MICROSTRUCTURAL  UNDERSTANDING  OF   HYDROCOLLOID  AND  MIXED  HYDROCOLLOID   SYSTEMS  FOR  BIOMEDICAL  APPLICATIONS       Abigail  Belinda  Norton       A  thesis  submitted  to   The  University  of  Birmingham   for  the  degree  of   DOCTOR  OF  PHILOSOPHY       Department  of  Chemical  Engineering     College  of  Physical  and  Engineering  Sciences     The  University  of  Birmingham   2016 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. 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Abstract     Hydrocolloid   materials   have   been   used   for   some   time   in   the   fields   of   regenerative  medicine  and  drug  delivery.  Despite  a  significant  body  of  work,   to  date  the  majority  of  research  in  the  area  has  focused  on  relatively  simple   compositions  and  microstructures.  In  comparison,  the  food  industry  has  long   used  refined  and  often  subtle  methods  to  structure  and  thereby  tailor  the   release  and  handling  properties  of  a  vast  range  of  similar  materials.  In  this   thesis,  a  range  of  processing  methodologies  has  been  used  to  generate  novel   materials  intended  for  use  in  the  regenerative  medicine  and  drug  delivery   using  gellan  and  kappa  carrageenan.  The  thesis  demonstrates  how  even  small   changes  in  process  conditions  can  result  in  significant  changes  in  the  way  a   material   handles   and   may   deliver   therapeutic   molecules.   It   has   been   demonstrated  that  gellan,  when  combined  with  poly  (vinyl  alcohol)  (PVA)   produces   a   material   of   enhanced   robustness.   However,   the   enhanced   robustness  was  subject  to  gellan  forming  the  dominant  phase.   By  imparting  a  shear  force  to  gelling  materials,  it  was  also  possible  to  produce   shear  thinning  fluid  materials  using  similar  biopolymers  (gellan  with  kappa   carrageenan).  These  polymers  typically  phase  separate,  however,  when  the   concentration  of  polymers  were  controlled,  simultaneous  gelation  could  be   achieved  suggesting  that  a  single  phase  system  was  formed.     Finally  it  was  demonstrated  that  it  was  possible  to  generate  a  novel  cell     I delivery  device  by  the  hydration  of  kappa  carrageenan  in  warm  biomedical   buffers,  with  no  high  temperatures  required.     Overall  this  thesis  demonstrates  the  range  and  complexity  of  structures  that   can  be  produced  using  the  relatively  small  number  of  polymers  that  can  be   used  in  the  clinic.         II Acknowledgements     I  would  firstly  like  to  thank  my  supervisors,  Professor  Liam  Grover  and  Dr.   Fotis   Spyropoulos   for   their   invaluable   guidance,   support   and   mentorship   during  my  research  time  and  the  writing  of  my  thesis.  I  will  be  always  grateful   for  your  constant  motivation  and  belief.     Special  thanks  goes  to  Lynn  Draper,  who  has  been  so  reassuring  and  helpful   throughout  my  PhD.  I  would  also  like  to  thank  the  EPSRC  for  funding  this   work.   My  time  in  Birmingham  has  been  so  gratifying,  and  I  have  my  friends  and   colleagues  in  Chemical  Engineering  to  thank  for  that.  A  big  thank  you  also   goes  to  my  closest  friends  outside  of  the  university;  you  have  all  been  so   supportive,  and  always  found  ways  of  taking  my  mind  off  the  stresses  of  PhD   life.   A   massive   thank   you   goes   to   my   family   for   all   their   love,   support   and   motivation.  My  sincerest  thanks  to  my  parents,  who  have  been  so  amazing,   especially  while  I  have  been  writing  my  thesis.  You  have  supported  me  in   everything  I  do  and  I  hope  I  will  continue  to  make  you  proud.   Finally  but  most  importantly,  to  Gary,  you  have  been  so  patient  and  loving   throughout  my  PhD.  I  could  not  have  done  this  without  you!  Thank  you.       III Table  of  Content     Abstract  .......................................................................................................................................  I   Acknowledgements  .............................................................................................................  III   Table  of  Content  ...................................................................................................................  IV   List  of  Figures  .....................................................................................................................  VIII   List  of  Tables  ........................................................................................................................  XVI   Chapter  1.  INTRODUCTION  ...............................................................................................  1   1.1  Context  of  the  study  ...............................................................................................................  2   1.2  Objectives  ...................................................................................................................................  5   1.3  Thesis  Structure  ......................................................................................................................  6   1.4  Publications  and  Presentations  ........................................................................................  7   Chapter  2.  LITERATURE  REVIEW  ...................................................................................  