[1]  Karuna*laka,  C.  et  al.  So2ening  and  hardening  of  macro-­‐  and  nano-­‐sized  organic  cocrystals  in  a  single-­‐crystal  transforma*on.  Angew.  Chem.  Int.  Ed.  Engl.  50,  8642–6  (2011).  [2]  Vekilov,  P.  G.  The  two-­‐step  mechanism  of  nuclea*on  of  crystals  in  solu*on.  Nanoscale  2,  2346–2357  (2010).  [3]  Gebauer,  D.,  Kellermeier,  M.,  Gale,  J.  D.,  Bergström,  L.  &  Cölfen,  H.  Pre-­‐nuclea*on  clusters  as  solute  precursors  in  crystallisa*on.  Chem.  Soc.  Rev.  43,  2348–2371  (2014).  [4]  Tr*k,  P.,  Kaufmann,  J.  &  Volz,  U.  On  the  use  of  peak-­‐force  tapping  atomic  force  microscopy  for  quan*fica*on  of  the  local  elas*c  modulus  in  hardened  cement  paste.  Cem.  Concr.  Res.  42,  215–221  (2012).  [5]  Bhardwaj,  R.  M.  et  al.  Exploring  the  Experimental  and  Computed  Crystal  Energy  Landscape  of  Olanzapine.  Cryst.  Growth  Des.  13,  1602–1617  (2013)  [6]  Mitchell,  C.  a,  Yu,  L.  &  Ward,  M.  D.  Selec*ve  nuclea*on  and  discovery  of  organic  polymorphs  through  epitaxy  with  single  crystal  substrates.  J.  Am.  Chem.  Soc.  123,  10830–9  (2001)    

the  nanoscale  open  new  opportuni*es  to  revise  and  ques*on  commonly  accepted  nuclea/on  and  crystal  growth  theories.  Atomic  Force  Microscopy  (AFM)  has  been  successfully  involved  in  various   aspects   of   ac*ve   pharmaceu*cal   ingredient   (API)   characteriza*on   including   crystal   growth,  stability   of   solid  dispersions,   surface  morphology,   phase   changes   and  dissolu*on   [1].   Recent   studies  conducted  on  proteins  crystalliza*on  at  nanoscale  show  new  evidence  disproving  generally  accepted  Classical  Nuclea/on  Theory  (CNT)  (Fig.1  a)  [2].  Currently,  ‘dense  liquid  droplets’  seen  in  protein   crystallisa*on   and   ‘pre-­‐nuclea*on   clusters’   (Fig.1   b)   [3]   seen   mostly   in   inorganic   salt  crystallisa*on,  are  two  main  concepts  of  non-­‐classical  nuclea*on  theory,  although  no  significant  progress  has  been  made  towards  beler  understanding    of    mechanisms  controlling  heterogeneous  nuclea*on  in  small  organic  molecules  systems,  what  is  in  par*cular  interest,  as  an  epitaxial  ordering  phenomenon  is  frequently  used  to  enhance  nuclea*on  rates  and  control  proper*es  of  materials.  Our  studies  present  a  new  light  on  heteronuclea*on  and  the  epitaxial  growth  mechanisms  based  epitaxial  growth  of  olanzapine  dihydrate  D  on  the  surface  of  olanzapine  form  I  (OZPN  I)  both  in  high  humidity  condi*ons  and  water  solu*on.  Results  obtained  from  Peak  Force  Quan/ta/ve  Nanomechanical  Mapping  Atomic  Force  Microscopy  (PF-­‐QNM-­‐AFM)  [4]  indicate  the  presence  of  intermediate  dense  liquid-­‐like  phase  in  process  of  dihydrate  D  nuclea*on.  

INTRODUCTION   Con*nues  advancement  and  rapid  development  of  techniques  opera*ng  at    

PeakForce-­‐QNM-­‐AFM  opens  new  opportuni*es  for  nanocharacterisa*on   of   the   mechanical   surface  proper*es.   This   mode   enables   force   curves  separa*on   in   order   to   obtain   Young’s   modulus,  adhesion   force   between   the   *p   and   the   sample,  energy  dissipa*on,  and  maximum  deforma*on  (Fig.  4)  [4]  .  

dihydrate D form I

2D lattice registry (100)OZPN/(001)D

-80 -60 -40 -20 0 20 40 60 80

0.5

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1.5

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θ=50°

θ=10°

E

Theta [Deg]

MOTIVATION   METHODS  

RESULTS  

Quan/ta/ve  Analysis  of  Observed  Droplets  

CONCLUSIONS  

-60 -40 -20 0 20 40 60

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[001]OZPN I

(100)OZPNI/ (001)D

y [Å

]

x [Å]

[010]OZPN I

Dense  nanodroplets  (ND)  visible  on  the  surface  of  (100)  OZPN  I  face  in   70   %   RH   were   characterised   by   PF-­‐QNM-­‐AFM.   Also   the   same  measurements   for   OZPN   I   crystal   placed   in   water   were   conducted.  Nanomechanical  characterisa*on  of  ND  by  PF-­‐QNM  AFM  reveals  that:  (i)  Three  different  phases  can  be  dis*nguished  (OZPN  I,  new  crystalline  

form  and  dense  droplets),  droplets  are  the  so2est  phase    (ii) ND  separate  from  water  as  a  new  stable  denser  phase,  (iii)   ND  undergo  transforma/on  to  a  new  solid  phase.    (iv)   Growing  new  crystalline  form  has  also  different  nanomechanical  

proper/es   then   OZPN   I   and   addi*onal   results   from   Raman  spectroscopy  shows  that  new  crystalline  form  is  OZPN  dihydrate  D.  

