Applied Science, Faculty of
Engineering, School of (Okanagan)
DSpace
UBCO
Earl, Emily Elizabeth
Master of Applied Science - MASc
University of British Columbia
Over the past 30 years, treatments for heartburn have primarily remained unchanged. Current medication focuses on increasing the pH of gastric reflux by altering the overall pH of gastric fluid. This is accomplished by using neutralizing agents or by impairing the body’s ability to produce gastric acid. While these methods are effective at providing symptom management, they can cause undesired side effects and do not specifically target the gastric acid pocket: a highly acidic layer of gastric acid that remains stagnant at the top of the stomach. This thesis explores the development of an alternative technique to treat heartburn that focuses on relocating the contents of the gastric acid pocket rather than neutralizing it. In order to effectively perform this relocation, high-density chitosan-based superporous hydrogels were developed to displace the fluid in the gastric acid pocket by quickly absorbing the contents and sinking to the bottom of the stomach, away from the esophagus. The efficacy of the hydrogels was determined based on four main factors: swelling ability, equilibrium swelling time, swollen density, and biocompatibility. Two main methods of hydrogel formation were compared: the gas blowing method and the freeze-drying method. Within these two methods, a variety of hydrogel formulations were prepared in which the concentrations of polymers, crosslinking agents, and high-density materials were varied to optimize the hydrogel for maximum swelling ability. Biocompatible ceramics such as titanium dioxide and zirconium dioxide were used as the high- density materials in the hydrogel, and their biocompatibility was confirmed through cell cytotoxicity studies. An optimal hydrogel formulation was developed using the freeze-drying procedure paired with density adaptation from zirconium dioxide. This formulation is capable of swelling more than 25 times its initial mass in simulated gastric fluid while maintaining a swollen density higher than that of gastric content. With this swelling ability, an estimated amount of 4 grams of hydrogel material would be required to relocate the entirety of the gastric acid pocket, outlining the potential of this technique for further study regarding heartburn management.
HIGH-DENSITY BIOCOMPATIBLE HYDROGELS FOR IN-VIVO GASTRIC ACID RELOCATION by Emily Elizabeth Earl B.A.Sc., The University of British Columbia (Okanagan), 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Mechanical Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) December 2021 © Emily Elizabeth Earl, 2021 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: HIGH-DENSITY BIOCOMPATIBLE HYDROGELS FOR IN-VIVO GASTRIC ACID RELOCATION submitted by Emily Elizabeth Earl in partial fulfillment of the requirements of the degree of Master of Applied Science. Dr. Mina Hoorfar, School of Engineering Supervisor Dr. Mohammad Arjmand, School of Engineering Supervisory Committee Member Dr. Julian Cheng, School of Engineering Supervisory Committee Member Dr. Kasun Hewage, School of Engineering University Examiner iii Abstract Over the past 30 years, treatments for heartburn have primarily remained unchanged. Current medication focuses on increasing the pH of gastric reflux by altering the overall pH of gastric fluid. This is accomplished by using neutralizing agents or by impairing the body’s ability to produce gastric acid. While these methods are effective at providing symptom management, they can cause undesired side effects and do not specifically target the gastric acid pocket: a highly acidic layer of gastric acid that remains stagnant at the top of the stomach. This thesis explores the development of an alternative technique to treat heartburn that focuses on relocating the contents of the gastric acid pocket rather than neutralizing it. In order to effectively perform this relocation, high-density chitosan-based superporous hydrogels were developed to displace the fluid in the gastric acid pocket by quickly absorbing the contents and sinking to the bottom of the stomach, away from the esophagus. The efficacy of the hydrogels was determined based on four main factors: swelling ability, equilibrium swelling time, swollen density, and biocompatibility. Two main methods of hydrogel formation were compared: the gas blowing method and the freeze-drying method. Within these two methods, a variety of hydrogel formulations were prepared in which the concentrations of polymers, crosslinking agents, and high-density materials were varied to optimize the hydrogel for maximum swelling ability. Biocompatible ceramics such as titanium dioxide and zirconium dioxide were used as the high-density materials in the hydrogel, and their biocompatibility was confirmed through cell cytotoxicity studies. An optimal hydrogel formulation was developed using the freeze-drying procedure paired with density adaptation from zirconium dioxide. This formulation is capable of swelling more than 25 times its initial mass in simulated gastric fluid while maintaining a swollen density higher than that of gastric content. With this swelling ability, an estimated amount of 4 grams of hydrogel material would be required to relocate the entirety of the gastric acid pocket, outlining the potential of this technique for further study regarding heartburn management. iv Lay Summary The treatments for heartburn have remained the same for many years and are primarily focused on reducing the acidity of the stomach contents, either through neutralizing agents (such as antacids), or drugs that inhibit the stomach’s ability to produce acid. These methods are effective at providing symptom management; however, increasing the pH of the gastric fluid can introduce a variety of negative effects that are still being studied. The work within this thesis focuses on the introduction of an alternative method to treat heartburn by better distributing the gastric acid within the stomach. To do this, a highly swellable material was developed to absorb the top, most acidic layer of gastric fluid and sink down to the bottom of the stomach. The material can swell with more than 25 times its own weight in gastric fluid and has passed initial biocompatibility screenings, outlining its potential for further study. v Table of Contents Abstract ...................................................................................................................................... iii Lay Summary ............................................................................................................................. iv Table of Contents ....................................................................................................................... v List of Tables .............................................................................................................................. ix List of Figures ............................................................................................................................. x List of Abbreviations ............................................................................................................... xiii Acknowledgements ................................................................................................................. xiv Dedication ................................................................................................................................. xv Chapter 1: Introduction .............................................................................................................. 1 1.1 Gastroesophageal Reflux Disease .............................................................................. 1 1.2 Gastric Acid Pocket ..................................................................................................... 3 1.3 Motivation .................................................................................................................... 4 1.4 Objectives ................................................................................................................... 5 1.5 Thesis Organization .................................................................................................... 6 Chapter 2: Background .............................................................................................................. 8 2.1 Gastric Acid Production and Inhibition ........................................................................ 8 2.1.1 Gastric Acid Production ..................................................................................... 8 2.1.2 Gastric Acid Neutralization ................................................................................ 9 2.1.3 Gastric Acid Suppression ................................................................................ 10 2.1.4 Lower Esophageal Sphincter Support and Protection ..................................... 11 2.2 Importance of Gastric Acid ........................................................................................ 13 2.2.1 Increased Risk of Fracture .............................................................................. 14 2.2.2 Hypomagnesemia ........................................................................................... 16 2.2.3 Clostridium Difficile .......................................................................................... 17 2.2.4 Vitamin B12 Deficiency ..................................................................................... 18 vi 2.3 Hydrogels and their Use in Gastrointestinal Drug Delivery ....................................... 19 2.3.1 What are Hydrogels? ....................................................................................... 19 2.3.2 Hydrogels in the Gastrointestinal Tract ........................................................... 20 2.3.3 Types of Drug Delivery Systems ..................................................................... 22 2.3.3.1 Expandable Systems ................................................................................... 23 2.3.3.2 Bioadhesive Systems .................................................................................. 24 2.3.3.3 Floating Systems ......................................................................................... 24 2.3.3.4 High-Density Systems ................................................................................. 25 2.3.3.5 Superporous Hydrogel Systems .................................................................. 26 2.4 Hydrogels for Use in Gastric Acid Relocation ........................................................... 27 2.4.1 Polymer Selection ........................................................................................... 27 2.4.2 High-Density Material Selection ...................................................................... 29 2.5 Chapter 2 Summary .................................................................................................. 31 Chapter 3: Methodology ........................................................................................................... 32 3.1 Gas Blowing Method ................................................................................................. 32 3.1.1 Materials .......................................................................................................... 32 3.1.2 Hydrogel Synthesis ......................................................................................... 32 3.2 Freeze-Drying Method .............................................................................................. 35 3.2.1 Materials .......................................................................................................... 35 3.2.2 First Generation ............................................................................................... 35 3.2.3 Second Generation ......................................................................................... 37 3.3 Characterization Techniques .................................................................................... 37 3.3.1 Swelling Studies .............................................................................................. 38 3.3.1.1 Initial Feasibility Assessment ...................................................................... 38 3.3.1.2 Additional Swelling Analysis ........................................................................ 39 3.3.2 Porosity Measurement and Calculation of Density .......................................... 39 vii 3.3.3 Scanning Electron Microscopy ........................................................................ 42 3.3.4 Fourier Transform Infrared Spectroscopy ....................................................... 43 3.3.5 X-Ray Diffraction Analysis ............................................................................... 44 3.3.6 Cell Toxicity Study ........................................................................................... 45 3.4 Chapter 3 Summary .................................................................................................. 46 Chapter 4: Results and Discussion ........................................................................................ 48 4.1 Gas Blowing Method ................................................................................................. 48 4.1.1 Hydrogel Synthesis ......................................................................................... 48 4.1.2 Swelling Studies .............................................................................................. 49 4.1.3 Porosity Measurement .................................................................................... 51 4.2 Freeze-Drying Method .............................................................................................. 52 4.2.1 First Generation ............................................................................................... 52 4.2.1.1 Hydrogel Synthesis ..................................................................................... 52 4.2.1.2 Swelling Studies .......................................................................................... 54 4.2.1.3 Porosity Measurement and Calculation of Density ...................................... 56 4.2.1.4 Scanning Electron Microscopy .................................................................... 58 4.2.1.5 Fourier Transform Infrared Spectroscopy ................................................... 60 4.2.1.6 Cell Cytotoxicity Study ................................................................................. 61 4.2.2 Second Generation ......................................................................................... 63 4.2.2.1 Hydrogel Synthesis ..................................................................................... 63 4.2.2.2 Swelling Studies .......................................................................................... 64 4.2.2.3 Porosity Measurement and Calculation of Density ...................................... 70 4.2.2.4 Scanning Electron Microscopy .................................................................... 72 4.2.2.5 Fourier Transform Infrared Spectroscopy ................................................... 73 4.2.2.6 X-Ray Diffraction ......................................................................................... 74 4.2.2.7 Cell Cytotoxicity Study ................................................................................. 75 viii 4.3 Chapter 4 Summary .................................................................................................. 76 Chapter 5: Conclusions and Future Work .............................................................................. 78 5.1 Summary ................................................................................................................... 78 5.2 Contributions ............................................................................................................. 79 5.3 Future Works ............................................................................................................. 79 Bibliography .............................................................................................................................. 81 ix List of Tables Table 3.1 Gas-blown hydrogel formulations: comparison of polymer percentage, crosslinker percentage, and percentage of titanium dioxide ......................................................................... 34 Table 3.2 Gas-blown hydrogel formulations: comparison of crosslinker percentage and effects of foam stabilizer ............................................................................................................................. 35 Table 3.3 Freeze-dried hydrogel formulations: chitosan percentage versus amount of zirconia . .................................................................................................................................................... 37 Table 4.1 Hydrogel porosity and density comparison of first-generation freeze-dried hydrogels . .................................................................................................................................................... 58 Table 4.2 Swelling ratios of second-generation freeze-dried hydrogel samples in pH 1.57 hydrochloric acid: glutaraldehyde study ...................................................................................... 65 Table 4.3 Swelling ratios of second-generation freeze-dried hydrogel samples in pH 1.57 hydrochloric acid: zirconia study ................................................................................................. 67 Table 4.4 Hydrogel porosity and density comparison of second-generation freeze-dried hydrogels .................................................................................................................................... 71 x List of Figures Figure 1.1 Hypothesized mechanism of gastric acid relocation .................................................... 6 Figure 1.2 Thesis layout ............................................................................................................... 7 Figure 2.1 Fully functional parietal cell, parietal cell under H2-receptor blockers, and parietal cell under proton-pump inhibitors from left to right, respectively, where “PP” refers to “proton pump” .................................................................................................................................................... 