Spherical Crystalline Anti-Retroviral Drug Particles with Tunable

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Spherical Crystalline Antiretroviral Drug Particles with Tunable Microstructure Eunice W.Q. Yeap, Denise Z. L. Ng, David Lai, Sonja Sharpe, Darryl Ertl, and Saif A. Khan Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00728 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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Crystal Growth & Design

Spherical Crystalline Antiretroviral Drug Particles with Tunable Microstructure Eunice W.Q. Yeap‡||, Denise Z.L. Ng‡||, David Lai†,ξ, Sonja Sharpe†, Darryl Ertlξ and Saif A. Khan‡* ‡ Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore † GlaxoSmithKline LLC, Advanced Manufacturing Technologies, 830 Winter Street, PC2000, Waltham, MA 02451, USA ξ GlaxoSmithKline LLC, Product and Process Engineering, 709 Swedeland Road, King of Prussia, PA 19406, USA

Abstract The crystallization of molecular solids is ubiquitous in various contexts, and forms the basis of pharmaceutical drug product design and manufacture. As drug molecules get more complex, their crystallization into well-controlled crystal forms becomes more challenging yet unquestionably important, from a process and product perspective. Here, we demonstrate the fabrication of molecular solids with exquisitely tunable crystalline microstructure by coconfinement of a highly supersaturated drug-colloidal dispersion within sub-millimeter droplets. Specifically, we show the evaporative solidification of an antiretroviral drug molecule (Lamivudine, 4-amino-1-[(2R,5S)-2- (hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)pyrimidinone) into spherical crystalline particles in the presence of colloidal silica or polystyrene, where we are able to tune the crystalline microstructure of the drug at the submicron level by using various colloid sizes. Confinement of the drug within droplets generated in a microfluidic device enables access to high degrees of supersaturation before the onset of crystal nucleation whilst allowing precise control over the amount of dispensed colloidal particles. The tunability of the microstructural length scale with colloid size and the surface-agnostic nature of the microstructure control are unprecedented observations that are not captured by currently available theories. Furthermore, differences in the polymorphic outcome of the crystallization conducted in the presence or absence of colloids were also observed. Our findings pave the way for the design and manufacturing of novel crystalline composites by colloid-induced microstructure control.

* Corresponding Author. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore. Email: [email protected]

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Spherical Crystalline Antiretroviral Drug Particles with Tunable Microstructure Eunice W.Q. Yeap‡||, Denise Z.L. Ng‡||, David Lai†,ξ, Sonja Sharpe†, Darryl Ertlξ and Saif A. Khan‡* ‡ Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore † GlaxoSmithKline LLC, Advanced Manufacturing Technologies, 830 Winter Street, PC2000, Waltham, MA 02451, USA ξ GlaxoSmithKline LLC, Product and Process Engineering, 709 Swedeland Road, King of Prussia, PA 19406, USA * Corresponding Author. Email: [email protected] KEYWORDS:

Crystallization, Microstructure, Colloids, Pharmaceutical Drugs,

Dosage Forms


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ABSTRACT The crystallization of molecular solids is ubiquitous in various contexts, and forms the basis of pharmaceutical drug product design and manufacture. As drug molecules get more complex, their crystallization into well-controlled crystal forms becomes more challenging yet unquestionably important, from a process and product perspective. Here, we demonstrate the fabrication of molecular solids with exquisitely tunable crystalline microstructure by co-confinement of a highly supersaturated drug-colloidal dispersion within sub-millimeter droplets. Specifically, we show the evaporative solidification of an antiretroviral drug molecule (Lamivudine, 4-amino-1-[(2R,5S)-2(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone) into spherical crystalline particles in the presence of colloidal silica or polystyrene, where we are able to tune the crystalline microstructure of the drug at the sub-micron level by using various colloid sizes. Confinement of the drug within droplets generated in a microfluidic device enables access to high degrees of supersaturation before the onset of crystal nucleation whilst allowing precise control over the amount of dispensed colloidal particles. The tunability of the microstructural length scale with colloid size and the surface-agnostic nature of the microstructure control are unprecedented observations that are not captured by currently available theories. Furthermore, differences in the polymorphic outcome of the crystallization conducted in the presence or absence of colloids were also observed.

