cell viability percentage decreased from 77.51% at the beginning of the experiment (at time zero) to 41.84% after one hour (at 1 hour), it then increased steadily to 59% at the ninth hour. This indicates that curcumin was released in a controlled manner from the 2.0 mg CNC/HAP composite which was used as a drug carrier throughout the experiment. From figure 4.15, the percentage cell viability at the second hour (21.43%) indicates a burst release of the curcumin from HAP only. This phenomenon compromises the effectiveness of treatment as drug is lost in an uncontrolled manner and there may lead to disease relapse as a result. From figure 10, the percentage cell viability showed a fluctuating form. This confirms the nature in which HAP behaves as a drug carrier; sorpti