11 Figure 2.2 Uncrosslinked polymer, physically crosslinked polymer, and chemically crosslinked polymer from left to right, respectively ........................................................................................ 20 Figure 2.3 pH-sensitive hydrogels .............................................................................................. 22 Figure 2.4 Segment of chitosan structure, a) uncrosslinked, b) crosslinked with glutaraldehyde .................................................................................................................................................... 29 Figure 3.1 Hydrogel formation using gas blowing method .......................................................... 33 Figure 3.2 Hydrogel formation using freeze-drying method ........................................................ 36 Figure 3.3 Micro-CT diagram ...................................................................................................... 40 Figure 3.4 SEM diagram, where BSE refers to the detector for backscattered electrons, SE refers to the detector for secondary electrons, and EDX refers to the detector for characteristic x-rays. Reproduced with permission [163]. ................................................................................. 43 Figure 3.5 X-ray diffractometer diagram ..................................................................................... 44 Figure 4.1 Gas-blown hydrogels, a) after crosslinking, b) after ethanol bath, c) after oven-drying .................................................................................................................................................... 48 Figure 4.2 Swelling ratios of gas-blown hydrogels (samples 1–6, Table 3.1) in pH 1.57 hydrochloric acid ......................................................................................................................... 50 Figure 4.3 Swelling ratios of gas-blown hydrogels (samples 7–10, Table 3.2) in pH 1.57 hydrochloric acid ......................................................................................................................... 51 Figure 4.4 Image analysis of a micro-CT scan of a gas-blown hydrogel, a) during mask selection, b) image converted to black and white, c) resulting image after applying threshold .. 52 xi Figure 4.5 Freeze-dried first-generation hydrogels (1% chitosan, 15% zirconia), a) without zirconia, b) with zirconia, c) without zirconia in hydrochloric acid, d) with zirconia in hydrochloric acid ............................................................................................................................................. 54 Figure 4.6 Swelling ratios of first-generation freeze-dried hydrogels (samples 1–6, Table 3.3) in pH 1.57 hydrochloric acid (± standard deviation). Solid bars indicate that the hydrogels possessed a density greater than the density of gastric fluid. .................................................... 55 Figure 4.7 Image analysis of a micro-CT scan of a first-generation freeze-dried hydrogel, a) unaltered image from micro-CT scan, b) image converted to black and white, c) image after threshold was applied ................................................................................................................. 57 Figure 4.8 SEM images of first-generation freeze-dried hydrogels without zirconia, a) 200x magnification, b) 1000x magnification, and with zirconia, c) 200x magnification, d) 1000x magnification ............................................................................................................................... 59 Figure 4.9 FTIR spectra of first-generation freeze-dried hydrogels with zirconia (15% w/v) and without zirconia ........................................................................................................................... 61 Figure 4.10 Cytotoxicity results, a) first-generation freeze-dried hydrogel sample without zirconia, b) first-generation freeze-dried hydrogel sample with zirconia ..................................... 62 Figure 4.11 First-generation and second-generation hydrogel comparison, a) first-generation hydrogel without zirconia, b) second-generation hydrogel without zirconia, c) first-generation hydrogel with zirconia, d) second-generation hydrogel with zirconia .......................................... 64 Figure 4.12 Expectant swelling ratios of second-generation freeze-dried hydrogels (samples 1–3, Table 4.2) in pH 1.57 hydrochloric acid (± standard deviation) .............................................. 66 Figure 4.13 Effect of solution pH on swelling ability of optimized hydrogel ................................ 68 Figure 4.14 Compressed versus uncompressed hydrogels, a) dimensions, b) swelling abilities in pH 1.57 hydrochloric acid (± standard deviation) ....................................................................... 70 xii Figure 4.15 Image analysis of a micro-CT scan of a second-generation freeze-dried hydrogel, a) unaltered image from micro-CT scan, b) image converted to black and white, c) image after threshold was applied ................................................................................................................. 72 Figure 4.16 SEM images of second-generation freeze-dried hydrogels without zirconia, a) 1000x magnification, c) 5000x magnification), and with zirconia, b) 1000x magnification, d) 5000x magnification ............................................................................................................................... 73 Figure 4.17 FTIR spectra of pure zirconia and second-generation freeze-dried hydrogels with zirconia (4.5% w/v) and without zirconia ..................................................................................... 74 Figure 4.18 XRD data of pure zirconia and second-generation freeze-dried hydrogels with zirconia (4.5% w/v) and without zirconia ..................................................................................... 75 Figure 4.19 Cytotoxicity results, a) live/dead staining of 0% and 4.5% zirconia hydrogels at day 1 and day 5, b) cell viability data (± standard deviation) ............................................................. 76 xiii List of Abbreviations GERD Gastroesophageal reflux disease PPI Proton-pump inhibitor LES Lower esophageal sphincter CDI Clostridium difficile infection RO Reverse osmosis PVA Poly(vinyl alcohol) Micro-CT Micro-computed tomography SEM Scanning electron microscope FTIR Fourier transform infrared spectroscopy XRD X-ray diffraction Ch Chitosan Zr Zirconia GA Glutaraldehyde xiv Acknowledgements I would like to acknowledge the Natural Sciences and Engineering Research Council of Canada for supporting this research, as well as the artists from Flaticon who developed the icons that were used to generate some of the figures within this work. I would also like to express my deepest gratitude to my supervisor Dr. Mina Hoorfar, for her encouragement and guidance have been extremely helpful throughout this project. Additionally, I would like to offer many thanks to my lab mates in the Advanced Thermo Fluidic Laboratory who have offered me support throughout this process, whether that be in terms of friendship, research advice, or otherwise. xv Dedication Thank you to my many friends and family, without whom, this would not be possible. 1 Chapter 1: Introduction Heartburn is a symptom that is caused by gastric fluid entering the esophagus and damaging the esophageal lining. When heartburn is an occasional occurrence, it is often treated with over-the-counter antacids to neutralize the acidity of the stomach contents, raising the pH of the refluxate closer to the native pH of the lower esophagus. If heartburn occurs more frequently, such as for those who have gastroesophageal reflux disease (GERD), the long-term use of pH-balancing antacids is not recommended due to unfavourable side effects. Instead, an alternative drug that inhibits the body’s ability to produce hydrochloric acid is prescribed. While these drugs are designed to alleviate the pain caused by acid reflux, they cause the natural pH of the stomach to be altered for time periods ranging from a few months to multiple decades. Although it was originally believed that one of the causes of frequent heartburn was the result of an individual’s heightened ability to produce gastric acid, it has since been noted that in the majority of individuals, this is not the case [1]. Often, it is the result of a dysfunction of the lower esophageal sphincter, causing it to relax at abnormal intervals. For individuals who experience esophageal malfunction and do not over-produce hydrochloric acid, the exact implications of the gastric acid reduction caused by acid inhibitors are unknown. 1.1 Gastroesophageal Reflux Disease In order for food to be absorbed into the body, it must be transferred from the mouth to the stomach, and finally to the intestines, where nutrients are separated from waste. In healthy individuals, the lower esophageal sphincter usually only relaxes to allow for the food that is traveling in the esophagus to pass into the stomach. Occasionally, healthy people may experience heartburn due to elevated pressure within the stomach, often a result of eating large meals [2]. The increased pressure forces the lower esophageal sphincter to open, causing heartburn. Although the exact reasoning is unknown, individuals with GERD experience transient lower esophageal sphincter relaxations where the lower esophageal sphincter relaxes 2 without being induced by the individual swallowing [3]. These unnecessary relaxations allow for gastric fluid to flow freely from the stomach into the esophagus, causing frequent and painful heartburn. In addition to heartburn, GERD can also cause a variety of other symptoms inclusive of regurgitation, difficulty swallowing, and even chronic coughing. GERD is a common condition, affecting approximately 20% of Canadians [4]; however, the diagnosis of GERD is frequently based solely on symptom assessment performed by clinicians [5]. The main symptoms required to be diagnosed with GERD include having minor heartburn at least twice a week or major heartburn at least once a week. As long as an individual is not presenting with warning signs of potential complications (such as weight loss, anemia, or dysphagia), additional testing may not be performed, and the individual will commonly be placed on acid-suppressive therapy [6]. While monitoring the patient’s response to gastric acid suppression is an inexpensive and non- invasive method to assess the likelihood of GERD, it has been estimated that this form of testing only has a specificity of 57% and a sensitivity of 80% [7]. This presents an issue because although heartburn is one of the main symptoms of GERD, it is also a symptom of a variety of other conditions, such as achlorhydria or gastroparesis, and can often be mistaken for angina or stomach ulcers [8]. Some of these other conditions may respond positively to gastric acid suppression and lead an individual and their doctor to believe that they have GERD. This individual may then be placed on long-term gastric acid inhibiting drugs because it aids in symptom relief; however, the long-term effects of these drugs are still being analyzed and relying solely on the acid inhibiting response test may lead to a larger percentage of people taking gastric acid inhibitors inappropriately. Even for those who genuinely have GERD, sustaining a gastric acid suppressed system for long periods of time (greater than one year) has potential side effects that have not yet been fully discovered or understood [9]. As treatments that specifically target the lower esophageal sphincter can be less favourable and arguably less 3 effective than gastric acid suppressants, there has not been significant improvement in the treatments for GERD [10]. Altering the pH of the gastric fluid is one of the simplest ways to eradicate heartburn, as the low pH of the fluid is what causes gastric reflux into the esophagus to be painful [11]. However, the entirety of the stomach contents does not need to have an increased pH, as only the topmost portion of the gastric fluid refluxes into the esophagus. The layer of fluid that rests at the top of the gastric contents is referred to as the gastric acid pocket and is thought to be the source of heartburn during gastric reflux [12]. 1.2 Gastric Acid Pocket After food passes into the stomach, it buffers the gastric fluid as a by-product of gastric digestion. This raises the overall pH of the gastric fluid to a value between 4 and 6. In 2001, Fletcher and colleagues performed a study to compare the different pH values of various areas within the stomach and the esophagus by slowly removing a pH catheter that was placed in the stomach of a participant. They discovered that while the pH of most of the stomach contents was between a value of 4 and 5, there was a segment of gastric fluid that had a much lower pH (approximately 2) immediately below the lower esophageal sphincter [13]. This layer of gastric fluid, dubbed the gastric acid pocket, is a highly acidic layer of gastric fluid that develops in both individuals with and without GERD. The development of the gastric acid pocket is thought to be a combination of the body’s increase in acid production in response to the ingestion of food and the lack of mixing of the gastric fluid [12], [14]. Although the gastric fluid that is present in the stomach prior to the ingestion of food becomes buffered once the food reaches the stomach, the parietal cells within the stomach become stimulated and produce acid to help with the digestive processes. The 4 parietal cells are located in the middle and upper regions of the stomach, known as the body and the fundus, respectively. While peristaltic action occurs in the stomach to mix and churn the gastric contents, there is limited peristalsis that occurs in the fundus, causing the upper portion of the gastric fluid to remain stagnant [15]. This creates the gastric acid pocket, as the newly secreted acid from the upper portion of the fundus does not get properly mixed. The gastric acid pocket appears roughly 15 minutes after eating, and it has a thickness of approximately 2 cm in healthy individuals and 5 cm in individuals with GERD [16]. This may lead one to believe that people with GERD possess a greater volume of fluid in the gastric acid pocket; however, studies have shown that the gastric secretion volume is not significantly different when comparing healthy individuals to those with GERD [17]. The reasoning for this elongation of the gastric acid pocket may be due to the occurrence of hiatal hernias, which are commonly seen in GERD patients [18]. A hiatal hernia is where a portion of the stomach is dislocated and resides above the diaphragm, often a result of trauma or intense pressure on the muscles surrounding the stomach. This causes the upper portion of the stomach to be much narrower than normal and could contribute to the lengthened gastric acid pocket observed in individuals with GERD. 1.3 Motivation Gastric acid suppressants are able to reduce the acidity and the size of the gastric acid pocket by limiting the amount of hydrochloric acid that is released by the stomach lining. This limitation can cause the pH of the gastric acid pocket to be raised to a value of 4 [19]; however, it results in the bulk of the gastric fluid being raised to a pH of between 6 and 7 on average [20]. The low pH environment of the stomach is essential for certain processes such as efficient nutrient absorption, protein breakdown, and the eradication of harmful bacteria. Although sustaining a high pH environment in the stomach is useful in controlling pain resulting from gastric reflux, 5 over time it may produce harmful effects due to the impairment of these functions. It is interesting to note that the use of gastric acid suppressants results in raising the pH of the gastric acid pocket to a value that is similar to the regular pH of the bulk of gastric fluid after it has been buffered by consumed meals. This suggests that if the stomach contents were properly mixed during the digestion process, the acidity of the topmost portion of gastric fluid would maintain a pH between 4 and 6. If this were to be achieved effectively, it would theoretically provide the same relief as gastric acid inhibitors without altering the natural pH of the gastric fluid. In addition, better incorporating the secreted gastric acid with the food to be digested within the stomach may increase the efficiency of digestion. This would allow for partially digested materials to be removed from the stomach at a faster pace, providing a shorter time period where extended pressure is placed on the lower esophageal sphincter and potentially reducing the length of reflux episodes. 1.4 Objectives The main objective of this thesis is to develop a technique in which the contents of the gastric acid pocket can be relocated to the bottom of the stomach to allow for better distribution of gastric acid, subsequently resulting in a higher pH of the gastric fluid nearest to the esophagus and reducing the likelihood of heartburn (Figure 1.1). Due to the frequent and successful use of hydrogels in gastrointestinal drug delivery systems, hydrogels were selected to achieve this effect [21]. Based on this, the sub-objectives of this thesis are as follows: • to develop a highly swellable hydrogel with a swollen density greater than that of gastric fluid (1.00 g/ml), • to maximize the swelling ability of the hydrogel while ensuring that equilibrium swelling is achieved in less than 30 minutes, • and to assess the cytocompatibility of the hydrogel to determine its potential for in-vivo biocompatibility. 