Our findings pave the way for the design and

manufacturing of novel crystalline composites by colloid-induced microstructure control. (215 words)



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The crystallization of molecular solids is ubiquitous in a variety of natural and technological contexts, from neurological diseases to the synthesis of high performance polymers, and forms the very basis of pharmaceutical drug product design and manufacture. Crystal growth under highly non-equilibrium conditions, whether from melts or supersaturated solutions, can exhibit morphological instabilities that give rise to complex spatial patterns - highly branched dendritic, spherulitic and fractal solidification patterns have been observed in a remarkably diverse array of systems, ranging from volcanic rocks to polymer melts.1-6 The nature of the patterns depends crucially on the distance from equilibrium, kinetics of molecular (and heat) diffusion, interfacial growth kinetics, crystalline anisotropy and the presence of molecular or particulate impurities.7 The adventitious presence of particulate ‘impurities’ is ubiquitous in nearly all crystallization processes, and typically leads to accelerated nucleation rates, since foreign solid surfaces can reduce thermal activation barriers for the formation of embryonic crystal nuclei.8, 9 In fact, microscopic solid particles are often deliberately added as seeding agents to promote nucleation in industrial applications, such as in pharmaceutical crystallization,8 and for microstructure control in metallic solidification from melts.10,

11

The role of

particulate additives in modulating the morphology of growing solidification fronts has been extensively investigated, and is yet incompletely understood. Under high driving forces, the formation of branched growth fronts is the most common consequence of the presence of particles; the structure of the growth front can then be dramatically altered, going from highly ordered single crystal dendrites to disordered and polycrystalline spherulites with increase in foreign particle concentration in the vicinity of the growing crystalline solid.12 Furthermore, the link between characteristic size of the crystallization microstructure and that of the impurity


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remains tenuous and poorly understood. Also relevant to this work is the effect of conducting crystallization in confinement, where fixed structures such as microchannels,13 mesoporous glass beads,14 controlled pore glass,15 microgels with nanopores,16, 17 or microdroplets,18, 19 confining supersaturated solutions were shown to influence morphological and polymorphic outcomes as compared to bulk crystallization. In particular, the formation and stabilization of metastable states within confined geometries is mainly attributed to competing surface and volume free energies when scales approach that of emerging crystal nuclei,20-22 which influences polymorphic outcomes.

Inspired by the classic experiments on solidification of molten droplets by Turnbull and co-workers in the 1950s,23 we use an experimental solution crystallization system in which it is possible to both access high degrees of supersaturation before the onset of spontaneous nucleation, whilst precisely controlling the amount and size of the added foreign particulates within the system. As a model, we demonstrate evaporative crystallization

of

a

molecular

drug

(Lamivudine,

4-amino-1-[(2R,5S)-2-

(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2-one) widely used in anti-retroviral therapeutic regimens, from highly supersaturated sub-millimeter aqueous droplets that also contain precise amounts of monodisperse spherical colloidal silica particles, and present the first instance of colloid-induced microstructure control in molecular crystallization. The presence of the colloidal particles within the droplet during crystallization leads to dramatic and, crucially, tunable alterations of the crystal morphology, from open lamellar spherulites to highly fibrous, tightly packed needles whose characteristic size can be tuned by the size of the colloid. Remarkably, this phenomenon is found to be independent of the nature of the colloid used; the



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substitution of silica with polystyrene leads to identical crystallization phenomena and morphologies. We also found polymorphic differences between the lamivudinecolloidal composites and the neat lamivudine particles, which indicates confinement effects induced by the presence of colloidal particles within the supersaturated drug solution during crystallisation.18 Powdered X-Ray Diffraction patterns for the particles indicated the formation of lamivudine Form I in the absence of colloids and the formation of anhydrous lamivudine Form V in the colloidal composite particles, which melted ~20 °C earlier than Form I, as confirmed via Differential Scanning Calorimetry.

