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[1] Karuna*laka, C. et al. So2ening and hardening of macro and nanosized organic cocrystals in a singlecrystal transforma*on. Angew. Chem. Int. Ed. Engl. 50, 8642–6 (2011). [2] Vekilov, P. G. The twostep 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. Prenuclea*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 peakforce 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 ‘prenuclea*on clusters’ (Fig.1 b) [3] seen mostly in inorganic salt crystallisa*on, are two main concepts of nonclassical 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 QNMAFM) [4] indicate the presence of intermediate dense liquidlike phase in process of dihydrate D nuclea*on. INTRODUCTION Con*nues advancement and rapid development of techniques opera*ng at PeakForceQNMAFM 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 1.0 1.5 2.0 2.5 3.0 3.5 4.0 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 -60 -40 -20 0 20 40 60 [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 PFQNMAFM. Also the same measurements for OZPN I crystal placed in water were conducted. Nanomechanical characterisa*on of ND by PFQNM 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 realspace analysis of crystal epitaxy (GRACE) [6] calcula*ons revealed significant 2D 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 peakforce 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 twostep nuclea/on theory described by Vekilov and co workers via denseliquid droplets [2]. New form growing on (100) OZPN I face was characterised as dihydrate D by Raman spectroscopy. Geometric realspace analysis of crystal epitaxy Surface of OZPN I (100)face was observed in 70 % RH condi*ons using PFQNM 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 TwoStep Nuclea/on and Epitaxial Growth Monika Warzecha 1 , Rajni M. Bhardwaj 1,2 , Susan Reutzel-Edens 2 , Dimitrios Lamprou 1, 3 , Alastair Florence 1, 3 1 Strathclyde 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. 3 EPSRC 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) twostep nuclea*on theory [2].
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