6 Figure 1.1 Hypothesized mechanism of gastric acid relocation 1.5 Thesis Organization This thesis is focused on the development of hydrogels for the effective relocation of gastric acid within the stomach. The overall structure of this thesis is represented by the diagram shown in Figure 1.2. In the following chapter, the fundamentals of gastric acid production, inhibition, and the importance of gastric acid are discussed. Additionally, an overview of hydrogels is presented and their use in the stomach is studied, primarily focused on gastroretentive drug delivery systems and how they can be adapted for this work. Chapter 3 presents the methodology for hydrogel development and testing, including various hydrogel synthesis procedures, experimental processes, and characterization techniques. In Chapter 4, the results of the different hydrogel analysis methods are discussed, various hydrogel formulations are compared, 7 and an optimized hydrogel is presented. Chapter 5 concludes the thesis with a summary of the achievements of the presented work, overall research findings, and potential concepts for further study. Figure 1.2 Thesis layout 8 Chapter 2: Background Primarily recognized by its main symptom - heartburn, gastroesophageal reflux disease has been treated with the same techniques for over 30 years [22]. The most common course of treatment is the inhibition of gastric acid production with the use of proton pump inhibitors or H2-receptor blockers. In addition to these treatments, a surgery known as fundoplication can be performed; however, it loses its effectiveness over time and occasionally needs to be redone [23]. If symptoms are persistent, traditional antacids are often taken with both treatments to further aid in symptom management. While all techniques aim to prevent the occurrence of heartburn, they achieve this through entirely different mechanisms, which come with a variety of advantages and disadvantages. 2.1 Gastric Acid Production and Inhibition 2.1.1 Gastric Acid Production Gastric acid is produced by parietal cells that line the inside of the upper portion of the stomach. The parietal cells have receptors for three hormones that stimulate the secretion of gastric acid: acetylcholine, gastrin, and histamine [24]. The acid production process begins with the release of acetylcholine from vagus nerve endings in response to an individual seeing, smelling, or chewing food. A portion of the acetylcholine binds to the parietal cells, acting as the initial stimulus for acid production; however, it also acts to stimulate the release of gastrin, which then stimulates the release of histamine [25]. Acid production is increased further after food reaches the stomach due to a heightened level of gastrin production in response to protein breakdown [26]. Once the hormones have activated the parietal cells, a series of reactions will take place, resulting in the secretion of gastric acid. The various reactions are as follows: 9 1. Water and carbon dioxide within the cell are combined using the carbonic anhydrase enzyme to produce carbonic acid, which is immediately dissociated into bicarbonate ions and hydrogen ions. 2. The bicarbonate ions leave the cell into the interstitial fluid through a chloride shift, resulting in an increase of chloride ions within the cell. 3. The hydrogen ions are forced out of the cell into the stomach using an H+/K+ ATPase pump, whereas the chloride ions leave the cell into the stomach via diffusion. 4. The chloride ions and the hydrogen ions combine to form hydrochloric acid in the stomach. 2.1.2 Gastric Acid Neutralization The simplest method to quickly resolve heartburn is to increase the pH of the gastric fluid. Even though low pH fluids aren’t necessarily harmful to the esophagus, the digestive enzyme pepsin that is used to break down proteins is most active at lower pH values, having maximum activity at a pH of 2 [27]. When fluid is refluxed into the esophagus, if pepsin is still in an activated state, it can begin to break down the proteins in the esophagus, causing pain. If acidic reflux continues to occur regularly, it can cause permanent damage to the esophagus and lead to precancerous conditions, such as Barrett’s esophagus [28]. To increase the pH, there are two main salts that are used: aluminum hydroxide and calcium carbonate. These drugs are effective at providing short term symptom management, but they can cause a variety of side effects. Some of the side effects include nausea, vomiting, abdominal cramps and acid rebound [29]. Acid rebound is a phenomenon where the stomach produces an increased amount of acid in response to some external factor. In the case of acid-neutralizing antacids, this rebound can be caused by the stimulation of calcium receptors, which then results in an increase in the production of gastrin [30]. Even though the stomach may be mostly empty, the increase in 10 gastrin will still stimulate the parietal cells to produce acid, rendering the antacids ineffective and encouraging repeated dosing. While certain individuals might find other gastrointestinal symptoms tolerable compared to heartburn, to have to trade one type of digestive pain for another is a serious drawback. Although gastric acid neutralizers provide almost immediate relief, this relief can wear off quickly. This is a result of the duration of action of antacids being reliant on the gastric emptying time of the individual. In a fasted state, an antacid may only provide relief for 30 minutes, which can lead to those with recurrent reflux consuming much higher than the recommended dosage [31]. 2.1.3 Gastric Acid Suppression In order to combat the heavy use of antacids, gastric acid suppressants such as H2-receptor blockers and proton pump inhibitors can be used to decrease the overall amount of hydrochloric acid produced in the stomach. To try and reduce the amount of acid produced, H2-receptor blockers reversibly bind to the histamine receptor on the parietal cells of the stomach and prevent the released histamine from stimulating acid production [32]. This binding allows for H2-receptor blockers to have a duration of action of up to 10 hours, but they can take up to 30 minutes to begin the suppression of acid production [33]. Although H2-receptor blockers target the most effective acid production receptor, they still leave two of the acid stimulating pathways (via acetylcholine and gastrin receptors) open, preventing the parietal cells from completely stopping acid production. The reversibility of the H2-receptor blockers also allows the parietal cells to continue acid production after the H2-receptor blockers wear off. The impaired functionality of a parietal cell affected by H2-receptor blockers is shown in Figure 2.1. 11 Figure 2.1 Fully functional parietal cell, parietal cell under H2-receptor blockers, and parietal cell under proton-pump inhibitors from left to right, respectively, where “PP” refers to “proton pump” In contrast, proton-pump inhibitors (PPIs) permanently halt the acid production of the parietal cells by preventing the H+/K+ ATPase pumps on the cells from functioning. Although PPIs still allow for chloride ions to pass through the cell, little to no hydrogen ions make it through to the stomach, preventing adequate amounts of hydrochloric acid from being formed. Unlike H2-receptor blockers, PPIs are active until the proton pumps they are attached to are replaced or the parietal cell dies [34]. Previous studies have indicated that approximately 25% of proton pumps on parietal cells are replaced within a one-day period [35], suggesting that the complete termination of acid production is never fully achieved. While PPIs are reportedly more effective at acid suppression than H2-receptor blockers [36], they can take up to four days to start relieving symptoms. 2.1.4 Lower Esophageal Sphincter Support and Protection The most commonly available treatment that specifically targets the lower esophageal sphincter (LES) to help reduce GERD symptoms is the fundoplication procedure. This procedure aims to strengthen the LES to prevent it from opening at undesired intervals. It involves taking a portion 12 of the fundus and wrapping it around the LES to support it. A 20-year study showed that although 90% of the participants who received the procedure were satisfied with the results, 45% percent of them were still taking PPIs to aid with symptom management and 16% required repeated fundoplication procedures within that time frame [37]. In addition, 26% of participants experienced dysphagia (difficulty swallowing) following the procedure. In addition to the fundoplication procedure, a variety of prokinetic agents have been tested to attempt and increase the strength of the LES without surgery by stimulating the muscles of the gastrointestinal tract; however, the results have been quite mixed. Although prokinetic agents appear to reduce the number of reflux episodes that occur [38], they are not widely used due to their non-selectivity. This causes them to create a variety of side effects, including involuntary movement and muscle spasms [39]. Another family of techniques that are used to protect the LES from gastric acid are alginate-based therapies. In this type of treatment, a floating raft of gel is created at the top of the gastric fluid due to an effervescent reaction between the alginate-based medication and gastric acid [40]. This raft acts as a barrier between the stomach and the esophagus that physically prevents gastric fluid from entering the lower esophageal sphincter. A large meta-analysis was done on the incorporation of alginates in heartburn treatments and found that alginate-based therapies significantly reduce the degree of heartburn and can, on occasion, be comparable to PPIs in terms of effectiveness [41]. However, the issue with this study was that 14 out of the 15 experiments in the analysis used alginate/antacid combination therapies as opposed to using pure alginates. The one study that did assess the efficacy of pure alginates only found them to be 11% more effective than placebo (22% vs. 33%) [42]. This indicates that while alginates may help reduce heartburn and can improve the efficacy of standard antacid treatments, their effects alone may not be significant enough to provide long-lasting heartburn relief. 13 Aside from the fundoplication procedure and the use of prokinetic drugs, all of the treatments to help reduce GERD symptoms are revolved around the reduction or neutralization of gastric acid. One of the major issues with both the neutralization and suppression strategies is that they partially rely on the misconception that those who have GERD produce a heightened amount of stomach acid compared with those who do not have GERD, but this is not the case. As previously mentioned, heartburn is primarily a result of the LES malfunctioning and although the pH of the gastric fluid is the reason that gastric reflux causes pain, the overproduction of gastric acid (hyperchlorhydria) is only seen in rare circumstances such as those with Zollinger-Ellison syndrome which occurs in approximately one in every million people [43]. Thus, in the majority of circumstances, using the previously mentioned acid inhibiting drugs alters the natural pH of the gastrointestinal environment. 2.2 Importance of Gastric Acid The low pH of gastric fluid allows for the body to properly digest essential nutrients and vitamins and eradicates certain bacteria; it is also the main initiating factor for chemical digestion within the stomach. While the small intestine is the key region for chemical digestion, the stomach maintains an important role regarding the breakdown of proteins and triglycerides. The two main digestive enzymes that are released into the stomach are pepsin, which is for protein denaturation, and gastric lipase, which is responsible for initiating the digestion of dietary fats. Although both enzymes are equipped to withstand the harsh environment of the stomach, they are not suited for identical conditions. In gastric juice, gastric lipase has been reported to have optimum activity between pH values of 2 and 6 and can maintain stability until a pH of approximately 8 [44]. Pepsin can also maintain stability until a pH of 8, but it has been shown to be most active at a pH of 2 [45]. The activity percentage for gastric lipase is roughly constant between pH values of 2 and 6, but pepsin quickly loses its effectiveness as the pH rises, transitioning from 100% activity at a pH of 2, to 70% between a pH of 3 and 5, and drops to 14 below 10% at a pH 5.5 or higher. This information suggests that while the breakdown of dietary fats may only be mildly affected by the use of gastric acid inhibitors, the digestion of proteins will be significantly delayed in areas of high acidity such as the acid pocket and may approach negligible function in the bulk of gastric fluid on gastric acid suppressants. When an individual under-produces gastric acid, this condition is known as hypochlorhydria and is much more common than hyperchlorhydria [46]. The use of PPIs and H2-receptor blockers induces hypochlorhydria in people with a healthy production of stomach acid and can cause an array of uncomfortable symptoms; however, the major concern stems from remaining in a state of hypochlorhydria for extended periods of time. Even though the recommended amount of time PPIs should be prescribed is for 4-8 weeks followed by a reassessment, the majority of people that are using PPIs continue to take them for much longer than the recommended treatment time due to fear of symptom recurrence [47]. As the long-term use of PPIs continues, the concern for long-term side effects follows. These concerns are focused on potential side effects such as an increased risk of fractures, hypomagnesemia, clostridium difficile infection, and vitamin B12 deficiency [48]. 2.2.1 Increased Risk of Fracture Although the cause of why long-term PPI use may increase the risk of bone fracture has not been officially determined, the reasoning that many researchers are attributing it to is the impact that gastric pH has on the body’s ability to properly absorb calcium and therefore support healthy bone structure. Various studies have been done to compare the ability to absorb calcium between people with hypochlorhydria and people with standard gastric acid production. Two different studies found that people with hypochlorhydria lose their ability to adequately absorb calcium carbonate [49], [50], similar to those who go through a gastric bypass procedure 15 [51]. As many gastric bypass patients get placed on PPI therapy after surgery to treat acid reflux symptoms [52], this calcium absorption deficiency may be a result of the PPI-induced hypochlorhydria as opposed to the gastric bypass surgery itself. In 2018 a meta-analysis was performed to attempt and correlate the increased fracture risk due to long-term PPI use with bone mineral density. The results of the analysis showed that although the risk of bone fracture may indeed increase as the length of PPI usage increases, that this increased risk could not be attributed to changes in bone mineral density as there were no significant differences in bone mineral density from those who were using PPIs and those who were not [53]. Another study also determined similar findings; however, it suggested that PPI use may have an effect on bone metabolism that primarily targets trabecular bone due to the specific fracture sites that commonly occur with PPI use [54]. Conversely, other studies have ignored the concept of calcium absorption entirely and have instead focused on PPIs increasing the likelihood of falling, therefore increasing the potential of bone fracture. A study that looked at long-term PPI use found that PPI users were much more likely to have decreased vitamin B12 compared to non-users (discussed further in Section 2.2.4) [55]. These researchers suggested that the decrease in vitamin B12 instilled mobility and balance problems which caused PPI users to fall more frequently than non-users, resulting in more fractures. Although PPIs have been noted to increase the risk of bone fracture by up to 1.27 times, other studies have determined that this risk is non-existent [56]. Almost half of all PPI users are above 65 years of age [57], resulting in an elevated risk of morbidity and mortality following a fracture [58]. Due to this, determining the connection between PPI use and fracture risk is incredibly important and should be strongly considered when justifying long-term PPI use in the elderly population. 16 2.2.2 Hypomagnesemia Magnesium is an essential mineral in the body; it helps in regulating proper nerve function, maintaining adequate blood sugar levels, and is involved in making proteins, amongst other entities. Hypomagnesemia is a state where the body has a decreased level of serum magnesium in the blood, which can result in twitching, tremors, exhaustion, and eventually muscle contractions and seizures. Although hypomagnesemia has only been reported in approximately 1% of PPI users [59], due to the potential severity of the symptoms that it causes and the lack of understanding regarding why it occurs makes it one of the more dangerous side effects. While the development of hypomagnesemia has been agreed upon by researchers [59], [60], [61], [62], the specific reasoning as to how PPIs cause hypomagnesemia is still unknown. A study was performed that compared blood magnesium levels of people who were taking PPIs against those who were taking H2-receptor blockers and against those who were not taking any form of anti-secretory medication. The researchers found that the blood magnesium levels of those who were taking the H2-receptor blockers were not significantly different from those who were not on any form of anti-secretory medication; however, the blood magnesium levels of those who were taking PPIs was reportedly lower than the other two groups [63]. They also found an inverse correlation between PPI dose and blood magnesium levels. This suggests that hypomagnesemia may not merely be a result of decreased stomach acid production because the H2-receptor blockers did not decrease the blood magnesium levels. As there have not been studies that compare blood magnesium levels of individuals who have pre-existing hypochlorhydria with blood magnesium levels of those with PPI-induced hypochlorhydria, it cannot be concluded whether hypomagnesemia is a function of increased gastric pH or is specifically related to PPI function. 