In a typical experiment, we first generate monodisperse aqueous droplets of ~0.5 mm diameter, containing lamivudine (8.0 w/v%) and colloidal silica (4.0 w/v%, diameter of 165 nm/450 nm), dispersed in an immiscible carrier fluid (Xiameter PMX-200 Silicone Fluid with 2 w/w% PEG-10 Dimethicone) using a microfluidic device (see Methods). An ensemble of ~1800 droplets is collected in a petri dish containing a thin film (1.0 mm) of PMX-200, and is heated at 65 °C under ambient conditions. Water slowly evaporates from the initially under-saturated droplets over the course of ~150 minutes (Figure 1(a)-(c)), until the droplets are roughly half their original size and do not observably shrink any further (Figure 1(c)). The droplet supersaturation in this terminal state is ~4, with mass fractions of 0.65, 0.32 and 0.03 for the drug, water and silica respectively. The droplet ensemble is then allowed to incubate in this highly non-equilibrium state; this is in effect analogous to the annealing of a liquid melt at a certain temperature below the melting point.25, 26 Stochastic nucleation and growth are observed to occur eventually within each droplet in the ensemble, and white spherical solid particles of ~0.2 mm diameter are obtained; the entire droplet ensemble 6 ACS Paragon Plus Environment

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solidifies in ~500 minutes (Figure 1(d)). We only observe a single nucleation event per droplet, either spontaneously occurring within an isolated droplet (‘primary’ nucleation) or, more commonly, due to contact with an adjacent particle that has already solidified (‘secondary’ nucleation).

Figure 1. (a) Monodisperse droplets loaded with colloidal silica (4.0 w/v) and lamivudine (8.0 w/v%) are generated in a microfluidic device and collected in a 1 mm thick film of silicone fluid at 65 °C. The droplets are initially under-saturated. (b) The droplets shrink due to loss of water by evaporation through the silicone fluid and into the ambient; this process continues for about 150 min, till the droplets shrink to nearly half their initial size, and reach a lamivudine supersaturation of ~4, after which no further observable shrinkage occurs. (d) Particle solidification is completed at ~500 min, and white spherical beads are obtained. All scale bars represent 500 µm.



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The phenomenology of particle solidification is depicted in the series of microscope images provided in Figure 2(a), in which a radially advancing solidification front is clearly observed. The growth is linear, indicating a spherulitic growth mechanism, with average growth rate in lamivudine-colloidal silica droplets (~0.36 µm/s) nearly half of that observed in neat lamivudine droplets (~0.78 µm/s). We also observe an opalescent irradiance ahead of the advancing solidification front in some droplets (refer to Figure S1 in the Supporting Information) – a signature of colloidal assembly into ordered, periodic photonic structures at the growth front.27 A spherulitic growth mechanism also occurs in the case of droplets that do not contain the colloidal silica (Figure 2(b)); however, the final particle microstructures are strikingly different, as we shall see below.


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Figure 2. Time lapse optical microscopy images featuring the propagation of radial advancing solidification fronts across droplets, and plots of the measured position of the front within each droplet versus time, for (a) lamivudine-silica (165 nm), and (b) neat lamivudine. X (µm) is the position of the advancing solidification front measured from the point of nucleation, and t (s) is the time elapsed from the onset of (secondary) nucleation at the droplet boundary. All scale bars represent 100 µm.

In the absence of silica, the spherical macrostructure is composed of loosely packed, lamellar plate-like microcrystals (Figure 3(a)-(c)). Digital image analysis of highresolution scanning electron microscope (SEM) images indicates that these microcrystals have an elliptical cross section of 365 nm x 915 nm and axial aspect ratios of ~100 (see Figure S2 for detailed histograms of size measurements in the Supporting Information). Such lamellar structures have been reported in thermally induced crystallization of polymers and metals from melt states,3,

25, 26

and in

molecular crystallization from supersaturated solutions.28 These structures are characterized by large angle branching of the unstable growth front, typically along crystallographic axes, giving rise to characteristic concentric, ring-like arrangements 9 ACS Paragon Plus Environment


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of lamellae, as seen in Figure 3(c).26 The particle microstructure is dramatically altered in the presence of colloidal silica. (Figure 3(d)-(f)) The spherical macrostructure is now composed of an extremely fine, fibrous and space-filling microstructure (Figure 3(d)-(e) for 165 nm silica), composed of sub-micron crystalline needles with a smaller elliptical cross-section as shown in Figure 4(c) (140 nm x 230 nm when grown in the presence of 165 nm silica) with extremely high axial aspect ratios of up to 300. The tight packing and nearly parallel radial alignment of these needles is apparent from the cross-sectional SEM image of Figure 3(f) (and also from Figure 5(c)). It is important to note here that this microstructure is a significant departure from commonly observed spherulitic morphologies in molecular crystallization from supersaturated solutions; it is rather more akin to the fibrous spherulitic habits observed in the thermal solidification of polymers and liquid metals.12

