Siddiqui, Nabil Ahmad (2017) Development of a boronic acid sensor-based glucose-responsive

77 Pages·2017·1.01 MB·English

by  Christine Middleton

DEVELOPMENT OF A BORONIC ACID SENSOR-BASED
GLUCOSE-RESPONSIVE NANOPARTICULATE
INSULIN DELIVERY SYSTEM

NABIL AHMAD SIDDIQUI
MASTER OF PHARMACY

Thesis submitted to The University of Nottingham for the degree of
Master of Philosophy

September 2016

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Abstract
Diabetes is one of the most common chronic diseases in the world and its
incidence is on the rise. Maintenance of continuous normoglycaemic conditions
is the key goal for the management of both type 1 and type 2 diabetes in
patients. Glucose responsive insulin delivery (GRID) systems have the potential
to act as artificial pancreas as they can modulate the insulin release relative to
external glucose concentrations. GRIDs can not only achieve tighter glycaemic
control by preventing both hypo- and hyperglycaemia, but also eliminate the
need for frequent finger-stick glucose tests and multiple daily insulin injections.
In this research, we examined the selectivity of and insulin release from two
boronic acids (2-formyl-3-thienylboronic acid (FTBA) and 4-formylphenylboronic
acid (FPBA)) to glucose when conjugated to chitosan as nanoparticles. Adsorption
of glucose to BA: chitosan conjugates was dose-dependent up to 1:1 at 35 and
42% for FPBA and FTBA respectively but the FTBA conjugates adsorbed more
glucose and fructose at respective FPBA ratios. The affinity of both BA conjugates
to glucose decreased with increase in BA ratio. On the other hand, the affinity
of both BA conjugates for fructose decreased from ratio 1:1 to 2:1 then rose
again at 3:1. Insulin release from FPBA nanoparticles (FPBAINP) and FTBA
nanoparticles (FTBAINP) were both concentration-dependent within
glyceamically relevant values (1-3mg/ml glucose and 0.002mg/ml fructose).
Furthermore, the total amounts of insulin released from FPBAINP in both the
media were higher than from FTBAINP. Both FPBAINP and FTBAINP have the
potential for development as a glucose-selective insulin delivery system in
physiological settings.

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Acknowledgements
I would like to express my utmost gratitude to Professor Nashiru Billa for choosing
me as one of his postgraduate students and providing me with guidance
throughout this exceptional research project. I also thank all the technicians for
providing me with the necessary training in handling various instruments
throughout my MPhil degree.

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List of contents
Abstract 2
Acknowledgements 3
Abbreviations 7

CHAPTER 1 – BACKGROUND AND OBJECTIVES 8
1.1. Overview of diabetes 8
1.2. Clinical manifestations of diabetes 9
1.3. Insulin 10
1.4. Current treatment options for diabetes 10
1.4.1. Improved technologies for insulin replacement therapy 11
1.4.2. Alternative routes of insulin delivery 12
1.5. Nanotechnology in medicine 13
1.5.1. Diabetes management with nanotechnology 13
1.6. Glucose-responsive insulin delivery (GRID) 14
1.6.1. Glucose oxidase 15
1.6.2. Glucose-binding protein (GBP) 15
1.6.3. Boronic acids 16
1.7. Polymer-based nanoparticulate insulin delivery systems 17
1.7.1. PLA nanoparticles 18
1.7.2. PLGA nanoparticles 18
1.7.3. Dextran nanoparticles 19
1.7.4. Chitosan nanoparticles 19
1.8. Objectives of the research 20

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CHAPTER 2 - SYNTHESIS AND CHARACTERISATION OF 22
CHITOSAN-BORONIC ACID CONJUGATES
2.1. Introduction 22
2.2. Materials and methods 26
2.2.1. Materials 26
2.2.2. Synthesis and purification of boronic acid- 26
chitosan conjugates
2.2.3. FTIR analyses 27
2.2.4. DSC analyses 27
2.2.5. Glucose adsorption studies 28
2.2.6. Investigation of boronic acid selectivity for 30
diols (glucose and fructose)
2.2.7. Statistical analyses 32
2.3. Results and Discussion 32
2.3.1. Characterisation of conjugates 32
2.3.2. Selectivity of boronic acids for glucose and fructose 42
2.4. Conclusions 48

CHAPTER 3 - INSULIN LOADED BORONIC ACID SENSOR-BASED 49
CHITOSAN NANOPARTICLES
3.1. Introduction 49
3.2. Materials and methods 50
3.2.1. Materials 50
3.2.2. Investigation of conditions for the formation of CSNP 51
3.2.3. Formulation of insulin loaded boronic acid- 51
functionalised chitosan-TPP nanoparticles
3.2.4. Physicochemical characterisation of nanoparticles 52
3.2.5. HPLC analyses for insulin content 52
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3.2.6. Evaluation of encapsulation efficiencies of nanoparticles 53
3.2.7. In vitro insulin release studies 53
3.3. Results and Discussion 54
3.3.1. Optimisation of conditions for CSNP preparation 54
3.3.2. Preparation of insulin loaded nanoparticles 56
3.3.3. Percentage encapsulation efficiency (EE%) 60
3.3.4. In vitro insulin release in various media 62
3.4. Conclusions 66