17 2.2.3 Clostridium Difficile Clostridium difficile (C. difficile) is a type of bacteria that enters the body through the mouth and can be contracted through contact with contaminated surfaces or individuals. Once inside the body, C. difficile can proliferate in the large intestine and can cause swelling and irritation of the organ if allowed to grow in excess, leading to diarrhea, fever, and dehydration. If accidentally ingested, the C. difficile bacteria are often prevented from substantially multiplying before they reach the large intestine because of the highly acidic environment of the stomach. Although C. difficile is not eradicated in low pH environments, pH values below 5.5 halt the growth of the bacteria [64]. In a healthy individual, this suggests that the bacteria would remain in an environment with a pH of approximately 3.5 while in the stomach, then shift to a pH of between 6 and 7.4 in the small intestine, and drop back to a pH of about 5.7 in the large intestine [65]. This would provide a time span of approximately 3-5 hours to allow for bacterial growth in the small intestine. For individuals who are on PPIs, with the gastric pH being raised to above 6 and gastric emptying time being delayed by 15-40% [66], this could provide an additional time span of up to 6 hours where the bacteria is freely able to grow, possibly leading to C. difficile infection (CDI) [67]. It has been determined that the use of PPIs can increase one’s chance of contracting CDI by up to two times [68]. The degree of risk is associated with the PPI dosage, as a study reported that individuals that were taking higher PPI doses were more likely to contract the infection [69]. This poses a particular issue regarding the frequent over-dosing of PPIs. In 2013 a study was performed that compared the efficacy of standard dose PPIs to double dose PPIs and found that the standard dose is just as effective as the double dose of PPI in individuals with uncomplicated GERD [70]. In addition to this, they also found that more than 90% of prescriptions from 2012-2013 for esomeprazole, lansoprazole, dexlansoprazole and pantoprazole (four of the most common PPIs) were for higher dosages, suggesting that many 18 people may be unnecessarily putting themselves at risk for CDI. The treatment for CDI is a round of antibiotics; however, even after this treatment, it is estimated that 20% of individuals will develop a recurrent infection which increases to 30% for those who are taking gastric acid suppressants [71]. It has been reported that taking PPIs and the required antibiotics to treat CDI does not negatively impact the function of the antibiotics [72]; however, it becomes increasingly difficult to treat recurring infections due to the toxicity of the required drugs [73]. 2.2.4 Vitamin B12 Deficiency Vitamin B12 is an essential vitamin that people gain primarily from the ingestion of dairy products, meat, and fish. It plays an important role in the development of red blood cells, the functionality of nerves, and the production of DNA. Inadequate amounts of vitamin B12 can result in a condition called pernicious anemia where the body is unable to make an appropriate amount of red blood cells. This results in the body not being able to transport oxygen effectively and can cause fatigue, headaches, heart palpitations, balance problems, disturbed vision, and a decline in mental ability [74]. When foods containing vitamin B12 are eaten, it must first be stripped from the various proteins before it can be effectively absorbed by the body. This stripping process occurs during the degradation of the proteins within the stomach, and thus is reliant on the enzyme pepsin to be in its active state. Many studies have reported that individuals who have taken PPIs for longer than a year have lower amounts of vitamin B12 in their system, which appears to decrease further with respect to the longevity of PPI use [75], [76, p. 2], [77]. A 12-month study suggested that although the vitamin B12 levels were decreased in PPI users, that approximately only 3% of individuals taking PPIs for that period actually became deficient in vitamin B12 [75]. This suggests that if individuals were to halt PPI use at a maximum time span of one year, as recommended, the likelihood of vitamin B12 deficiency is quite low. However, an alternate study that assessed 25,956 individuals that had been 19 diagnosed with vitamin B12 deficiency found that 16.2% of them had been dispensed a 2 year or longer supply of acid suppressant medication [76, p. 2]. Although some of the previously mentioned symptoms appear to be more so linked to PPI usage specifically rather than gastric acid reduction, the need for an alternative solution to long-term recurrent heartburn is vividly apparent. Certain researchers have attempted to improve the efficacy of various heartburn medications by increasing the time that the medication spends in the stomach; this involves using technologies to prevent the medication from being removed from the stomach upon gastric emptying. These technologies are referred to as gastroretentive drug delivery systems and are often composed of hydrogels. 2.3 Hydrogels and their Use in Gastrointestinal Drug Delivery 2.3.1 What are Hydrogels? Hydrogels are three-dimensional structures composed of crosslinked hydrophilic polymers that allow the hydrogel to swell with large quantities of water or other fluid [78]. Even though swollen hydrogels can end up being more than 99% water by weight, they can still maintain a semi-solid structure due to their crosslinked nature. Polymers are groups of extremely large molecules consisting of many small repeating units. Individual polymer chains can become intertwined, either through the introduction of new covalent bonds between different molecular groups or through physical entanglements between and within the polymer chains (Figure 2.2). These entanglements and bonds are referred to as crosslinks and they prevent the different polymer molecules from moving with respect to each other, changing the overall form and structure of the polymer material. When crosslinks are made up of covalent bonds, this is referred to as chemical crosslinking and is done through the introduction of a crosslinking agent [79]. Physical crosslinks are often the result of weak interactions in the polymeric network, such as electrostatic interactions or hydrogen bonding; however, they can also be formed from forcing 20 the polymer to take a specific structure, which can occur due to crystallite formation after freeze-thaw cycles [80], [81]. Figure 2.2 Uncrosslinked polymer, physically crosslinked polymer, and chemically crosslinked polymer from left to right, respectively The swelling ability of a hydrogel is largely dependent on the polymer used and how the hydrogel network is formed. By changing the different polymers, crosslinking agents, and other entities in a hydrogel network, hydrogels with specific properties can be developed. While these factors alter standard traits such as swelling capacity, elasticity, strength, and porosity, they can also help to create stimuli-responsive hydrogels that exhibit light-sensitivity, temperature-sensitivity, electro-sensitivity, or pH-sensitivity [82]. The excellent swelling capabilities of hydrogels combined with the ability to tailor hydrogel properties make them an ideal candidate for use in the biomedical field [83]. 2.3.2 Hydrogels in the Gastrointestinal Tract There are a variety of reasons a hydrogel may be developed to tolerate the environments within the gastrointestinal tract. Some of the common implementations are in edible coatings, food 21 packaging, and drug delivery systems [84]. When considering a hydrogel for use within the gastrointestinal tract, pH-sensitivity is a factor that should be highly considered due to the continual variance of pH between the gastrointestinal fluids. The development of pH-sensitive hydrogels is done using ionic polymers. Hydrophilic polymers contain polar functional groups on their polymer chains, including hydroxyl groups (-OH), carboxyl groups (-COOH), and amino groups (NH2), giving them the ability to swell in media. Certain polar groups can also be positively or negatively charged in addition to being polar which can enhance the swelling capacity of a hydrogel even further depending on the pH of the media it is in [85]. When considering pH-sensitive hydrogels, these groups can either be anionic (containing negative ions) or cationic (containing positive ions). In response to a change in pH, these ionic groups can either become protonated or deprotonated, resulting in the addition or removal of extra charges within the polymer network (Figure 2.3) [86]. These like charges generate repulsive forces within the polymer, resulting in increased swelling. In low pH solutions, anionic hydrogels are protonated and become neutrally charged, whereas cationic hydrogels are protonated but become positively charged [87]. Based on this, a cationic polymer is necessary for maximized swelling in a low pH environment and an anionic polymer is necessary for maximized swelling in a high pH environment. 22 Figure 2.3 pH-sensitive hydrogels One of the most recognizable uses of pH-sensitive polymers in the gastrointestinal tract is for enteric coatings on drug tablets that prevent a drug from being released before reaching the small intestine [88]. Enteric coatings are used to either improve the bioavailability of a drug by preventing it from being degraded in the stomach or to protect the stomach from certain undesirable effects a drug may produce [89]. In a similar fashion, hydrogels have been developed to help increase the safety and bioavailability of certain drugs using gastroprotective drug delivery systems and gastroretentive drug delivery systems, respectively. Gastroprotective drug delivery systems are mainly focused on withholding drug-related action until leaving the stomach, whereas gastroretentive drug delivery systems aim to increase the time a drug spends in the stomach [90], [91], [92, p.], [93]. 2.3.3 Types of Drug Delivery Systems Similar to the goals of this thesis, gastroretentive drug delivery systems based on hydrogels are designed to have optimal swelling in low pH environments. They are often used to control the release of drugs into the stomach, providing drugs that are preferentially absorbed in the upper 23 gastrointestinal tract a longer absorption window that is not dependent on gastric emptying time [94]. There are a variety of different forms of gastroretentive drug delivery systems; however, the most common include expandable systems, bioadhesive systems, floating systems, high-density systems, and superporous hydrogel systems. 2.3.3.1 Expandable Systems Expandable drug delivery systems are designed so that they can be folded or compressed inside of a capsule for easy ingestion and then expand to sizes greater than the pyloric sphincter upon contact with gastric fluid [95]. These systems need to be tailored to disintegrate, shrink, or be enzymatically digested after the drug has been released from the system to continue moving through the gastrointestinal tract [96]. Expandable systems usually rely on one of two primary actions to remain in the stomach, the first being unfolding and the second being swelling [97]. Unfolding expandable systems have been based on a variety of different geometric shapes, including tetrahedrons, rings, clovers, disks, pellet/sphere arrangements, and snowflakes [98]. The systems are often composed of multiple polymers, one that encourages swelling and generates the overall shape of the device and another that acts as a degradable glue that is dissolved in the stomach, allowing the device to be split into pieces and pass through the pyloric sphincter. Because these systems focus more on being able to maintain a specific shape within the stomach rather than expand volumetrically, the swelling ratios of these hydrogels are often not considered or are comparatively small [99], [100], [101], [102]. Expandable systems that primarily focus on swelling have been shown to have incredibly high swelling capabilities (> 5000%); however, these systems are limited by their swelling time, some taking up to 24 hours to swell to their maximum potential [103], [104], [105]. Some issues with expandable systems are that they can be difficult to manufacture, there are challenges involved in maintaining the structural integrity of the device in the stomach, and there 24 is always a risk of bowel obstruction [106]. Additionally, it has been found that they do not work well in a fasted state and end up getting expelled from the stomach earlier than intended [107]. 2.3.3.2 Bioadhesive Systems Bioadhesive drug delivery systems have been developed to stick to the mucosal lining of the stomach through the use of adhesive polymers and different coatings [108]. The most common polymers used for adhesion in the gastrointestinal tract are chitosan, poly(acrylic acid), alginate, poly(methacrylic acid), and sodium carboxymethyl cellulose, the majority of which being ionic, resulting in increased mucoadhesive abilities [109]. Swelling is an essential factor for mucoadhesive systems, as a higher degree of swelling suggests increased flexibility and an elevated surface area, which is important for adequate adhesion to mucous linings [110]. Even so, similar to unfolding systems, maximizing the swelling ability of the hydrogel is not the main goal, resulting in the swelling ratio being comparatively minimal or not studied [111], [112], [113], [114]. Additionally, while bioadhesive systems offer excellent advantages regarding gastroretention, their usage is limited for long-term drug delivery due to the rapid mucus turnover in the stomach [115]. There is also concern that the hydrogel may accidentally become attached to the esophagus and cause drug-related damage due to the non-specific binding of the systems. 2.3.3.3 Floating Systems Floating drug delivery systems have densities less than gastric fluid, allowing them to rest on the top of the gastric contents. The main concept behind these systems is that if the hydrogel can stay floating for extended periods of time, it will not be expelled from the stomach upon gastric emptying. Floating gastroretentive drug delivery systems can be split into two categories: effervescent or non-effervescent, depending on their mechanism of buoyancy. Effervescent systems incorporate materials such as sodium bicarbonate into the hydrogel matrix that react 25 with gastric acid and generate carbon dioxide [116]. This carbon dioxide becomes trapped within the hydrogel and lowers the density of the overall structure, causing it to float to the surface of the gastric fluid. These effervescent materials end up consuming between 5% and 50% of the total weight of the system to ensure that enough gas is formed, limiting the amount of drug that these systems can hold [117]. Additionally, the gas generation that occurs due to the effervescent materials is the result of a neutralization reaction in the stomach, suggesting that these types of gastroretentive systems may have an impact on the gastric pH. As effervescent systems are developed primarily based on the gas-generation reaction, the degree of swelling is not a priority and is often not significant [118], [119], [120], [121]. Non-effervescent systems use swellable hydrophilic polymers to form floating hydrogels within the stomach that are further supported by the entrapment of air or incorporation of low-density materials, such as oil [106]. The swelling achieved using these forms of gastroretentive devices is better than some of the previously discussed methods; however, the swelling is still not dramatic (~200 - 400%) and in some methods tested over a very long time span (> 12 hours) [122], [123], [124]. 2.3.3.4 High-Density Systems In contrast to floating systems, high-density systems have a density greater than that of gastric fluid and sink to the bottom of the stomach, where they become temporarily implanted in the folds of the lower stomach. While a density of greater than ~1.00 g/ml is required to initiate sinking in the stomach, it has been estimated that a density of between 2.5 and 3 g/ml is required to prevent materials from being ejected from the stomach during peristalsis [110]. High-density materials such as titanium dioxide and iron powder were used to increase the density of these systems; however, achieving a density of ~2.5 g/ml while also containing an appropriate amount of drug proved to be very difficult [106]. Owing to this, high-density systems are more of 26 a concept and have not been successfully implemented for commercially available drugs. Select studies have been done on high-density systems which proved that the increased density also increased gastric retention time; however, recent concerns have developed regarding the toxicity of the high-density materials used (in this case, iron) [125]. Although there is no information on the swelling abilities of previously researched high-density drug delivery systems, it is likely that the swelling abilities of these systems were negligible because of their stringent density requirements. 2.3.3.5 Superporous Hydrogel Systems Superporous hydrogel systems are some of the most popular gastroretentive drug delivery systems to use. They are usually a combination of a type of expanding system and a type of floating system where volumetric change occurs through swelling and a low density is maintained through the use of a highly porous structure. Standard, non-porous hydrogels can take many hours to swell to their equilibrium point, making them difficult to use for swelling-based gastroretention; however, superporous hydrogels are able to swell up to 100 times faster than their non-porous equivalents [126]. In order to avoid being pre-emptively removed from the stomach, a swellable gastroretentive system needs to swell to a size larger than the pyloric sphincter or float to the top of the gastric fluid within 20-minutes [127]. The incredibly rapid swelling time and low density of superporous hydrogels make it easy for them to fulfil either one or both of these tasks within the time frame. As suggested in the name, superporous hydrogels are different from standard hydrogels in that they are much more porous. Superporous hydrogels have large interconnected pores that are greater than 100 μm in size, whereas standard hydrogels only have pores that are between 10 nm and 10 μm [128]. Opposed to being limited by the rate of diffusion, this interconnected porous structure allows for liquid to flow through the open channels within the hydrogel, 27 resulting in extremely fast swelling based on capillary action. Pores can be introduced into hydrogels through a variety of techniques, but the methods that have gained the most popularity are the gas blowing method and the freeze-drying method [129]. The gas blowing method involves introducing a foaming agent into the solution during the hydrogel formation process, which generates bubbles that become stuck in the hydrogel structure. The freeze-drying method does not change the formation of the hydrogel but changes the process in which excess water is removed from the hydrogel after the formation is complete. In the freeze-drying process, excess water in the hydrogel is removed via sublimation, which replaces small grains of ice with free space, creating pores [130]. Superporous hydrogels developed for gastroretentive drug delivery have been reported to have high swelling ratios (> 1000%), extremely fast swelling times, and low enough densities to float on top of gastric fluid [131], [132], [133]. 