development and characterisation of medicated wound dressings for chronic wound healing

259 Pages·2014·10.09 MB·English

DEVELOPMENT AND CHARACTERISATION OF
MEDICATED WOUND DRESSINGS FOR CHRONIC WOUND
HEALING

By

HARSHAVARDHAN VILASRAO PAWAR {BPharm, MRes (Pharm. Sci.)}

A thesis submitted in partial fulfilment of the requirements of the University of
Greenwich for the degree of Doctor of Philosophy

July, 2013

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DECLARATION

“I certify that this work has not been accepted in substance for any degree, and is not
concurrently being submitted for any degree other than that of Doctor of Philosophy being
studied at the University of Greenwich. I also declare that this work is the result of my own
investigations except where otherwise identified by references and that I have not plagiarised the
work of others”.

………………………………

HARSHAVARDHAN PAWAR (CANDIDATE)

___________________________________________________________________________

PHD SUPERVISORS

DR. JOSHUA BOATENG …………………………………………..

DR JOHN TETTEH …………………………………………...

FEBRUARY 2014

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ACKNOWLEDGEMENTS
First and foremost, I would like to take this opportunity to thank the almighty God for
His loving kindness and faithfulness in sustaining my life all these years. To the greatest extent
thanks to Dr. Joshua Boateng for giving me such opportunity to work on this project and thanks
for your steadfast support, encouragement, understanding and guidance. Your insight was pivotal
in allowing me to complete my PhD. I would like to extend my deepest gratitude and
appreciation to my second supervisor Dr. John Tetteh for his advice, guidance and many critical
discussions. I would like to thank my parents who always provided constant love, freedom and
support for doing the PhD.
I would also like to acknowledge the University of Greenwich for nominating me for
ORSAS funding and the School of Science for making up the difference in my tuition fees to
complete my PhD. I am grateful to Dr Isaac Ayensu and Dr Farnoosh Kianfar, for being my
second pair of eyes giving me suggestions in exploring new ideas. I would like to acknowledge
Dr Concetta Giovino, Dr Nazanin Zand and Dr Arun Kumar Kotha for their selfless help
throughout my PhD. I am also grateful to Dr. Ian Slipper for all the SEM and XRPD techniques
and Devyani Amin for the HPLC analysis and troubleshooting of the HPLC and Samantha Lewis
for her help during microbiological studies. Thanks also to all other staff and technicians for
supporting me during the experimental work.
Last but not least I would like to thank to Amit Patil, Vilas Beloshe, Nilesh Manikwar,
Anniruddha Bhagurkar, Shankar Manjare and Prithviraj Patil and roommates who have given me
constant support and financial help when I asked. Big thanks to Amit Bhosale for cheering me up
every time throughout my PhD. You guys are amazing, without all of you it will not be possible
to pass this journey. I am obliged to express my gratitude to my research group members and all
the persons who met me on the path of this journey and helped me directly and indirectly to
complete my studies.

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ABSTRACT
Chronic wounds are difficult to heal and exhibit physiological features including
prolonged inflammatory phase, mixed bacterial flora resistance and formation of biofilms,
ineffectiveness of topical antimicrobials and high volumes of wound exudate. Polymeric gels of
Polyox (POL) and blends of POL with carrageenan (CAR), chitosan (CS),
hydroxypropylmethylcellulose (HPMC) and sodium alginate (SA) in different weight ratios were
used to prepare films by the solvent casting technique and evaluated using scanning electron
microscopy (SEM), X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC)
and Fourier transform spectroscopy (FTIR). The same gels were analysed using DSC to develop
an optimum lyophilisation cycle with or without an annealing step to obtain freeze dried wafers
of POL-CAR and POL-SA. Films prepared from POL were non-transparent and showed
spherulitic crystallisation, however POL blends with CAR and SA (75/25 and 50/50 weight
ratios respectively) showed improved flexibility and transparency with reduced spherulitic
crystallisation (i.e. homogeneous surface) through hydrogen bonding between POL and/or CAR
and SA. Addition of annealing step -25°C resulted in formulations with porous surface
morphology for both POL-CAR and POL-SA wafers. Annealing (wafers) and addition of
glycerol (GLY) (films) resulted in improved mechanical properties expected to withstand the
mechanical stresses occurring during day-to-day activities and whilst flexible enough to prevent
potential damage to newly formed tissue. Tough and flexible films were obtained by the addition
of 9%w/w and 20% w/w GLY in POL-SA and POL-CAR respectively. Further, the POL-CAR
and POL-SA films and wafers were loaded with 5-15%w/w of diclofenac (DLF) and 15-30%
w/w of streptomycin (STP). Furthermore POL-CAR and POL-SA blank (BLK) and drug loaded
(DL) films and wafers were analysed for swelling, mucoadhesion (in presence of normal and
viscous simulated wound fluid), in vitro drug dissolution and anti-bacterial activity and
compared against marketed medicated wound dressings. Addition of drug (STP and DLF)
resulted in fair transparency of films and decreased porosity of wafers with existence of sodium
sulphate which affected general performance of the films and wafers in terms of swelling,
mucoadhesion, and antimicrobial activity. BLK plasticised (GLY) films and BLK annealed
wafers showed higher swelling capacities compared to DL films and wafers. Neither DL films
nor wafers showed 100 % release of the incorporated STP and DLF due to the formation of
sodium sulphate which reduced hydration. Findings also showed that POL-SA films and wafers
were effective against normal exudate whereas POL-CAR films and wafers were effective for
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viscous exudate to achieve better bioavailability and prolonged retention time. Multivariate data
analysis of mucoadhesion showed slower rate of mucin diffusion into POL-CAR films and
wafers compared to POL-SA films and wafers. The formulated films, wafers and marketed
dressing showed antibacterial efficacy against 105 CFU/ml of S. aureus, P. aeruginosa and E.
coli. STP and DLF present in both films and wafers acted synergistically and showed better
antimicrobial activity than marketed dressings. Film dressing allows ease of application and due
to fair transparency and flexibility whereas wafer dressings are useful to control exudate and
both can maintain a moist environment. Combination of STP and DLF within a single dressing is
expected to help to treat and prevent wound infections whereas DLF can help to relieve pain and
inflammation associated with injury.