Figure 3. Scanning Electron Microscopy (SEM) images of (a-c) neat lamivudine microparticles from emulsions containing lamivudine at 80 mg/mL, and (d-f) lamivudine-silica (165 nm) microparticles from emulsions containing lamivudine at 10 ACS Paragon Plus Environment

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80 mg/mL and silica nanoparticles at 40 mg/mL. (a-c) Lamivudine microparticles are irregularly shaped, and are composed of large, loosely packed and lamellar needles. (d-e) Lamivudine-silica microparticles are uniformly sized and have a well-controlled spherical macrostructure. The microparticles are composed of tightly packed, fine, fibrous lamivudine needles of high aspect ratio (up to 300), spanning the extent of the microparticles. (f) These needles are of sub-micron cross-sectional dimension, and highly aligned, being nearly parallel towards the droplet periphery.

Next, we demonstrate how the characteristic size of the particle microstructure can be tuned by that of the colloidal particles added. Histograms of SEM measurements of needle cross-sectional dimensions are compared and contrasted in Figure 4(a-c) for colloidal silica particles of 450 nm, 330 nm and 165 nm diameters respectively; the cross-sectional dimensions of the crystals are seen to increase with colloid size. Finally, and equally strikingly, we demonstrate that these phenomena are observed even when the silica is substituted by another colloid, polystyrene. The differences between the colloidal silica and polystyrene can be characterized in term of zeta potential and terminal functional groups on the surface. The measured zeta potential for the silica nanoparticles and polystyrene nanoparticles are -44.7 mV and -27.4 mV respectively. The native terminal groups of the silica nanoparticles are silanol moeities whereas the terminal surface groups of the polystyrene colloidal particles are sulfate moeities. This hints at the surface-independent nature of the mechanisms at play. Figure 5 shows spherical particles obtained from the same method, but with the addition of negatively charged colloidal polystyrene particles (375 nm); an identical fibrous microstructure is observed (Figure 5(a)-(c)), with phenomenologically identical (linear) growth front kinetics (Figure 5(d)) and sub-micron needle-like



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microcrystals of dimensions 175 nm x 300 nm (Figure 5(e)). Equally strikingly, the measured cross-sectional dimensions of the microcrystals once again reflect the sizes of the colloidal particles used, while also matching the scale of crystals obtained using 330 nm colloidal silica.


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Figure 4. Histograms of cross-sectional dimensions of the lamivudine needles in the microparticles, when colloidal silica of diameter (a) 450 nm, (b) 330 nm and (c) 165 nm is used. The needles are typically of elliptical cross-section, and the reported measurements are of width along the two principal axes (x and y), made from topdown SEM micrographs (see inset of (a)). The cross-sectional dimensions of the needles are seen to increase with colloid size. Scale bars represent 1 µm.

Figure 5. SEM images of lamivudine-colloidal polystyrene (375 nm) microparticles. (a-c) Both particle macro- and microstructures are exactly analogous to the case of lamivudine-colloidal silica, as shown in Figure 3 (d-f). The microstructure is composed of tightly packed and highly aligned fine, fibrous sub-micron needles spanning the entire particle. (d) Position of the advancing solidification in lamivudine-colloidal polystyrene droplets; a higher linear growth rate is measured compared to neat lamivudine. (e) A histogram of lamivudine crystal dimensions measured as described in Figure 4; strikingly, the measured dimensions for 375 nm



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colloidal polystyrene are in between those measured for 165 nm and 450 nm colloidal silica respectively and are consistent with the crystal sizes for the 330 nm colloidal silica case, providing further evidence of the generality of microstructural dimensions tunability with the size of colloidal particle used. A representative top-down SEM micrograph is included in the inset, with the scale bar representing 1 µm.