2.4 Hydrogels for Use in Gastric Acid Relocation In order to relocate the gastric acid pocket, a combination of a superporous hydrogel system and a high-density system is required. Superporous hydrogel systems on their own will either be too light to sink to the bottom of the stomach or will have swelling times that are not fast enough to fully absorb the contents of the gastric acid pocket before sinking. The two requirements for the proposed hydrogel are that it must have a density higher than that of gastric fluid to be able to sink to the bottom of the stomach and it must be able to swell with a relatively large amount of fluid in a short period of time to be effective. Based on these requirements, two materials need to be selected: 1) a polymer ideal for fast swelling in a low pH environment and 2) a bioinert material that can be used to increase the density of the hydrogel. 2.4.1 Polymer Selection Although the majority of the superporous hydrogels that are used on the market are composed of synthetic polymers due to their improved water absorption capacity and higher gel strength, 28 the continued desire to use natural and environmentally friendly materials is pushing research towards the use of natural polymers [134], [135]. Gastroretentive devices are often made with synthetic polymer composites or hybrids to be able to withstand the pressure and forces that occur within the stomach. These pressure limitations restrict the use of natural polymers, often resulting in them being excluded from hydrogel formulations or used simply as a composite agent [136], [137]. As gastroretention is not needed in this study, the limitation of using strong, synthetic polymers is removed. The most popular cationic polymers used for biocompatible hydrogels include chitosan, poly(ethyleneimine), poly(amino-co-ester)s, poly(amidoamine)s and poly-L-(lysine) [138]. Among these, chitosan is the only naturally occurring polymer. Chitosan is prepared from the deacetylation of chitin, a major component of the shells of crustaceans and the second-most abundant biopolymer on the planet. It is currently available as a dietary supplement for the promotion of weight loss; however, claims regarding its effectiveness are mixed [139], [140]. In addition to being biodegradable and biocompatible, it is also digestible by various bodily enzymes [141], [142]. This, in combination with its excellent mucoadhesive abilities, cationic nature, and natural source, makes it an excellent candidate for gastric acid relocation and was selected as the primary polymer for this research. For chitosan-based hydrogels that are used in the stomach, dialdehydes are the most common crosslinkers used, namely glutaraldehyde and glyoxal [143]. These are chemical crosslinkers that connect the monomers of the chitosan polymer through their amine groups (Figure 2.4). As previously mentioned, by tailoring the concentrations and ratios of chitosan to crosslinker, hydrogels with different strengths and properties can be attained. Both glyoxal and glutaraldehyde were tested in the following experiments as listed in Chapter 3, and the relationship between polymer and crosslinker concentrations is explored throughout this work. 29 Figure 2.4 Segment of chitosan structure, a) uncrosslinked, b) crosslinked with glutaraldehyde 2.4.2 High-Density Material Selection The high-density materials that were originally used for high-density gastroretentive drug delivery systems include titanium dioxide, barium sulfate, zinc oxide, and iron powder. Some of these materials, such as zinc oxide and iron powder, are absorbed by the body more readily than the other materials, making them unideal candidates due to the potential of excess absorption [144]. Others, such as titanium dioxide, have been used in many biomedical areas, including use in bone prosthetics, dental treatments, and drug delivery systems [145], [146]. Titanium dioxide has been shown to be able to bond to various different polymers for use in hydrogels. Al-Taweel et al. and Xing et al. have both reported the presence of weak hydrogen bonds developed between TiO2 molecules and the -OH and -NH2 groups of chitosan chains in 30 titanium dioxide/chitosan composite hydrogel films [147], [148]. Chitosan/titanium dioxide hydrogel composites have shown significant promise in research, primarily in the areas of wound healing and food preservation [149]. Safari et al. have also shown that chitosan/titanium dioxide hydrogel composites could improve the release rate of dopamine into the gastrointestinal tract for the optimized treatment of conditions such as Parkinson’s disease [150]. Titanium dioxide is a bioceramic: a ceramic material that possesses excellent biocompatible properties. Titanium dioxide happens to be bioinert as well, meaning that it does not elicit a response when in contact with biological tissue. A similar bioceramic that has gained recent attention is zirconium dioxide which is being used as an alternative to titanium in dental crowns. While it has not been used in as many chitosan composites as titanium dioxide, zirconium dioxide has been used in chitosan-based biocomposites for nucleic acid detection and bone tissue engineering applications [151], [152]. Titanium dioxide and zirconium dioxide are both metal oxides that possess similar structures: two oxygen molecules both double-bonded to a central metal atom. This indicates that zirconium dioxide may be able to maintain the same weak bonding between chitosan that is achieved through using titanium dioxide. Even though zirconium dioxide has not been used in as many biomaterials as titanium dioxide, its biocompatibility has been proven by many studies [153], [154]. The particular benefit that zirconium dioxide has over titanium dioxide regarding this work is the density. The density of titanium dioxide is ~4.23 g/ml, whereas the density of zirconium dioxide is ~5.68 g/ml, suggesting that less density-altering agent could be added to the hydrogel if using zirconium dioxide rather than titanium dioxide. In this work, titanium dioxide was first used as a density-altering agent to identify if the previously discovered hydrogen bonds were strong enough to hold the ceramic within the hydrogel structure during swelling. After this, zirconium dioxide was introduced and its potential for similar weak bonding to chitosan was explored. 31 2.5 Chapter 2 Summary Gastric acid is essential for many different functions, including the eradication of various bacteria, the breakdown of proteins and fats, and the effective absorption of vitamins and minerals. When people experience recurrent heartburn, often seen in individuals with GERD, heartburn is alleviated by using gastric acid neutralizers or suppressants to increase the pH of the gastric fluid. Although these drugs are very effective at increasing the pH of the gastric fluid, the use of gastric acid suppressants for extended periods of time has shown adverse side effects such as hypomagnesemia, vitamin B12 deficiency, and an increased risk for contracting CDI and for fracturing bone. An alternative treatment for heartburn that does not alter the natural pH of the gastrointestinal environment, such as the one described in this thesis, would likely prevent these gastric pH-related effects. In order to develop a hydrogel to act as a carrier for gastric acid in the stomach, hydrogels used in gastroretentive drug delivery systems were discussed and compared. The results of this analysis suggested that to absorb a significant amount of gastric acid quickly, a type of hydrogel known as a superporous hydrogel would be best for this task. A primary polymer for the hydrogel was selected by considering the low pH environment of the stomach, the desired biocompatibility of the hydrogel, and the necessary swelling requirements. The polymer chitosan, derived from the shells of crustaceans, was selected to be the primary polymer for this research due to its natural source, excellent biocompatibility, and cationic nature. To adapt the density of the hydrogel to be able to sink to the bottom of the stomach after being swollen with gastric fluid, the bioceramics titanium dioxide and zirconium dioxide were selected based on their frequent use in the medical and food industries. 32 Chapter 3: Methodology Two different methods of creating superporous hydrogels were tested and compared: the gas blowing method and the freeze-drying method. The gas blowing method involves introducing a foaming agent during the crosslinking process to generate pores within the hydrogel, whereas in the freeze-drying method, the pores are generated during the sublimation process. 3.1 Gas Blowing Method 3.1.1 Materials Chitosan (medium molecular weight, 75-85% deacetylated), titanium(iv) oxide nanopowder (21nm), glyoxal (40% in water), poly(vinyl alcohol) (99+% hydrolyzed), and sodium bicarbonate were purchased from Sigma Aldrich. Hydrochloric acid (2.0N) and glacial acetic acid were purchased from VWR. All aqueous solutions were prepared with reverse osmosis (RO) purified water and all other chemicals used were of analytical grade. 3.1.2 Hydrogel Synthesis The hydrogels made using the gas blowing method were based on the hydrogels prepared by Gupta and Shivakumar [155] and Chavda and Patel [136]. A 2% w/w solution of chitosan was prepared by dissolving chitosan in 1% v/v acetic acid at 60 degrees Celsius under magnetic stirring for a minimum of 5 hours. Additionally, a 10% w/w solution of poly(vinyl alcohol) (PVA) was prepared by dissolving PVA in RO water at 95 degrees Celsius under magnetic stirring for 30 minutes. A solution of 10% w/w glyoxal was used as the crosslinker, titanium dioxide nanoparticles were used as the high-density material, and sodium bicarbonate was used as the foaming agent. The chitosan, PVA, and titanium dioxide particles were combined under magnetic stirring for a minimum of 30 minutes to ensure that the solution was well mixed. The pH of the solution was then lowered to 5.0 by the dropwise addition of 0.1M acetic acid to 33 ensure the reaction between glyoxal and chitosan would occur. The glyoxal was then added to the mixture which was shaken vigorously and after 30 seconds sodium bicarbonate was added to induce bubble generation. The hydrogel was left to rest for 30 minutes and then placed into an ethanol bath for at least 5 hours before being dried in an oven overnight at 55 degrees Celsius (Figure 3.1). Figure 3.1 Hydrogel formation using gas blowing method The chitosan, PVA, glyoxal, and titanium dioxide were combined in varying amounts, as listed in Table 3.1. Titanium dioxide was only added to Samples 5 and 6 to compare how the titanium dioxide affected the swelling ability of the hydrogels. 34 Table 3.1 Gas-blown hydrogel formulations: comparison of polymer percentage, crosslinker percentage, and percentage of titanium dioxide Chitosan (mg) PVA (mg) Glyoxal (mg) Titanium Dioxide (mg) Sodium Bicarbonate (mg) Sample 1 400 160 400 0 80 Sample 2 400 160 160 0 80 Sample 3 160 400 400 0 80 Sample 4 160 400 160 0 80 Sample 5 160 400 400 20 80 Sample 6 160 400 400 50 80 After the first experiment, another experiment was prepared to try and increase the swelling ability of the hydrogels further. In the second experiment, the PVA was removed from the hydrogels, a foam stabilizer, Pluronic F127, was added into one of the mixtures, and the amount of glyoxal was varied. In this experiment, the titanium dioxide was excluded from the study in an attempt to identify the ideal crosslinker percentage as well as to determine the effects of the foam stabilizer. The individual sample volumes were also increased to increase the ease of handling. The different sample sets are shown in Table 3.2. 35 Table 3.2 Gas-blown hydrogel formulations: comparison of crosslinker percentage and effects of foam stabilizer Chitosan (ml) Glyoxal (ml) Pluronic F 127 (%w/w) Sodium Bicarbonate (mg) Sample 7 3 0.12 0 50 Sample 8 3 0.12 0.5 50 Sample 9 3 0.9 0 50 Sample 10 3 0.6 0 50 3.2 Freeze-Drying Method 3.2.1 Materials Chitosan (medium molecular weight, 75-85% deacetylated), zirconium (IV) oxide (5 micron diameter), glutaraldehyde solution (25% in water), sodium chloride, and pepsin (from porcine gastric mucosa) were purchased from Sigma Aldrich. Hydrochloric acid (2.0N), glacial acetic acid, and the Cytotoxicity Assay Kit were purchased from VWR. All aqueous solutions were prepared with RO purified water, and all other chemicals used were of analytical grade. 3.2.2 First Generation The first generation of freeze-dried hydrogels was developed based on the formulas used by Mirzaei et al. [156]. As it is has been reported that using an increased concentration of chitosan can raise the swelling ratio of chitosan-based hydrogels, two solutions of chitosan with different concentrations were tested and compared [157], [158]. Both 1% w/v and 2% w/v solutions of chitosan were prepared by dissolving chitosan in 1% v/v acetic acid. These chitosan solutions were combined with 1% aqueous glutaraldehyde and various quantities of zirconia (zirconium dioxide) before being mixed using a vortex mixer. After the mixtures were well combined, they were left to sit for 30 minutes to crosslink before being placed into a -80-degree Celsius freezer. 36 Once the hydrogels were adequately frozen, they were placed into a freeze dryer (Labconco FreeZone 1L Benchtop Freeze Dry System) and lyophilized for 12 to 24 hours depending on the sample size (Figure 3.2). Figure 3.2 Hydrogel formation using freeze-drying method The different hydrogel formulations used for the initial test are shown in Table 3.3. The mass of zirconia added to the samples was incremented by units of 5% in relation to the total volume of the sample. This was done to determine the mass of zirconia required to raise the density of the hydrogels above the density of gastric fluid. Based on preliminary trials, it was found that testing 1% w/v chitosan samples with zirconia percentages higher than 20% was excessive and that testing 2% w/v chitosan samples with zirconia percentages less than 25% was insufficient, which is why the samples were allocated accordingly. A standardized volume of 0.12 ml of 1% glutaraldehyde solution was added to each 1 ml sample to act as the crosslinking agent (12% w/w glutaraldehyde to chitosan for 1% chitosan samples, and 6% w/w glutaraldehyde to 37 chitosan for 2% chitosan samples). All experiments were performed in triplicate, and the data presented in the results section are the average values of each of the three trials. Table 3.3 Freeze-dried hydrogel formulations: chitosan percentage versus amount of zirconia . 10% Zirconia (w/v) 15% Zirconia (w/v) 20% Zirconia (w/v) 25% Zirconia (w/v) 30% Zirconia (w/v) 35% Zirconia (w/v) 1% Chitosan Sample 1 Sample 2 Sample 3 - - - 2% Chitosan - - - Sample 4 Sample 5 Sample 6 3.2.3 Second Generation The second generation of freeze-dried hydrogels was developed based on the results from the first generation of freeze-dried hydrogels. The methodology used to prepare the hydrogels was kept identical to Section 3.2.2; however, the concentrations of glutaraldehyde and zirconia were modified to attempt and improve the biocompatibility and swelling abilities of the hydrogels. An initial test was performed using reduced quantities of 1% aqueous glutaraldehyde solution, comparing 2% w/w, 1% w/w, 0.5% w/w, and 0.25% w/w concentrations of glutaraldehyde to chitosan. After the ideal percentage of glutaraldehyde was determined, a final test was performed to identify the amount of zirconia required to raise the density of the improved swollen hydrogel above the density of gastric fluid. 3.3 Characterization Techniques The efficacy of the hydrogels was determined based on four main factors: maximum swelling ability, equilibrium swelling time, swollen density, and biocompatibility. Based on these factors, the studies done on the swelling abilities of the hydrogels were deemed the most important and 38 were used to identify potential candidates for further study. After a sample was selected to move forward with the experimentation process, it was examined through various techniques such as micro-computed tomography imaging, scanning electron microscopy, Fourier transform infrared spectroscopy, x-ray diffraction analysis, and cell cytotoxicity live/dead assays. Formulations that showed limited promise were rejected after only a few characterization techniques, whereas formulations that showed increasing promise were fully studied. 3.3.1 Swelling Studies 3.3.1.1 Initial Feasibility Assessment The initial feasibility test that was performed on all samples was a swelling experiment in pH 1.57 hydrochloric acid. This specific media was selected to replicate the environment of the gastric acid pocket. The hydrogel samples were weighed and placed in hydrochloric acid for 30 minutes to allow for the hydrogels to swell. Based on the hydrogel type, the samples were periodically extracted, blotted to remove excess fluid, and re-weighed to determine their swollen mass. Hydrogels that were made using the gas blowing method were extracted between one- and ten-minute intervals, whereas hydrogels made using the freeze-drying method were left undisturbed for the full 30 minutes. The reasoning for this is that some of the hydrogels that were made with the freeze-drying method were fragile and tended to break upon repeated removal from the solution. The swelling ratios of the hydrogel samples were calculated according to (1) as shown below: 𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑅𝑎𝑡𝑖𝑜 (%) = 𝑀! − 𝑀"𝑀" ∗ 100 (1) where Ms represents the swollen mass of the hydrogel and Md represents the initial dry mass of the hydrogel. 