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TABLE OF CONTENTS
DECLARATION ............................................................................................................................ ii
ACKNOWLEDGEMENTS........................................................................................................... iii
ABSTRACT ...................................................................................................................................iv
TABLE OF CONTENTS ...............................................................................................................vi
LIST OF FIGURES ....................................................................................................................... xv
LIST OF TABLES........................................................................................................................xxi
ABBREVIATIONS .................................................................................................................. XXII
LIST OF PUBLICATIONS AND CONFERENCE PROCEEDINGS ....................................... xxv
CHAPTER 1 : GENERAL INTRODUCTION AND LITERATURE REVIEW ........................... 1
1.1 OVERVIEW ......................................................................................................................... 1
1.2 TYPES OF WOUNDS ......................................................................................................... 3
1.2.1 Clinical context ................................................................................................................. 4
1.2.1.1 Simple wounds .......................................................................................................... 4
1.2.1.2 Complex wounds ....................................................................................................... 4
1.2.2 Nature of repair process .................................................................................................... 5
1.2.2.1 Acute wounds ............................................................................................................ 5
1.2.2.2 Chronic wounds ......................................................................................................... 5
1.2.2.3 Exuding wounds ........................................................................................................ 7
1.2.3 Skin layers and area of skin layers .................................................................................... 8
1.2.3.1 Superficial wounds .................................................................................................... 8
1.2.3.2 Partial thickness wounds ........................................................................................... 8
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1.2.3.3 Full thickness wounds ............................................................................................... 8
1.2.4 Wounds based on contamination ...................................................................................... 9
1.2.4.1 Clean wounds ............................................................................................................ 9
1.2.4.2 Clean contaminated wounds .................................................................................... 10
1.2.4.3 Contaminated wounds ............................................................................................. 10
1.2.4.4 Dirty wounds ........................................................................................................... 10
1.3 WOUND HEALING AND STAGES INVOLVED IN HEALING .............................. 10
1.3.1 Moist wound healing....................................................................................................... 10
1.3.2 Haemostasis .................................................................................................................... 11
1.3.3 Inflammation ................................................................................................................... 12
1.3.4 Proliferation .................................................................................................................... 13
1.3.5 Remodelling and scar maturation ................................................................................... 14
1.4 BACTERIAL WOUND INFECTION ............................................................................ 14
1.4.1 Management of wound infection .................................................................................... 17
1.4.2 Role of topical antimicrobials ......................................................................................... 17
1.4.3 Exudate associated with acute and chronic wounds ....................................................... 25
1.5 WOUND DRESSINGS ..................................................................................................... 27
1.5.1 Traditional wound dressings ........................................................................................... 27
1.5.2 Modern wound dressings ................................................................................................ 29
1.5.2.1 Film dressings .......................................................................................................... 30
1.5.2.2 Hydrogel dressings .................................................................................................. 30
1.5.2.3 Foam dressings ........................................................................................................ 30
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1.5.2.4 Hydrocolloid dressings ............................................................................................ 31
1.5.2.5 Alginate dressings.................................................................................................... 31
1.5.2.6 Medicated dressings ................................................................................................ 32
1.5.2.7 Freeze dried wafers .................................................................................................. 33
1.5.3 Biological wound dressings ............................................................................................ 35
1.6 FUNCTIONAL PROPERTIES OF MOIST WOUND DRESSINGS ......................... 35
1.6.1 Bio (muco)adhesion ........................................................................................................ 35
1.6.2 Swelling and drug release ............................................................................................... 37
1.6.2.1 Theories of polymer erosion .................................................................................... 37
1.6.2.2 Theories of polymer erosion/degradation ................................................................ 38