Lastly, besides structural observations, polymorphic characterization of the neat lamivudine and lamivudine-colloidal composite particles conducted using a combination of powdered X-ray Diffraction and Differential Scanning Calorimetry analysis yielded interesting results. There are five known forms of lamivudine reported in literature.29-31 The crystal form obtained within neat lamivudine particles was found to be the 0.2 hydrate lamivudine polymorph Form I, as shown from the characteristic peaks in the XRD spectra at 10.1°, 11.6°, 12.1°, 15.2°, 18.5°, 24.9° and 28.3° (marked with red dots in Figure 6(a)), and the endotherm at 176°C in Figure 6(b), which are both consistent with the reported values for Form I in literature.30 In contrast, both lamivudine-colloidal silica and lamivudine-colloidal polystyrene particles displayed indicative peaks at 14.1°, 18.1°, 19.4° and 27.6°, and two distinct characteristic peaks at 20.4° and 27.3° which corresponded to an anhydrous lamivudine polymorph Form V.31 The melting point of lamivudine in these lamivudine-colloidal particles was also found to be significantly lowered, at 159.3°C and 160.6°C in the particles with silica and polystyrene respectively, and is close to the reported melting point of 161.5°C for Form V.31 Reference XRD patterns for all known lamivudine polymorphs and the obtained lamivudine forms from the different particles in this work are provided in Figure S3 of the Supporting Information. Our findings are in line with the outcomes of nanoconfinement previously seen within 14 ACS Paragon Plus Environment

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hard templates provided by controlled glass pores14 or mesoporous silica,15 or soft templates such as within pores of hydrogels,17 where metastable or new polymorphic forms were obtained.

Figure 6. (a) Powder X-Ray Diffraction spectra of lamivudine from neat lamivudine particles, lamivudine-silica (330 nm) particles and lamivudine-polystyrene (375 nm) particles. The red dots above the neat lamivudine spectra mark out characteristic 15 ACS Paragon Plus Environment


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peaks for lamivudine polymorphic Form I. Two distinct characteristic peaks at 17.7° and 23.4° (marked with vertical dotted lines) were seen for both lamivudine-silica and lamivudine-polystyrene spectra, matched those of lamivudine Form V. (b) Differential Scanning Calorimetry (DSC) thermograms of lamivudine from neat lamivudine particles, lamivudine-silica (330 nm) particles and lamivudinepolystyrene (375 nm) particles. The melting point of lamivudine in the neat lamivudine particles at 176.8 °C corresponds to the Form I melting point.30 There lowered melting point for the lamivudine within the lamivudine-silica and lamivudine-polystyrene particles at 159.4 °C and 160.6 °C respectively corresponds to the Form V melting point.31

The role of ‘impurities’ or ‘seeds’ in crystallization phenomena has been a source of debate (and often controversy) for several decades; impurities affect not only the rates of nucleation but also impact the structure and morphology of the growing crystal front.4,

8

While the former effect has been thoroughly studied, the latter has not,

particularly due to the considerable difficulty involved in setting up well-controlled experiments that are not confounded by the effects of solid surfaces besides those of interest. However, amongst the various attendant factors, the role of high supersaturation/undercooling as a prerequisite for fibrillar growth is well accepted. The seminal work of Keith and Padden made the first, albeit controversial case, for the role of impurity segregation ahead of the growth front in triggering morphological instabilities and fibrillar crystal growth fronts.3 More recently, Granasy and coworkers have used phase field theory to simulate the effect of particulate impurities in the solidification of the Ni-Cu eutectic system.12,

32

Their results reveal the role of

foreign solid particles as agents for growth front nucleation (GFN), and predict 16 ACS Paragon Plus Environment