39 3.3.1.2 Additional Swelling Analysis If a particular formulation of hydrogel exhibited a high degree of swelling in the initial feasibility experiment, additional swelling tests were performed on that sample set. These additional swelling tests included assessing the response to pH variance, the swelling ability in simulated gastric fluid, the swelling rate over time (for freeze-dried formulations), and the response to compression. Three forms of media were used to assess the pH sensitivity of the hydrogels: hydrochloric acid with a pH value of 1.57, hydrochloric acid with a pH value of 1.2, and water with a pH value of 7.0. Hydrochloric acid with a pH of 1.2 was selected to compare with simulated gastric fluid (pH 1.2). This was done to determine the effects that sodium chloride and pepsin have on the swelling ability of the hydrogels. Compressing the hydrogels before analyzing their swelling ability was done to simulate the effects of being compacted inside a gel capsule for easier transfer to the stomach. Hydrogel samples were flattened between two Petri dishes prior to submersion in hydrochloric acid to determine how the swelling abilities of the hydrogel were altered due to the reduced initial volume. 3.3.2 Porosity Measurement and Calculation of Density The porosities of the hydrogels were measured in two ways: 1) the solvent displacement method and 2) applying image processing to micro-computed tomography scans. For the solvent displacement method, dried hydrogel samples were weighed and placed in hexane for 30 minutes to allow for the hexane to permeate through the porous structure. After 30 minutes, the hydrogels were weighed again, and their porosities were calculated as shown in (2): 𝑃 = 𝑀# −𝑀" 𝜌# ∗ 𝑉$ ∗ 100 (2) where Mh represents the mass of the hydrogel after being immersed in hexane, 𝜌# is the density of hexane, and Vi is the volume of the hydrogel. Using this method, the porosities of hydrogels that contained zirconia were compared against the porosities of hydrogels that did not have 40 zirconia but were otherwise identical. This was done to determine the extent to which the zirconia impacts the porous nature of the hydrogels. Due to the difficulty of working with hexane because of its high evaporation rate, these values were compared against those calculated using the micro-computed tomography image analysis method. Micro-computed tomography, also referred to as micro-CT, is similar to the type of tomography that is frequently used in hospitals for CAT scans. It is a non-destructive technique that is used to observe the internal structure of an object of interest and has been typically used in the fields of tissue engineering, medicine, and food chemistry, amongst others [159], [160]. Micro-CT uses a beam of x-rays that is repeatedly transmitted through a rotating sample to acquire a series of 2D x-ray projections (Figure 3.3). These projections can be reconstructed using a filtered back-projection algorithm to create cross-sectional images of the sample [161]. Materials that have a high density will be able to block or absorb most of the x-rays, resulting in a white colouration on the x-ray image, whereas materials that have a lower density will not, causing them to appear grey or black. Figure 3.3 Micro-CT diagram 41 The Zeiss microXCT 400 scanner was used to obtain cross-sectional images of the samples. Three of the images from each sample set were selected and analyzed in MATLAB where an intensity threshold was placed on the images to separate hydrogel material from free space. Using the binary images gained from the thresholding effect, the number of white pixels, black pixels, and total pixels in the hydrogel area were determined. The porosity was calculated using (3): 𝑃 = 𝑁%𝑁& ∗ 100 (3) where NB is the number of black pixels in the hydrogel area and NT is the total number of pixels in the hydrogel area. Freeze-dried hydrogel samples made without zirconia did not provide enough x-ray opacity to be visible on the micro-CT scans, as a result, only freeze-dried hydrogels with zirconia were analyzed using this method. Using the determined porosity values, the densities of the hydrogels could be calculated. Three different densities were calculated for the hydrogels: apparent density, actual density, and swollen density, calculated using (4), (5), and (6), respectively: 𝑑' = 𝑀"𝑉$ (4) 𝑑 = 𝑀"𝑉# = 𝑀"(1 − 𝑃/100) ∗ 𝑉$ (5) 𝑑! = 𝑀!𝑉$ (6) where Vh represents the volume of the hydrogel, excluding the free space within the hydrogel structure. 42 3.3.3 Scanning Electron Microscopy The morphologies of the hydrogel samples were studied using a scanning electron microscope (SEM). SEMs are a type of microscope that utilize a beam of electrons instead of photons to form an image, resulting in a much higher resolution than traditional optical microscopes [162]. Electrons are produced at the top of the microscope using an electron gun and are sent down the microscope through a series of apertures and lenses to focus the electrons into a beam. This beam of electrons makes contact with the sample, and as it does, secondary electrons, backscattered electrons, and characteristic x-rays are produced, which can be detected to create images. An example of how an SEM works is presented in Figure 3.4. Samples both with and without zirconia were analyzed to identify any differences in hydrogel structure caused by the zirconia. The cylindrical samples were cut in half to expose the inner structure of the cross-linked polymer and were coated with a thin layer of gold or iridium (Leica EM ACE600). The first-generation samples were observed using backscattered electrons at a voltage of 20 kV (MIRA3 TESCAN) and the second-generation samples were observed using secondary electrons at a voltage of 5 kV (FEI Helios). 43 Figure 3.4 SEM diagram, where BSE refers to the detector for backscattered electrons, SE refers to the detector for secondary electrons, and EDX refers to the detector for characteristic x-rays. Reproduced with permission [163]. 3.3.4 Fourier Transform Infrared Spectroscopy Fourier transform infrared spectroscopy (FTIR) was used to investigate the compatibility between the chitosan hydrogel and the zirconia particles. FTIR is used to study how infrared light interacts with matter, providing insight into the different molecules present within a sample as well as their respective concentrations [164]. In FTIR, infrared radiation is applied to a sample, which is selectively absorbed at different wavelengths depending on the molecules present within the material. This absorption data can be recorded and converted to an infrared spectrum where different absorption peaks can be observed, corresponding to the molecular fingerprint of the material [165]. The absorption spectra of hydrogels containing zirconia, 44 hydrogels without zirconia, and pure zirconia were compared using a Nicolet™ iS20 FTIR Spectrometer. 3.3.5 X-Ray Diffraction Analysis X-ray diffraction analysis (XRD) is a common technique used to identify the crystallographic structure of a material. When an x-ray contacts a solid crystalline object, it is reflected in a particular direction based on the structural orientation of the atoms within the object. The direction and number of these x-rays can be detected, giving light to the different crystal structures within the material (Figure 3.5). One of the more powerful uses of XRD is the detection of crystalline phases within an amorphous sample, as amorphous materials, such as chitosan, do not produce diffraction patterns with identifiable peaks [166]. In this study, XRD was used to identify the crystal structure of zirconia and determine if incorporating it into the hydrogel resulted in any changes in the XRD pattern. The freeze-dried hydrogel samples were ground down to a fine powder and were analyzed over a 2-theta range of 5 -70 degrees using a Rigaku Rapid Axis X-ray Diffractometer. Figure 3.5 X-ray diffractometer diagram 45 3.3.6 Cell Toxicity Study Some of the preliminary tests used to determine if a drug is safe for human interaction are cell cytotoxicity assays [167]. In cell cytotoxicity assays, cells are used to determine the likelihood that a material possesses toxic properties based on the percentage of cells that survive while in contact with the material. A common version of these assays are live/dead viability stains, where one type of dye specifically stains living cells (often resulting in green fluorescence) and another type of dye specifically stains dead cells (resulting in red fluorescence) [168]. After being given nutrients and time to grow, the cells can be stained with both dyes and observed under fluorescence, where the number of living and dead cells can be determined. When these values are compared against those of the control group, it can be ascertained if the material causes an increase in cell death, therefore maintaining cytotoxic properties. In order to assess the biocompatibility of the hydrogels, a cell toxicity study was performed with 3T3 mouse embryonic fibroblast cells. A 24-well tissue culture plate was placed into a biosafety cabinet and was sterilized by UV for 12 hours. After sterilization, 100 μL of pre-crosslinked hydrogel mixture was added to each of the chosen wells, yielding hydrogel layers approximately 1 mm thick. Six wells were filled with hydrogel mixture containing zirconia and six wells were filled with hydrogel mixture that did not have zirconia, resulting in three hydrogels for each test. The culture plate was frozen at -80 degrees Celsius overnight and subsequently placed in a freeze dryer to lyophilize the hydrogel discs. The freeze-dried hydrogel discs were then washed twice with PBS solution and 3T3 cells were seeded onto the surface of the hydrogels at a seeding density of 2X106 cells per well. 300 μL of Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin-streptomycin was added to each well and was changed every 2 days. The cell-seeded well plate was incubated at 37 degrees Celsius and 5% CO2 for a total of five days. 46 After the first day, the cell culture media was replaced, and the viability of the cells was assessed using a Live/Dead cytotoxicity kit (Viability/Cytotoxicity Assay Kit for Animal Live and Dead Cells). A staining solution of 2 μM calcein AM and 4 μM EthD-III was prepared as listed in the cytotoxicity kit protocol and added to half of the cell-seeded wells on the culture plate. The well plate was covered in aluminum foil and the cells were incubated for 30 minutes at room temperature. Following the incubation period, the cell-bound hydrogel surfaces were washed several times with PBS, observed using fluorescence microscopy, and returned to the incubator for the remainder of the five days. On the fifth day, the well plate was once again removed, and the live/dead assay was repeated as previously described. Cell viability data was measured using ImageJ to count the number of live and dead cells. 3.4 Chapter 3 Summary The gas blowing method and the freeze-drying method are two commonly used methods that are used to prepare superporous hydrogels. The gas blowing method introduces pores into the hydrogel structure by adding a foaming agent into the hydrogel during the hydrogel formation process and the freeze-drying method introduces pores into the hydrogel structure through lyophilization after the hydrogel formation process is complete. In this chapter, the processes for creating both gas-blown hydrogels and freeze-dried hydrogels were discussed and the different experiments within each hydrogel type were presented. Using the gas blowing hydrogel preparation method, experiments were prepared to determine the effects that the amounts of polymer, crosslinking agent, titanium dioxide, and foam stabilizer have on the swelling capabilities of the hydrogels. Hydrogels prepared using the freeze-drying method were split into two generations. The experiments with the first generation of hydrogels were selected to determine the effects that different concentrations of chitosan and zirconia have on the hydrogel properties, whereas the experiments with the second generation of hydrogels were prepared to identify how the amount of crosslinking agent influences the hydrogel properties. Different 47 characterization techniques for the hydrogels were also introduced, including the methods used to calculate the swelling ratios, porosities, and densities of the hydrogels, as well as the use of micro-CT, SEM analysis, FTIR, XRD, and cell cytotoxicity assays. 48 Chapter 4: Results and Discussion 4.1 Gas Blowing Method 4.1.1 Hydrogel Synthesis Immediately after crosslinking, the hydrogels congealed within the solutions. The hydrogels that were made with a high amount of glyoxal formed a singular solid mass that floated at the top of the solution (Figure 4.1, a)), whereas the hydrogels that were made with a low amount of glyoxal turned the solution into a slurry of semi-solid material. After drying in the oven overnight, the samples shrunk significantly and changed from a clear, gelatinous substance to a brown, brittle material (Figure 4.1, c)). The reduction in size occurs due to the removal of liquid from the hydrogel and the change in colour was a result of the heat treatment of the chitosan [169]. When the hydrogels were placed into hydrochloric acid to swell, they did not return to their original form that was expressed after the crosslinking process; the hydrogels remained small and brittle for the full 30-minute testing period and no visible changes in size were observed for any of the samples. Figure 4.1 Gas-blown hydrogels, a) after crosslinking, b) after ethanol bath, c) after oven-drying 49 4.1.2 Swelling Studies The results from the initial swelling tests done with the gas-blown hydrogels in pH 1.57 hydrochloric acid solution are shown in Figure 4.2. Both Sample 2 and Sample 4 performed very poorly compared to the other hydrogels, yielding a final swelling ratio of just under 20% after the 30-minute period. Sample 2 and Sample 4 were the only samples that had the lower amount of glyoxal used during hydrogel formation, indicating that a higher amount of crosslinker was ideal for maximized swelling. Usually, an increased amount of glyoxal would lead to decreased swelling ability due to a more tightly bound hydrogel network [170]. While the observed trend went against what was to be expected, it was assumed to occur as a result of the drying process. With oven-dried hydrogels, it is common to observe a more wrinkled and collapsed hydrogel structure due to the volume change in the hydrogel [171]. The increased strength of the polymer network in the hydrogels made with a larger amount of glyoxal may have prevented the generated pores within the hydrogel from collapsing completely during the drying process; however, in the samples synthesized with a lower amount of glyoxal, the hydrogel network may not have been strong enough to sustain the porous structure while drying, resulting in a complete collapse of the pores and limited swelling ability. Out of the six samples, Sample 1 performed the best, yielding a swelling ratio just above 70%. Sample 1 had the highest amount of both chitosan and glyoxal, suggesting that an increase in chitosan also increased the swelling ability of the hydrogels. The formulations of Samples 3, 5, and 6 were identical aside from the mass of titanium dioxide added which increased with sample number. As Sample 6 showed the highest swelling ability of the three, this indicated that the titanium dioxide may also help in maintaining the porous structure of the hydrogel. Additionally, none of the titanium dioxide was observed to separate from the hydrogel during the swelling process, indicating that the titanium dioxide was successfully integrated with the chitosan hydrogel. 50 Figure 4.2 Swelling ratios of gas-blown hydrogels (samples 1–6, Table 3.1) in pH 1.57 hydrochloric acid The results from the second experiment in pH 1.57 hydrochloric acid are shown in Figure 4.3. The results follow the same trend as the first experiment: an increase in the concentration of glyoxal in the hydrogel resulted in increased swelling. Additionally, while adding foam stabilizer appeared to increase the swelling ratio at the beginning of the test, it did not seem to significantly affect the swelling of the hydrogel overall. This suggests that the foam stabilizer may not be able to combat the pore destruction from the drying process. Based on the swelling patterns visible from both tests one and two, it seems as if the hydrogels may have swollen further given additional time in the media; however, yielding a swelling ratio of only approximately 70% in 30 minutes is not acceptable for the purposes of this study. 51 Figure 4.3 Swelling ratios of gas-blown hydrogels (samples 7–10, Table 3.2) in pH 1.57 hydrochloric acid 4.1.3 Porosity Measurement To try and identify why the hydrogels had such limited swelling, the porosity of the Sample 6 hydrogel was analyzed using the micro-CT method, as shown in Figure 4.4. There are a few large pores that can be seen on the inside of the hydrogel, likely due to the bubbles generated from the addition of sodium bicarbonate during the formation process. Examining the entire set of micro-CT scan images made it clear that the majority of these pores were closed pores: pores that do not connect to other pores or to the outside of the hydrogel. While these closed pores contribute to the overall porosity of the hydrogel, they are largely inaccessible to the swelling media and offer minimal support in decreasing the equilibrium swelling time [172]. Aside from the large pores, the remainder of the hydrogel appears to be very dense, showing little to no porosity in the actual gel structure. The total porosity of the sample was calculated to be approximately 49%, including the closed pores. This lack of porosity was probably what caused the very slow swelling speed of the hydrogels. This led to the conclusion that in order to 52 be effective for acid relocation, a hydrogel with a much higher porosity must be used, shifting the research from gas-blown hydrogels to freeze-dried hydrogels. Figure 4.4 Image analysis of a micro-CT scan of a gas-blown hydrogel, a) during mask selection, b) image converted to black and white, c) resulting image after applying threshold 4.2 Freeze-Drying Method 4.2.1 First Generation 4.2.1.1 Hydrogel Synthesis The first-generation freeze-dried hydrogels presented with varying degrees of gelation prior to the lyophilization process, depending on the percentage of chitosan. The 1% chitosan samples, both with and without zirconia, remained in the liquid phase after the addition of the glutaraldehyde, whereas the 2% chitosan samples congealed into a solid mass after approximately 10 minutes of continual stirring. To determine if the gelation was time based, three 1% chitosan samples were kept at room temperature for 24 hours after the stirring process and reassessed. Even after the 24-hour crosslinking period, the 1% chitosan samples maintained a liquid phase and did not present with any visible change in viscosity. Based on this and the gelation time of the 2% percent samples, the 30-minute gelation time was deemed 53 satisfactory for further tests. The combination of these gelation responses implied that the amount of glutaraldehyde present in the formulations was more than what was required for the adequate crosslinking of the chitosan within the hydrogels. This also suggested that the 1% chitosan samples did not have enough polymer to completely gel during the crosslinking process. After lyophilization, structural differences could be identified between the various samples. Unlike the gas-blown hydrogels, all of the freeze-dried samples maintained their original volumes during the water removal process and had very spongy appearances (Figure 4.5, a) and b)). Depending on the quantity of chitosan and zirconia in the hydrogel, the samples presented with varying degrees of brittleness and rigidity. The 1% samples without zirconia portrayed the lowest rigidity and the 2% samples with 35% zirconia portrayed the highest rigidity. The addition of zirconia into the polymer network increased the structural integrity of the 1% samples, preventing them from being easily ripped apart while in hydrochloric acid (Figure 4.5, c) and d)); however, in the higher concentration 2% samples, the large amounts of zirconia resulted in extremely brittle and hard hydrogels. 