9   2.1  Introduction  ............................................................................................................................  10   2.2  Hydrocolloids  .........................................................................................................................  12   2.2.1  Gellan  Gum  ......................................................................................................................  15   2.2.2  Kappa  Carrageenan  .....................................................................................................  18   2.2.3  Poly  (vinyl  alcohol)  (PVA)  ........................................................................................  21   2.3  Gelation  .....................................................................................................................................  22   2.3.1  Quiescent  gels  ................................................................................................................  22   2.3.2  Fluid  gels  ..........................................................................................................................  25   2.4  Mixed  hydrocolloid  systems  ............................................................................................  28   2.5  Hydrocolloids  in  emulsions  ..............................................................................................  33     IV 2.5.1  Emulsion  formation  ....................................................................................................  33   2.5.2  Role  of  Hydrocolloids  .................................................................................................  37   Chapter  3.  FORMATION  AND  CHARACTERISATION  OF  QUIESCENT   GELLAN  GELS  AND  MIXED  GELLAN/PVA  GELS  ....................................................  39   3.1  Background  ..............................................................................................................................  40   3.2  Materials  and  methods  .......................................................................................................  41   3.2.1  Materials  ..........................................................................................................................  41   3.2.2  Methods  ............................................................................................................................  41   3.2.2.1  Preparation  of  gellan  PVA  gels  ....................................................................................  41   3.2.2.2  Gellan  stained  with  5-­‐‑(4,6-­‐‑Dichlorotriazinyl)  Aminofluorescein  (DTAF)  42   3.2.2.3  Mechanical  Testing  ...........................................................................................................  42   3.2.2.4  Rheological  Analysis  ........................................................................................................  44   3.2.2.5  Confocal  Scanning  Laser  Microscopy  (CSLM)  .......................................................  45   3.3  Results  and  Discussion  .......................................................................................................  45   3.3.1  Understanding  poly  (vinyl  alcohol)  (PVA)  modification  of  gellan  gels  45   3.3.2  Visualisation  of  gellan/PVA  microstructures  ..................................................  61   3.4  Conclusions  ..............................................................................................................................  68   Chapter  4.  THE  FORMATION  AND  CHARACTERISATION  OF  LOW  ACYL   GELLAN/  KAPPA  CARRAGEENAN  MIXED  FLUID  GELS  .....................................  71   4.1  Background  ..............................................................................................................................  72   4.2  Materials  and  Methods  .......................................................................................................  72   4.2.1  Materials  ..........................................................................................................................  72   4.2.2  Fluid  gel  preparation  ..................................................................................................  73   4.2.3  Fluid  gel  characterisation  .........................................................................................  74   4.2.3.1  Rheological  characterisation  ........................................................................................  74   4.2.3.2  Differential  scanning  calorimetry  (DSC)  .................................................................  74     V 4.3  Results  and  discussion  ........................................................................................................  75   4.3.1  Single  polymer  fluid  gels  ...........................................................................................  75   4.3.2  Mixed  polymer  fluid  gels  ..........................................................................................  85   4.4  Conclusions  ..............................................................................................................................  99   Chapter  5.  WARM  (30  OC)  HYDRATION  OF  POTASSIUM  KAPPA   CARRAGEENAN  AND  ITS  INCLUSION  IN  EMULSIONS  AND  COMPLEX   EMULSIONS  .........................................................................................................................  101   5.1  Background  ...........................................................................................................................  