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microstructure transitions upon increasing particle concentration, from open, lamellar microstructures to highly fibrous space-filling microstructures characterized by noncrystallographic small angle branching. Our results are the first instance of controlled, impurity-induced microstructural transitions of this kind in molecular crystallization from supersaturated solution, and are a striking confirmation of these in silico observations, albeit for a very different system than the metallic eutectics simulated. Further, both the tunability of the microstructural length scale with impurity size (Figure 4) and the surface-agnostic nature of the structural transition phenomena (Figure 5) are hitherto unprecedented observations that are not captured by this theory, or indeed by any others currently available. Finally, it is worth noting the remarkable phenomenological parallels between our results and the freeze-casting of ceramic materials.33 In freeze-casting under very high thermal gradients, suspended microparticles in a slurry are rejected from the moving solidification front and trapped between growing cellular solvent crystals, leading to ordered lamellar microstructural morphologies. The controlled confinement of a highly supersaturated molecular melt and a colloidal suspension inside sub-millimeter droplets in our work effectively accesses the same physical regimes, albeit under markedly different conditions. Indeed, we have probed similar parameters such as colloid volume fractions and seen interesting modifications to the aspect ratio of the crystals obtained within the microparticles (shown in Figure S4 of the Supporting Information). We leave a detailed examination of the templating mechanism and confinement effects exerted by the colloids on the growing crystals to a separate, ongoing study. Our results are of particular importance in drug product design, in which the crystalline solid state is by far the most frequently employed in pharmaceutical dosage forms. As drug molecules get larger and more complex, their crystallization 17 ACS Paragon Plus Environment


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into well-controlled crystal habits becomes all the more challenging yet unquestionably important, both from a process and product perspective. Furthermore, there is considerable interest in the development of nanocrystalline drugs, which dramatically increase the bioavailability of poorly soluble drugs. From all these perspectives, our findings, which describe how controlled colloid addition and confinement within droplets can be used to tune crystalline microstructure at the submicron level as well as polymorphic outcome, while still retaining control over the macrostructure, pave the way for the design and manufacturing of crystalline solids for application in next-generation pharmaceutical dosage forms.

ACKNOWLEDGEMENTS We gratefully acknowledge funding from the GSK-EDB Fund for Green and Sustainable Manufacturing and an intra-CREATE grant from the National Research Foundation of Singapore.

ASSOCIATED CONTENT Supporting Information (I) Irradiance observed at advancing solidification front within droplets, (II) size measurements of lamivudine crystals in the absence of colloidal impurities, (III) reference powdered X-ray Diffraction patterns for lamivudine forms, (IV) effect of colloid concentration on crystal morphology within microparticles, and (V) experimental section. This material is available free of charge via the Internet at http://pubs.acs.org.


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Corresponding Author *Email: [email protected]

Author Contributions ||

Eunice W.Q. Yeap and Denise Z.L. Ng contributed equally to this work.

Notes The authors declare no competing financial interest.

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FOR TABLE OF CONTENTS USE ONLY

Spherical Crystalline Antiretroviral Drug Particles with Tunable Microstructure Eunice W.Q. Yeap‡||, Denise Z.L. Ng‡||, David Lai†,ξ, Sonja Sharpe†, Darryl Ertlξ and Saif A. Khan‡*

We demonstrate the fabrication of antiretroviral drug particles with tunable crystalline microstructure, by using microfluidic droplets to confine a drug-colloid suspension followed by evaporative solidification. The tunability of the microstructure within the spherical crystalline composite particles is affected by the colloids’ sizes and insensitive to the nature of the colloids’ surface. Our findings pave the way for the design and manufacturing of novel crystalline solids by colloid-induced microstructure control.



Figure 1. (a) Monodisperse droplets loaded with colloidal silica (4.0 w/v%) and lamivudine (8.0 w/v%) are generated in a microfluidic device and collected in a 1 mm thick film of silicone fluid at 65 °C. The droplets are initially under-saturated. (b) The droplets shrink due to loss of water by evaporation through the silicone fluid and into the ambient; this process continues for about 150 min, till the droplets shrink to nearly half their initial size, and reach a lamivudine supersaturation of ~4, after which no further observable shrinkage occurs. (d) Particle solidification is completed at ~500 min, and white spherical beads are obtained. All scale bars represent 500 µm. 140x257mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2. Time lapse optical microscopy images featuring the propagation of radial advancing solidification fronts across droplets, and plots of the measured position of the front within each droplet versus time, for (a) lamivudine-silica (165 nm), and (b) neat lamivudine. X (µm) is the position of the advancing solidification front measured from the point of nucleation, and t (s) is the time elapsed from the onset of (secondary) nucleation at the droplet boundary. All scale bars represent 100 µm 441x216mm (300 x 300 DPI)