54 Figure 4.5 Freeze-dried first-generation hydrogels (1% chitosan, 15% zirconia), a) without zirconia, b) with zirconia, c) without zirconia in hydrochloric acid, d) with zirconia in hydrochloric acid 4.2.1.2 Swelling Studies The results of the initial swelling test in pH 1.57 hydrochloric acid (Figure 4.6) outlined two potential candidates: Sample 2 and Sample 3, as these samples were the only ones that possessed a density higher than gastric fluid. A clear relationship can be observed between the amount of zirconia added to the hydrogel and the swelling ability, but as the swelling ratio is calculated based on weight, this is to be expected. The difference in hydrogel mass before and after swelling was compared with ANOVA and was found to be insignificant within each chitosan percentage group (𝛼 = 0.05). This suggests that the addition of zirconia has no impact on the swelling capabilities of the hydrogels. It can also be seen that an increased percentage of chitosan has a negative impact on the swollen density of the hydrogels, as none of the 2% samples were capable of sinking in gastric fluid even with an additional amount of zirconia up to 35%. This is likely a result of the amplified crosslinking density of the polymer structure that occurs with the use of a greater concentration of chitosan. Due to the strengthened attachments 55 that develop between polymer chains, the pores generated during the lyophilization process of a highly crosslinked hydrogel will be smaller than the pores generated in a hydrogel with a lower crosslinking density [155]. This makes it more difficult for media to penetrate the hydrogel structure, resulting in reduced swelling abilities [173]. Additionally, the chitosan consumes more of the sample volume in the 2% chitosan hydrogels, decreasing the overall density of the hydrogel due to the low density of chitosan (0.2 – 0.38 g/ml [174]). Based on the findings from this experiment, Sample 2 appeared to have optimal polymer and zirconia concentrations for maximized gastric acid relocation and was selected for further analysis. These results also indicated that an additional mass of 15% w/v was required to cause the necessary density increase in the hydrogels and that the maximum swelling ratio that could be attained was approximately 435%. Figure 4.6 Swelling ratios of first-generation freeze-dried hydrogels (samples 1–6, Table 3.3) in pH 1.57 hydrochloric acid (± standard deviation). Solid bars indicate that the hydrogels possessed a density greater than the density of gastric fluid. 56 The swelling response of the Sample 2 hydrogel was also analyzed in pH 1.2 hydrochloric acid, pH 1.2 simulated gastric fluid, and pH 7.0 water. The average swelling ratios of the samples in pH 1.2 hydrochloric acid and simulated gastric fluid yielded a difference in swelling response of less than 0.2% and ANOVA determined that this difference was insignificant (𝛼 = 0.05). This suggests that the presence of pepsin and sodium chloride in gastric fluid had a negligible effect on the swelling capabilities of the hydrogel. The overall swelling ratio in pH 1.2 media was calculated to be approximately 437%, only 2% larger than in pH 1.57 media; however, the swelling ratio of the hydrogel in water was reduced by ~18%. At low pH values, the amine groups on the chitosan polymer are protonated, resulting in the polymer becoming positively charged. These positive charges within the hydrogel network cause segments of the polymer to repel against one another, encouraging further swelling in the hydrogel. As this effect only occurs at low pH values, the swelling was reduced in pure water. 4.2.1.3 Porosity Measurement and Calculation of Density The freeze-dried hydrogels had highly porous structures as depicted in Figure 4.7. Scanning through the micro-CT images outlined that the pores within the freeze-dried hydrogels were interconnected and open. This allows for fluid to permeate the hydrogels through capillary wetting instead of diffusion, resulting in extremely fast and significant swelling [175]. This interconnected porous structure was likely the reason for the dramatic increase in swelling ability compared to the gas-blown hydrogels. Large, stacked pores can be observed running down the length of the hydrogel in the axial direction. Although the pores are quite narrow, they appear to vary in length from ~500 μm to ~4000 μm, allowing for the freeze-dried hydrogels to be classified as superporous hydrogels [176]. 57 Figure 4.7 Image analysis of a micro-CT scan of a first-generation freeze-dried hydrogel, a) unaltered image from micro-CT scan, b) image converted to black and white, c) image after threshold was applied The results from both the hexane-based porosity test and micro-CT-based porosity analysis are shown in Table 4.1. The results from the hexane-based porosity test indicate that the addition of zirconia into the hydrogel increased its porosity. This is a common finding in hydrogels that have metal oxides incorporated into their structure, and is specifically noted for titanium dioxide [177]. It is hypothesized to occur from the inorganic material disrupting the crosslinking process, resulting in a more porous hydrogel overall, even though there is a decrease in pore width [178]. While the hexane-based porosity values indicated how the zirconia impacts the porosity of the hydrogel, the micro-CT-based values were considered to be more accurate due to the high evaporation rate of hexane. Due to this, the micro-CT-based values were selected for further calculations for the 15% zirconia hydrogel. The densities of the hydrogels with and without zirconia were calculated using the optimal porosity values. As expected, the swollen density of the Sample 2 hydrogel was calculated to be 1.01 g/ml, confirming its ability to sink in gastric fluid. 58 Table 4.1 Hydrogel porosity and density comparison of first-generation freeze-dried hydrogels . Porosity (%) Apparent Density (g/ml) Density (g/ml) Swollen Density (g/ml) Sample 2, without Zirconia 71.2* 0.01 0.033* 0.858 Sample 2, with Zirconia 90.5* | 72.1** 0.153 0.550** 1.01 * Calculated using the hexane-based method ** Calculated using the micro-CT-based method 4.2.1.4 Scanning Electron Microscopy The SEM images (Figure 4.8) show how the zirconia impacted the inner structure of the hydrogel. Highly porous structures are clearly visible in both types of hydrogels; however, it was observed that the zirconia altered the pore structure that developed during the lyophilization process. The hydrogels without zirconia expressed a honeycomb or sponge-like pore structure, whereas the hydrogels with zirconia expressed a flatter and more compressed pore structure, which was assembled in thin layers. Sponge-like pores are a common occurrence in chitosan-based freeze-dried hydrogels [171]. Sponge-like pores occur due to the lyophilization process; as a hydrogel sample freezes, ice crystals will nucleate and grow along the lines of thermal gradients, resulting in a specific pore pattern after the ice has been removed from the sample [179]. While the hydrogels with zirconia still appeared to form based on these thermal gradients, the pores were much less wide (a change of ~200 μm to ~25 μm). 59 Figure 4.8 SEM images of first-generation freeze-dried hydrogels without zirconia, a) 200x magnification, b) 1000x magnification, and with zirconia, c) 200x magnification, d) 1000x magnification Instead of resting inside the pores of the hydrogel, the zirconia particles appeared to be bonded to the polymer network. This may have occurred because the zirconia was weakly bonded to the chitosan through hydrogen bonds, an effect that has been previously witnessed for titanium dioxide [147], [148]. The observation is consistent with the experimental results because if the zirconia was not adequately connected to the polymer network, it would precipitate out of the hydrogel upon submersion in liquid. The effective incorporation of zirconia into the hydrogel is 60 extremely beneficial because even in this highly porous state, additional chemicals or techniques are not required to help bond the zirconia to the polymer network. 4.2.1.5 Fourier Transform Infrared Spectroscopy The FTIR results are shown in Figure 4.9. In the pure chitosan hydrogel, a broad band could be noted at 3417 cm-1 which correlates to O-H and N-H stretching and small sharper bands were seen at 2935 cm-1 and 2365 cm-1 which are attributed to stretching in the CH2 aliphatic groups. The small shoulders at 1153 cm-1 and 1028 cm-1 as well as the peak at 1070 cm-1 are due to the asymmetric stretching of the C-O-C bridge in the glycosidic bonds. C=N stretching in the imine bonds is responsible for the small shoulder present at 1641 cm-1 and the peak present at 1562 cm-1 is due to stretching in the C=C bonds. Additionally, the peak at 1406 cm-1 is from stretching vibrations in the C-N bonds. These various peaks are commonly noted in glutaraldehyde-crosslinked chitosan hydrogels, suggesting that the general hydrogel preparation methodology used was appropriate and that the hydrogels were chemically crosslinked [180], [181], [182]. The sharp peak at 743 cm-1 that was only present in the hydrogel with zirconia is caused by Zr-O bending vibrations [183]. Although small, many of the chitosan-glutaraldehyde peaks could be recognized in the hydrogel with zirconia. The reasoning that the chitosan-glutaraldehyde peaks had very low amplitudes in the hydrogel with zirconia may be that the specific sample of hydrogel taken for analysis was primarily composed of zirconia. At 15% w/v, the mass of the sample after freeze-drying was more than 90% due to the added zirconia. Since the sample must be broken down into a powder for FTIR analysis, there is a high likelihood that the majority of this powder was zirconia, resulting in very little of the chitosan-glutaraldehyde polymer actually being analyzed. 61 Figure 4.9 FTIR spectra of first-generation freeze-dried hydrogels with zirconia (15% w/v) and without zirconia 4.2.1.6 Cell Cytotoxicity Study The cytocompatibility of the hydrogels was assessed under fluorescence microscopy, where living cells were viewed as the colour green and dead cells were viewed as the colour orange or red. The hydrogels were first observed under an optical microscope, where the cells were evaluated for their elongation and growth. This evaluation took place one day after the cells had been seeded onto the surfaces of the hydrogels. It was found that while the cells in the control group had elongated, the cells that were seeded onto the hydrogels had not elongated or multiplied. To determine if the hydrogels merely hindered the multiplication process of the cells causing them to grow slower than normal, the samples were stained with fluorescent dye and the cells were observed under a fluorescent microscope (Figure 4.10). It can be seen that the cells did not elongate in either sample, noted by their small circular shapes. In addition, the 62 colour of the cells was light orange in both samples, indicating that the cells had died. As both samples demonstrated similar cytotoxicity, it can be inferred that the addition of the zirconia was not the sole factor that resulted in the death of the cells. Figure 4.10 Cytotoxicity results, a) first-generation freeze-dried hydrogel sample without zirconia, b) first-generation freeze-dried hydrogel sample with zirconia Based on other studies conducted using glutaraldehyde-crosslinked chitosan, it was concluded that the likely cause of the failed cell toxicity study was excess glutaraldehyde [184], [185]. The hydrogels presented in this subchapter were made with a 1% solution of glutaraldehyde, however, the mass of the glutaraldehyde was calculated to be 12% of the mass of the chitosan when using a 1% chitosan solution. Studies performed by Martínez-Mejía et al. suggest that amounts of glutaraldehyde between 2% and 6% of the mass of the chitosan can be used with no cytotoxic results. Hence, a second generation of freeze-dried hydrogels was developed in which the amount of glutaraldehyde was greatly reduced. 63 4.2.2 Second Generation 4.2.2.1 Hydrogel Synthesis Prior to lyophilization, the hydrogel samples appeared to show similar degrees of gelation after the crosslinking process. Due to the limited amount of glutaraldehyde used and low concentration of chitosan, solid hydrogels were never formed and the samples remained completely liquid until placement in the freezer. After lyophilization, the hydrogels were extracted from the falcon tubes and physical differences could be observed between the different sample sets. While the hydrogels all expressed the same general appearance, some of the zirconia had pooled at the bottom of the falcon tubes and separated from the hydrogels. Hydrogels that were made with higher percentages of glutaraldehyde were able to support larger amounts of zirconia within the hydrogel structure, resulting in an initial difference in weight between the hydrogel samples. There was a noticeable difference in colour that could be observed between the first-generation and second-generation hydrogels as shown in Figure 4.11. The crosslinked chitosan in the first-generation of hydrogels expressed a pink/orange tone, whereas the second-generation of hydrogels had a very pale-yellow colour. It was originally thought that this pink hue developed from a reaction within the crosslinking process, causing the natural colour of chitin to be expressed [186]. As the percentage of chitosan used in both versions of the hydrogel was identical, this colour change must have been due to the difference in glutaraldehyde concentration, suggesting that excess glutaraldehyde created pink and orange tones within the chitosan structure. 64 Figure 4.11 First-generation and second-generation hydrogel comparison, a) first-generation hydrogel without zirconia, b) second-generation hydrogel without zirconia, c) first-generation hydrogel with zirconia, d) second-generation hydrogel with zirconia 4.2.2.2 Swelling Studies The results from the initial 30-minute swelling experiment are listed in Table 4.2. Each of the samples possessed a density higher than that of gastric fluid, even after the excess zirconia had been removed from the hydrogel samples. As the swelling ratio is largely dependent on the initial weight of the sample, the fact that the swelling ability of the hydrogels increased as the amount of zirconia remaining in the hydrogels decreased was to be expected; however, a relationship between the swelling ability and the degree of crosslinking was also present. It can be noted that regardless of the initial weight of the hydrogel, the swollen mass of the hydrogels increased as the amount of the glutaraldehyde decreased, suggesting that samples with less glutaraldehyde could swell more. This confirms our previously stated hypothesis from Section 4.2.1.2 suggesting that hydrogels with a lower crosslinking density have greater swelling capabilities. As it was found that zirconia does not impact the swelling abilities of the hydrogels (Section 4.2.1.2), a new value termed the expectant swelling ratio was calculated for each of the 65 samples to better visualize the relationship between swelling ability and the volume of crosslinker (Figure 4.12). The expectant swelling ratio was defined as the swelling ratio that the hydrogel would have achieved if zirconia was not introduced into the hydrogel sample. The results from Figure 4.12 clearly outline the inverse relationship between the amount of glutaraldehyde and the swelling ratio of the hydrogels. Table 4.2 Swelling ratios of second-generation freeze-dried hydrogel samples in pH 1.57 hydrochloric acid: glutaraldehyde study Volume of Chitosan (ml) Volume of Glutaraldehyde (ml) Mass of Zirconia Added (g) Initial Mass (g) Swollen Mass (g) Swelling Ratio (%) Sample 1 1 0.02 0.15 0.0857 0.83 868 Sample 2 1 0.01 0.15 0.0885 0.9238 943 Sample 3 1 0.005 0.15 0.0609 1.0508 1625 Sample 4 1 0.0025 0.15 0.0533 -* -** * & ** The swollen mass and swelling ratio of Sample 4 is left blank because the sample disintegrated upon submersion in the hydrochloric acid solution. 66 Figure 4.12 Expectant swelling ratios of second-generation freeze-dried hydrogels (samples 1–3, Table 4.2) in pH 1.57 hydrochloric acid (± standard deviation) The degree of crosslinking in Samples 1, 2, and 3 was found to be sufficient as the hydrogels maintained a solid structure within the hydrochloric acid, but this was not the case for Sample 4, which immediately disintegrated upon contact with the solution. Based on these results, it was concluded that the ideal volume of glutaraldehyde for the 1 ml samples was 0.005 ml, or a 0.5% w/w (glutaraldehyde to chitosan) mixture. It was also concluded that the amount of zirconia in the hydrogel network could likely be further decreased as a means of increasing the swelling ratio of the hydrogels. This inspired a second set of experiments to determine the ideal amount of zirconia, the results of which are shown in Table 4.3. 