1.6.2.3 Drug release mechanisms ........................................................................................ 39
1.7 POLYMERS, EXCIPIENTS AND DRUGS USED IN THIS PROJECT ................... 39
1.7.1 Polyethylene oxide (Polyox™) ........................................................................................ 39
1.7.2 Carrageenan (CAR) ........................................................................................................ 40
1.7.3 Sodium alginate (SA) ...................................................................................................... 41
1.7.4 Chitosan (CS) .................................................................................................................. 42
1.7.5 Hydroxypropylmethylcellulose (HPMC)........................................................................ 43
1.7.6 Plasticizer ........................................................................................................................ 43
1.7.7 Model drugs .................................................................................................................... 45
1.7.7.1 Diclofenac sodium (DLF) ........................................................................................ 45
1.7.7.3 Streptomycin sulphate (STP) ................................................................................... 45
1.8 FORMULATION AND CHARACTERISATION ........................................................ 46
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1.8.1 Freeze drying .................................................................................................................. 46
1.8.2 Solvent film casting ........................................................................................................ 47
1.8.3 Texture analysis characterisation .................................................................................... 48
1.8.4 Spectroscopic and chemometric data analysis ................................................................ 50
1.9 AIMS AND OBJECTIVES .............................................................................................. 51
CHAPTER 2 : PREPARATION AND PHYSICO-CHEMICAL CHARACTERISATION
OF POLYOX™ BASED SOLVENT CAST FILMS ................................................................ 53
2.1 INTRODUCTION ............................................................................................................ 53
2.2 MATERIALS .................................................................................................................... 54
2.3 METHODS ........................................................................................................................ 55
2.3.1 Preliminary formulation development ............................................................................ 55
2.3.2 Preparation of blank (BLK) POL-CAR and POL-SA films ........................................... 56
2.3.3 Preparation of drug loaded (DL) POL-CAR and POL-SA films .................................... 56
2.3.4 Scanning electron microscopy (SEM) ............................................................................ 57
2.3.5 Differential scanning calorimetry (DSC) ........................................................................ 58
2.3.6 X-ray powder diffraction (XRPD) .................................................................................. 58
2.3.7 Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) ...... 59
2.3.8 Texture analysis (TA) ..................................................................................................... 59
2.4 RESULTS AND DISCUSSION ....................................................................................... 61
2.4.1 Preliminary formulation development ............................................................................ 61
2.4.2 Scanning electron microscopy (SEM) ............................................................................ 66
2.4.3 Differential scanning calorimetry (DSC) ........................................................................ 69
2.4.4 X-ray powder diffraction (XRPD) .................................................................................. 73
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2.4.5 Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) ...... 75
2.4.6 Texture analysis (TA) ..................................................................................................... 80
2.5 CONCLUSIONS ............................................................................................................... 84
CHAPTER 3 : DEVELOPMENT, OPTIMISATION AND PHYSICO-CHEMICAL
CHARACTERISATION OF MEDICATED LYOPHILISED WAFER DRESSING. ......... 86
3.1 INTRODUCTION ............................................................................................................ 86
3.2 MATERIALS .................................................................................................................... 87
3.3 METHODS ........................................................................................................................ 87
3.3.1 Preparation of POL-CAR and POL-SA [blank (BLK) and drug loaded (DL)] gels ...... 87
3.3.2 Freeze drying cycle development with annealing step ................................................... 87
3.3.3 Lyophilisation ................................................................................................................. 88
3.3.4 Scanning electron microscopy (SEM) ............................................................................ 89
3.3.5 Mechanical properties of wafers (TA) ............................................................................ 90
3.3.6 X-ray diffraction (XRD) ................................................................................................. 90
3.3.7 Differential scanning calorimetry (DSC) ........................................................................ 91
3.3.8 Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) ..... 91
3.4 RESULTS AND DISCUSSION ....................................................................................... 92
3.4.1 Freeze drying cycle development with annealing step ................................................... 92
3.4.2 Freeze-drying formulation development and optimisation ............................................. 93
3.4.3 Scanning electron microscopy (SEM) ............................................................................ 94
3.4.4 Mechanical properties of wafers (TA) ............................................................................ 96
3.4.5 X-ray diffraction (XRD) ................................................................................................. 99
3.4.6 Differential scanning calorimetry (DSC) ...................................................................... 100
x