102   5.2  Materials  and  Methods  ....................................................................................................  103   5.2.1  Materials  .......................................................................................................................  103   5.2.2  Formation  of  kappa  carrageenan  samples  .....................................................  104   5.2.2.1  Formation  of  Kappa  carrageenan  in  water  and  buffers  .................................  104   5.2.2.2  Formation  of  primary  emulsions  ..............................................................................  104   5.2.2.3  Formation  of  secondary  emulsions  .........................................................................  105   5.2.3  Sample  measurements  ...........................................................................................  105   5.2.3.1  Rheology  ..............................................................................................................................  105   5.2.3.2  Differential  Scanning  Calorimetry  (DSC)  ..............................................................  106   5.2.3.3  Mastersizer  .........................................................................................................................  106   5.2.3.4  Microscopy  .........................................................................................................................  107   5.3  Results  and  Discussion  ....................................................................................................  107   5.3.1  Hydration  and  swelling  of  kappa  carrageenan  in  water  and  HEPES  and   DMEM  buffer  ..........................................................................................................................  107   5.3.2  Inclusion  of  the  kappa  carrageenan  low  temperature  hydrated  gel   networks  within  a  water  in  oil  emulsion  ...................................................................  126   5.3.3  Inclusion  of  kappa  carrageenan  stabilised  primary  emulsions  for  the   production  of  duplex  emulsions  ....................................................................................  137     VI 5.4  Conclusions  ...........................................................................................................................  150   Chapter  6.  CONCLUSIONS  AND  RECOMMENDATIONS  FOR  THE  FUTURE ...................................................................................................................................................  152   6.1  Hydrocolloid  structuring  for  biomedical  applications  ......................................  153   6.2  Formation  and  characterisation  of  quiescent  mixed  gels  ................................  153   6.3  Visualisation  of  hydrocolloid  microstructures  .....................................................  154   6.4  The  formation  and  characterisation  of  multicomponent  fluid  gels  .............  155   6.5  Warm  hydration  of  kappa  carrageenan  ...................................................................  156   Chapter  7.  REFERENCES  ................................................................................................  158           VII List  of  Figures   Figure  2.1  –  Schematic  representation  of  the  microstructural  approach,  linking  the   materials  and  processing  parameters  with  the  resultant  sensory  response.  Adapted   from  (Norton  and  Foster,  2002)……………………………………………………………………………...11   Figure  2.2  –  Comparison  of  the  textures,  from  soft  and  flexible  to  firm  and  brittle,   exhibited  by  a  range  of  hydrocolloids.  Image  adapted  from  Phillips  and  Williams   (2009)………………………………………………………………………………………………………………………14   Figure  2.3  –  Schematic  representation  of  the  viscosity  dependence  of  polysaccharide   systems,  showing  the  transition  from  dilute  regions,  past  the  critical  concentration   (C*)   into   a   concentrated   region.   Image   adapted   from   Phillips   and   Williams   (2009)………………………………………………………………………………………………………………………15   Figure  2.4  –  Tetrasaccharide  repeating  unit  of  deacylated  gellan.  The  presence  of  an   acetyl  (*)  and  a  glyceryl  (**)  groups  are  found  in  native  gellan  (also  known  as  high   acyl  gellan)………………………………………………………………………………………………………………16   Figure  2.5  –  Chemical  structure  of  the  kappa  carrageenan  showing  the  anydro  bridge   with  a  single  sulphate  group……………………………………………………………………………………20   Figure  2.6  –  Schematic  representation  of  the  coil-­‐helix  transition  and  the  sol-­‐gel   transition   of   hydrocolloids   (Miyoshi   et   al.,   1996).   This   highlights   how   thermo   reversible  gels  behave  during  the  cooling  process  (a  &  b)  and  then  transition  back   through  the  phases  to  a  random  network  (d  &  c)  during  heating.  Temperatures  of  the   transitions  are  dependent  on  the  hydrocolloid………………………………………………………..23   Figure  2.7  –  Low  acyl  gellan  formed  under  quiescent  conditions  (a)  and  sheared   cooling  to  form  a  fluid  gel  (b)  of  low  acyl  gellan……………………………………………………….26   Figure   2.8   -­‐   Examples   of   possible   structures   for   double   polymer   networks:   interpenetrating  networks  (a),  coupled  networks  (b)  and  phase  separated  networks   (c)…………………………………………………………………………………………………………………………….30   Figure  2.9  –  Phase  diagram  for  gelatin  and  maltodextrin  mixed  systems  (Norton  and   Frith,  2001b).  Norton  &  Frith  show  the  bimodal,  phase  inversion  concentration  line     VIII