67 Table 4.3 Swelling ratios of second-generation freeze-dried hydrogel samples in pH 1.57 hydrochloric acid: zirconia study Volume of Chitosan (ml) Volume of Glutaraldehyde (ml) Mass of Zirconia Added (g) Initial Mass (g) Swollen Mass (g) Swelling Ratio (%) Sample 5 1 0.005 0.055 0.0605 1.07 1673 Sample 6 1 0.005 0.05 0.05198 1.08 1987 Sample 7 1 0.005 0.045 0.05065 1.12 2107 The results of this set of experiments show that an additional mass of 0.045 grams of zirconia (4.5% w/v) is the ideal amount to add to the hydrogels for maximum swelling efficiency. Efforts made to use less than 0.045 grams resulted in hydrogels with a density lower than gastric fluid and were deemed unsuccessful. Thus, the hydrogel in sample set 7 was determined to have the optimized hydrogel formula and was selected for further study. Three additional swelling studies were performed on the Sample 7 hydrogel: determination of equilibrium swelling time, pH sensitivity, and response to compression. The equilibrium swelling time and pH sensitivity tests were combined into one experiment where the swelling of the hydrogel over time was compared for three different forms of media, the results of which are shown in Figure 4.13. This experiment indicated that the hydrogel samples were fully swollen after less than a minute in solution. Based on this observation, as well as the change in appearance of the hydrogels during the swelling process, it is likely that the hydrogels were completely swollen by the time the sinking process was complete. After the first minute the hydrogel appears to fluctuate mildly in weight; however, this may be due to the continual interference from removing the hydrogel from the solution to take accurate measurements. A difference in swelling ability can be noticed when comparing the pH 7.0 and pH 1.2 and 1.57 solutions. Initially, there is a 328% difference in swelling ratio from the low pH solutions to the 68 high pH solution, representing the same trend that was depicted in the first-generation hydrogels. The results of the trials in simulated gastric fluid were excluded from Figure 4.13 because the pepsin caused the hydrogel to slowly disintegrate due to its chitosanolytic nature [142]. After approximately one minute in the simulated gastric fluid, the pepsin began to dissolve the hydrogel, causing small segments of zirconia to separate from the hydrogel sample. While this indicated that the hydrogel was performing as desired and could be broken down in the stomach prior to moving through the rest of the gastrointestinal tract, it made the data from re-weighing the hydrogel sample unreliable. For the purposes of this study, it was assumed that the swelling response of the hydrogel in simulated gastric fluid was similar to that of the hydrogel in pH 1.2 hydrochloric acid based on the studies done with the first-generation hydrogels and the identical pH values. Figure 4.13 Effect of solution pH on swelling ability of optimized hydrogel To assess the hydrogel’s response to compression, hydrogel samples were flattened to approximately 15% of their original volume (as shown in Figure 4.14, a)) and assessed for 69 swelling ability in pH 1.57 hydrochloric acid (Figure 4.14, b)). It was originally hypothesized that removing the free air from the porous network would impede the swelling abilities of the hydrogels; however, experimental results demonstrated that their swelling abilities improved as a result of the compression. The compressed hydrogels were able to swell ~400% more than the uncompressed hydrogels in the same time span. Based on similar findings from other researchers, it is thought that this increase in swelling ability occurs due to a change in the porous structure of the hydrogels that happens during the compression process [187]. Additionally, the zirconia may be preventing the capillary channels within the hydrogel from completely sealing shut as the hydrogel is flattened, similar to how disintegrants support compressed superporous hydrogels used for drug delivery [188]. This additional support is likely the reasoning as to why the swelling times of the hydrogels were not increased after being compressed. The improved swelling ability of the compressed hydrogels is extremely beneficial when considering placing the hydrogels inside of gel capsules for easier transport to the stomach. 70 Figure 4.14 Compressed versus uncompressed hydrogels, a) dimensions, b) swelling abilities in pH 1.57 hydrochloric acid (± standard deviation) 4.2.2.3 Porosity Measurement and Calculation of Density The hydrogels both with and without zirconia were highly porous, showing average porosities of 91% and 88%, respectively, using the hexane-based method and 87% using the micro-CT-based method (Table 4.4). The different porosity values were compared using ANOVA and the differences between the means were not found to be significant (𝛼 = 0.05), suggesting that the introduction of zirconia into the hydrogel network at a 4.5% w/v concentration did not greatly impact the porosity of the hydrogel. The estimated swollen density for the hydrogel samples without zirconia implied that the hydrogels did not need the addition of zirconia to sink in gastric fluid; however, experimental results proved that this was not the case. This suggests that the estimated values for the swollen hydrogel densities are likely incorrect by approximately 4%. The minor discrepancies between the predicted and observed swelling densities can probably 71 be attributed to the small initial masses of the hydrogel samples, causing minor variations in weight to be amplified in the results. Table 4.4 Hydrogel porosity and density comparison of second-generation freeze-dried hydrogels Porosity (%) Apparent Density (g/ml) Density (g/ml) Swollen Density (g/ml) Sample 7, without Zirconia 88* 0.0132 0.11* 1.04 Sample 7, with Zirconia 91* | 87** 0.0507 0.39** 1.09 * Calculated using the hexane-based method ** Calculated using the micro-CT-based method An image from the micro-CT scan is shown in Figure 4.15. It can be seen that the pores developed in the second-generation hydrogels were less linear and more open than in the first-generation of hydrogels, possessing pores ranging from 100 – 300 μm in size. This change in pore shape was likely primarily due to the decrease in zirconia from the first-generation hydrogels to the second-generation hydrogels, as the cross-section depicted here seems to resemble the SEM image of the first-generation hydrogel without zirconia. The increase in pore width likely contributed to the increased swelling abilities of the second-generation hydrogels [189]. 72 Figure 4.15 Image analysis of a micro-CT scan of a second-generation freeze-dried hydrogel, a) unaltered image from micro-CT scan, b) image converted to black and white, c) image after threshold was applied 4.2.2.4 Scanning Electron Microscopy The inner structures of the hydrogels are shown in Figure 4.16. Both types of hydrogels are shown to have highly interconnected porous structures made with incredibly thin polymer layers. Unlike the first generation of hydrogels, the addition of zirconia did not seem to change the type of pores that developed in the hydrogel, likely due to the dramatic decrease in zirconia concentration. Although the hydrogels still possessed an incredibly spongy structure, the hydrogel walls appeared to be much thinner and flakier in the second generation of hydrogels. As the only difference between the first- and second-generation hydrogels without zirconia was the concentration of glutaraldehyde, this difference in pore structure demonstrated how the degree of crosslinking affects the overall structure of the hydrogel. 73 Figure 4.16 SEM images of second-generation freeze-dried hydrogels without zirconia, a) 1000x magnification, c) 5000x magnification), and with zirconia, b) 1000x magnification, d) 5000x magnification 4.2.2.5 Fourier Transform Infrared Spectroscopy Figure 4.17 shows the FTIR results of the second-generation hydrogels and the zirconia powder. The same peaks that were recognized in the original FTIR results of the first-generation hydrogels are all present, but the chitosan-glutaraldehyde peaks are more pronounced in the second-generation zirconia filled hydrogel compared to the first. Additionally, the same peak at 743 cm-1 due to the Zr-O bending vibrations is visible in the 4.5% zirconia hydrogel and confirmed by the analysis of pure ZrO2. Minor noise in the FTIR spectra occurred at approximately 2300 cm-1, which is likely due to the asymmetric stretching mode of CO2 [190]. 74 Figure 4.17 FTIR spectra of pure zirconia and second-generation freeze-dried hydrogels with zirconia (4.5% w/v) and without zirconia 4.2.2.6 X-Ray Diffraction Figure 4.18 shows the XRD patterns of pure zirconia, the chitosan hydrogel without zirconia, and the chitosan hydrogel with zirconia. Zirconia has a crystal structure, represented by many sharp peaks over the 2-theta range. These peaks in the zirconia spectrum can be identified at 24.2º, 28.7º, 31.5º, 34.8º, 38.5º, 40.8º, 44.4º, 49.5º, 54.0º, 56.0º, and 60.5º, corresponding to [011], [-111], [111], [200], [120], [-112], [211], [022], [220], [310], [131], and [120] planes, respectively, which are indicative of monoclinic zirconium dioxide [191]. The XRD spectrum of the chitosan hydrogel made without zirconia does not show any peaks due to its amorphous structure and follows the standard general shape for glutaraldehyde-crosslinked chitosan [180]. The peaks associated with the crystal structure of the zirconia can be observed in the XRD spectra of the hydrogel with 4.5% zirconia. As the peaks did not appear to differ in any way, it 75 implied that the crystalline structure of the zirconia was maintained during the hydrogel preparation process. Figure 4.18 XRD data of pure zirconia and second-generation freeze-dried hydrogels with zirconia (4.5% w/v) and without zirconia 4.2.2.7 Cell Cytotoxicity Study The cytocompatibility of the hydrogels was assessed under fluorescence microscopy where living cells were visible as the colour green and dead cells were visible as the colour orange/red. Images of the stained hydrogels at days 1 and 5 are present in Figure 4.19, a). The cells in both hydrogels with and without zirconia experienced elongation and growth over the 5-day period. The percentage of cell viability of the 3T3 cells was determined for both samples by image analysis and was calculated to be upwards of 80% (Figure 4.19, b)). The viability of the 3T3 cells in the control group did not significantly differ from the viability of the cells within the hydrogel samples, indicating that both hydrogel varieties are cytologically compatible. 76 Additionally, there was no significant difference in cell viability between hydrogels containing zirconia and hydrogels not containing zirconia, confirming that zirconia was not impacting the cytotoxicity of the samples. These results suggested that the hydrogel has strong potential for in-vivo biocompatibility and may be considered for further study. Figure 4.19 Cytotoxicity results, a) live/dead staining of 0% and 4.5% zirconia hydrogels at day 1 and day 5, b) cell viability data (± standard deviation) 4.3 Chapter 4 Summary In this chapter, the results from the various hydrogel swelling tests and characterization methods were discussed. It was found that the swelling capabilities of the gas-blown hydrogels were very poor due to the dense, low-porosity hydrogel structure that occurred as a result of the oven-based drying. The porosity and swelling capabilities of the hydrogels dramatically increased by using the freeze-drying technique, initially increasing the swelling abilities by more than 350% and the porosity by ~20%. It was found that by increasing the concentration of chitosan used in the hydrogel, the swelling capabilities and the density were negatively 77 impaired, resulting in a 1% w/v chitosan concentration being selected as the ideal concentration for this study. In the first-generation hydrogels, a zirconia concentration of 15% w/v was required to satisfy the density requirements and a maximum swelling ratio of ~430% was achieved. Cell cytotoxicity analysis outlined that the concentration of glutaraldehyde in the first-generation hydrogels was too high, resulting in the hydrogels both with and without zirconia exhibiting cytotoxic behaviour. To combat this, the hydrogels in the second generation were formed with significantly reduced quantities of glutaraldehyde, and the impact of glutaraldehyde was studied. It was found that by decreasing the quantity of glutaraldehyde, the porosities and swelling abilities of the hydrogels were dramatically increased, and less zirconia was required to satisfy the density requirements. The maximum swelling ratio increased to ~2100%, which increased even further to ~2500% if the hydrogel was compressed prior to testing. The amount of zirconia was reduced from a 15% w/v concentration to a 4.5% w/v concentration, and the porosity increased to ~90%. A cell cytotoxicity assay on the optimized second-generation hydrogels indicated that the hydrogels did not exhibit any cytotoxic effects, suggesting their potential for in-vivo biocompatibility. 78 Chapter 5: Conclusions and Future Work 5.1 Summary The goal of this study was to develop a strategy for relocating the contents of the gastric acid pocket away from the LES to prevent heartburn. To achieve this, a hydrogel composite was developed that is capable of swelling with the highly acidic contents of the gastric acid pocket and subsequently sinking to the bottom of the stomach to relocate the contents further away from the esophagus. The hydrogel composite is a chitosan/zirconia-based hydrogel that has a swollen density of ~1.05 g/ml and a swelling capacity of ~2575% after being in a compressed state. It was found that the bioceramics titanium dioxide and zirconium dioxide could be effectively incorporated into the hydrogel structure without the need for additional polymer coatings or bonding techniques. The bonding of these high-density materials was not affected by the degree of swelling of the hydrogels, as these materials remained bound within the hydrogels during and following the swelling process. It was also discovered that the crosslinking density of the hydrogels could be reduced to a 0.5% w/w concentration of glutaraldehyde to chitosan before the hydrogel composites lost structural integrity in gastric pH. The optimized hydrogels were found to swell to their maximum abilities after less than a minute of submersion within hydrochloric acid solutions, demonstrating their incredibly fast response times. Additionally, the pH-sensitive nature of chitosan was observed through a difference in swelling of ~300% between neutral and acidic media. The biocompatibilities of the hydrogels were assessed through a live/dead cell cytotoxicity study using 3T3 mouse fibroblast cells and it was found that the optimized hydrogels both with and without zirconia exhibit excellent biocompatibility, yielding cell viability of above 85% after five days. As the average volume of the gastric acid pocket is 100 ml [17], this suggests that a hydrogel mass of approximately 4 grams would be required to completely relocate the entirety of the gastric acid pocket, equivalent to three times the mass of a standard calcium carbonate antacid tablet. This study outlines the potential for this hydrogel formulation to be further studied regarding heartburn 79 management, specifically acting as an alternative treatment for GERD in which the natural pH of the gastric fluid can be maintained and potential long-term side effects of continual PPI use can be avoided. 5.2 Contributions The contributions from this thesis are as follows: • A potential alternative technique for the treatment of heartburn involving the relocation of the gastric acid pocket was introduced that, to the best of the author’s knowledge, has not been previously researched. • Zirconium dioxide was successfully incorporated into a highly swellable chitosan-based hydrogel without the need for additional chemicals or techniques. • A high-density, biocompatible, chitosan-based hydrogel able to swell with 25 g/g of gastric fluid in one minute was developed to act as an alternative treatment for heartburn. 5.3 Future Works Although this work introduces a hydrogel formulation for the potential relocation of gastric acid within the stomach, there are a few limitations that should be mentioned and researched further in future studies. First, while achieving a swelling ratio of > 2500% is considered excellent for this work, if compressed into pills, it would require approximately seven #000-sized capsules of hydrogel to relocate the entirety of the gastric acid pocket. Improving the swelling ratio would reduce the amount of hydrogel required to enact this effect and could subsequently increase consumer acceptance. This could potentially be accomplished by combining the gas blowing and freeze-drying techniques or by adding additional swelling compounds into the hydrogel. Alternatively, waterproof sprays or coatings that are dissolvable at low pH values could be explored to evaluate the potential for creating an oral suspension of hydrogel particles, which 80 may be preferable to certain individuals. Second, due to the source of chitosan, there is a potential that this hydrogel would elicit an allergic response from those who have shellfish allergies. The potential for this response should be studied, and if the hydrogel proves to be efficacious in the future, a chitosan-free version should be produced to provide an alternative for those who are afflicted. Finally, the effectiveness of the hydrogel to relocate the gastric acid pocket and alleviate heartburn cannot be reliably studied without in-vivo testing. Due to this, additional in-vitro testing should be done to confirm that the amount of zirconia and glutaraldehyde will not cause distress to the gastrointestinal system. The hydrogels should also be studied using a human gastric simulator (such as the one developed by Kong and Singh [192]) to observe how the peristaltic forces within the stomach affect the rate of digestion of the hydrogels. After both are complete, in-vivo studies using animal models could be performed and the variance in pH between different regions of the stomach could be examined to observe the relocation of the gastric acid pocket. 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Thesis/Dissertation
eng
Mechanical Engineering
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Hoorfar, Mina
High-density biocompatible hydrogels for in-vivo gastric acid relocation
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