Peak  Force  micrographs  

Young’s  Modulus  maps  

MAX  

MIN  

Geometric   real-­‐space   analysis   of   crystal   epitaxy  (GRACE)   [6]   calcula*ons   revealed   significant   2-­‐D  la[ce   registry  between   (100)OZPN   and   (001)D,   that   is  responsible  for  nuclea/on  and  epitaxial  growth  of  dihydrate  D  on  the  surface  of  OZPN  I    

Fig.  4  a)  Principle  of  AFM  opera*on,  detec*ng  the  bending  of  the  can*lever  with  a  photodetector  and  laser  beam,  b)  Diagram  presen*ng  a  force  vs.  separa*on  curve  for  one  cycle  of  the  peak-­‐force  tapping  AFM  

Olanzapine   (OZPN,   Zyperxa®)   (Fig.   2)   is   a   BCS   class   II   drug   (low  solubility,   high   permeability)   used   in   schizophrenia     (bipolar   disorder)  treatment.  OZPN  exhibits  rich  solid  state  diversity,  so  far  60  dis*nct  forms  were   iden*fied   including   3   polymorphic   forms   (I,   II,   III),   52   crystalline  solvates,   3   polymorphic   dihydrates   B,   D,   E,   and   disordered   higher  hydrate  plus  an  amorphous  form  [5].    

OZPN   I   is   stable   from   under   ambient   condi*ons,   although  significant   surface   changes   were   observed   when   OZPN   I   single  crystal  was  stored  for  2  days  in  quiescent  water.  Epitaxial  growth  of  a  new  form  was  observed  on  (100)  OZPN  I  face.  

Fig.  2.  Structure  of  Olanzapine  [5].    

Fig.3  a)  Crystal  structure  of  OZPN  I  showing  the  distance  between  (100)OZPN  planes,  b)  AFM    micrograph  represen*ng  OZPN  I  (100)  face  showing  the  layered  structure.    c)  OZPN  I  single  crystal  stored  in  water  for  2  days,  d)  AFM  characterisa*on  of  the  surface  of  OZPN  I  (100)  face  a2er  storing  the  crystal  2  days  in  water  

The  main  objec/ve   is  to  characterise  new  form  growing  on  the   surface   of   (100)   OZPN   I   face   and   study   nuclea/on   and  growth  mechanism.    

70  %  RH   water  

(a)   (b)   (c)  

(d)  

(a)   (b)  

00h:00min   01h:00min   09h:00min  

Obtained   informa*on   about   observed  nanodroplets   both   in  water   and  70%  RH  agrees  with  the  two-­‐step  nuclea/on  theory  described  by  Vekilov  and  co-­‐workers  via  dense-­‐liquid  droplets   [2].    New   form  growing  on   (100)  OZPN   I  face  was  characterised  as  dihydrate  D  by  Raman  spectroscopy.  

Geometric  real-­‐space  analysis  of  crystal  epitaxy    

Surface  of  OZPN  I  (100)face  was  observed  in  70  %  RH  condi*ons  using  PF-­‐QNM-­‐AFM.   Surface   of   (100)OZPN   with   visible   ledges   becomes   covered   with   large  number  of  small  nanodroplets.  

Fig.  5  AFM  height  mode  micrographs  of  OZPN  I  surface  (100)  face  in  70  %  RH,  room  temperature  condi*ons,  a)  0  min,  b)  1  hour,  c)  9  hours.    

(a)   (b)   (c)  

Fig.  7  a)Moiré  palerns  between  (100)OZPN/(001)D  showing  2D  epitaxial  match,  b)  Epitaxy  score  between  (100)OZPN/(001)D,  c)    Face  indexed  Dihydrate  D  crystal  growing  on  the  surface  of  (100)OZPN,    c)  crystal  structure  of  OZPN  I  and  OZPN  dihydrate  D.  

Fig.  6  AFM  micrographs  and  corresponding  Young’s  Modulus  Maps  for  a)  Dense  droplet  observed  in  70%  RH  condi*ons;  b,c)  dense  droplets  observed  in  water;  d)  new  crystalline  form  growing  on  the  surface  of  OZPN  I  (100)  face.  

(b)  (a)   (c)   (d)  

(a)  

(b)   (c)   (d)  

Atomic  Force  Microscopy  Studies    on  Two-­‐Step  Nuclea/on  and  Epitaxial  Growth      

Monika Warzecha1, Rajni M. Bhardwaj1,2, Susan Reutzel-Edens2, Dimitrios Lamprou1, 3,  Alastair Florence1, 3    

1Strathclyde  Ins*tute  of  Pharmacy  and  Biomedical  Sciences,  University  of  Strathclyde,  Glasgow,  G4  0RE,  UK    2  Small  Molecule  Design  &  Development,  Eli  Lilly  and  Company,  Indianapolis,  IN  46285,  U.S.A.    3EPSRC  Centre  for  Innova*ve  Manufacturing  in  Con*nuous  Manufacturing  and  Crystallisa*on  c/o  Technology  and  Innova*on  Centre,  99  George  Street,  Glasgow,  G1  1RD,  U.K  

Fig.  1  Free  energy  diagram  according  to  a)  Classical  nuclea*on  theory,  b)  two-­‐step  nuclea*on  theory  [2].