Future Work 67
References 68

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Abbreviations
GRID Glucose-responsive insulin delivery
BA Boronic acid
FPBA 4-formylphenylboronic acid
FTBA 2-formyl-3-thienylboronic
FTIR Fourier transform infrared spectroscopy
DSC Differential scanning calorimetry
HPLC High Performance Liquid Chromatography
NAD+ Nicotinamide adenine dinucleotide (oxidised)
NADH Nicotinamide adenine dinucleotide (reduced)
UV Ultraviolet (spectroscopy)
CS Chitosan
TPP Tripolyphosphate
INP Insulin Nanoparticle

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CHAPTER 1
BACKGROUND AND OBJECTIVES
1.1. Overview of diabetes
Diabetes mellitus is a medical disorder involving high blood glucose levels –
hyperglycaemia [1]. As one of the most common chronic diseases in almost every
country in the world, the incidence of diabetes is on the rise. The number of
diabetic patients is expected to cross 400 million [2] and the total worldwide
health expenditure for diabetes is estimated to be in excess of US$560 billion
per year by 2030 [3]. This shows how much of an economic burden diabetes poses
to be for national healthcare systems globally. Diabetes mellitus is broadly
categorised into the three most common types – type 1, type 2, and gestational
diabetes.
Type 1 diabetes, formerly known as juvenile diabetes, is usually diagnosed in
children and young adults, although it can appear at any age. It accounts for 10%
of all diabetes mellitus cases. Type 1 diabetes is the result of insulin deficiency
in the body which is caused by an autoimmune condition in the affected
individuals, whereby insulin-producing β-cells in the pancreas are destroyed by
T-cells, eventually leading to hyperglycaemia [4]. People with type 1 diabetes
need to take insulin every day to stay alive.
Type 2 diabetes is often categorized as a ‘lifestyle disease’ and manifests as a
result of the body’s inability to effectively utilise insulin (insulin resistance) and
lack of insulin production, leading to high blood glucose levels. This form of
diabetes is often associated with lack of physical activity and obesity [5]. For
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type 2 diabetes, initial treatment focuses on delaying disease progression via
exercise and control of meals. Furthermore, patients are prescribed oral and/or
injectable medications to improve their insulin production and function.
However, insulin is required as the body’s own insulin production diminishes
eventually.
Gestational diabetes develops in some pregnant women, however, most of the
time, this type of diabetes goes away after the baby is born. People with
gestational diabetes have a greater chance of developing type 2 diabetes later
in life. Sometimes diabetes diagnosed during pregnancy is actually type 2
diabetes.
1.2. Clinical manifestations of diabetes
Typical symptoms of diabetes are listed as follows:
 Increased frequency of urination
 Excessive thirst
 Extreme hunger – even though patient is having sufficient food
 Feeling very tired all the time
 Cloudy vision
 Slow healing of minor injuries
 Unexplained weight loss even though patient is eating more
 Tingling or numbness in the hands and/or feet
Some people may experience only a few of the symptoms listed above. A lot of
people with type 2 diabetes experience very mild to no symptoms and hence
remain incognizant of their medical condition.
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1.3. Insulin
It is a 51-amino acid polypeptide hormone produced by the β-cells of the islets
of Langerhans in the pancreas. The amino acids are distributed in two chains of
21 and 30 amino acids. The chains are linked together by two disulphide bridges.
Insulin controls the glucose levels in blood by inducing the liver and muscle cells
to take up glucose from the blood [6]. In type 1 diabetes, an autoimmune
response causes the destruction of β-cells by T-cells which leads to insulin
deficiency and high blood glucose levels. Insulin, like other proteins and
peptides, is unstable in the gastrointestinal tract and hence oral delivery is not
feasible. Consequently, subcutaneous injections of insulin are used [7]
1.4. Current treatment options for diabetes
Early detection and treatment of diabetes can reduce the risk of developing the
complications of diabetes. Maintenance of continuous normoglycaemic
conditions (70–140 mg per dl or 4–8 mM of glucose) is the key goal for the
management of both type 1 and type 2 diabetes in patients [8]. This level of
glucose is currently managed by subcutaneous injections, usually by the patients
themselves or by trained professionals. Hyperglycaemia (high blood glucose
levels), if left untreated for a prolonged period of time, can lead to medical
complications such as blindness, heart and kidney failure, nerve degeneration,
and foot ulceration leading to amputation [9]. Contrarily, an excess of insulin in
blood can lead to hypoglycaemia (low blood glucose levels) which can result in
seizures, unconsciousness or even death [10].
Insulin replacement therapy is prescribed for the management of Type 1 diabetes
[11]. For type 2 diabetes, lifestyle changes and oral medications are the initial
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