Figure 3. Scanning Electron Microscopy (SEM) images of (a-c) neat lamivudine microparticles from emulsions containing lamivudine at 80 mg/mL, and (d-f) lamivudine-silica (165 nm) microparticles from emulsions containing lamivudine at 80 mg/mL and silica nanoparticles at 40 mg/mL. (a-c) Lamivudine microparticles are irregularly shaped, and are composed of large, loosely packed and lamellar needles. (d-e) Lamivudine-silica microparticles are uniformly sized and have a well-controlled spherical macrostructure. The microparticles are composed of tightly packed, fine, fibrous lamivudine needles of high aspect ratio (up to 300), spanning the extent of the microparticles. (f) These needles are of sub-micron cross-sectional dimension, and highly aligned, being nearly parallel towards the droplet periphery. 162x84mm (300 x 300 DPI)


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Figure 4. Histograms of cross-sectional dimensions of the lamivudine needles in the microparticles, when colloidal silica of diameter (a) 450 nm, (b) 330 nm and (c) 165 nm is used. The needles are typically of elliptical cross-section, and the reported measurements are of width along the two principal axes (x and y), made from top-down SEM micrographs (see inset of (a)). The cross-sectional dimensions of the needles are seen to increase with colloid size. Scale bars represent 1 µm. 176x281mm (300 x 300 DPI)



Figure 5. SEM images of lamivudine-colloidal polystyrene (375 nm) microparticles. (a-c) Both particle macro- and microstructures are exactly analogous to the case of lamivudine-colloidal silica, as shown in Figure 3 (d-f). The microstructure is composed of tightly packed and highly aligned fine, fibrous sub-micron needles spanning the entire particle. (d) Position of the advancing solidification in lamivudine-colloidal polystyrene droplets; a higher linear growth rate is measured compared to neat lamivudine. (e) A histogram of lamivudine crystal dimensions measured as described in Figure 4; strikingly, the measured dimensions for 375 nm colloidal polystyrene are in between those measured for 165 nm and 450 nm colloidal silica respectively and are consistent with the crystal sizes for the 330 nm colloidal silica case, providing further evidence of the generality of microstructural dimensions tunability with the size of colloidal particle used. A representative top-down SEM micrograph is included in the inset, with the scale bar representing 1 µm. 462x298mm (300 x 300 DPI)


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Figure 6. (a) powder X-Ray Diffraction spectra of lamivudine from neat lamivudine particles, lamivudinesilica (330 nm) particles and lamivudine-polystyrene (375 nm) particles. The red dots above the neat lamivudine spectra mark out characteristic peaks for lamivudine polymorphic Form I. Two distinct characteristic peaks at 17.7° and 23.4° (marked with vertical dotted lines) were seen for both lamivudinesilica and lamivudine-polystyrene spectra, matched those of lamivudine Form V. (b) Differential Scanning Calorimetry (DSC) thermograms of lamivudine from neat lamivudine particles, lamivudine-silica (330 nm) particles and lamivudine-polystyrene (375 nm) particles. The melting point of lamivudine in the neat lamivudine particles at 176.8 °C corresponds to the Form I melting point.30 There lowered melting point for the lamivudine within the lamivudine-silica and lamivudine-polystyrene particles at 159.4 °C and 160.6 °C respectively corresponds to the Form V melting point. 31 104x151mm (300 x 300 DPI)




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Figure S1. An optical microscopy image of a droplet undergoing crystallization, where an opalescent irradiance is seen at the advancing solidification front. 117x110mm (300 x 300 DPI)



Figure S2. (a) FESEM image of lamivudine crystals in neat lamivudine microparticles. (b) Histograms of lamivudine crystal dimensions when formulated without silica. A SEM micrograph of the lamivudine needles is shown at the top right of the histogram, including an illustration of the reported lamivudine needle measurements along the two principal axes (x and y). The scale bar represents 5 µm. 252x96mm (300 x 300 DPI)


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Figure S3. powder X-Ray Diffraction spectra of known lamivudine forms – Form I to Form V from literature,1, 2 and from neat lamivudine particles, lamivudine-silica (330 nm) particles and lamivudinepolystyrene (375 nm) particles from this work. Reproduced and adapted from Ref. [1] with permission from the Royal Society of Chemistry. 119x176mm (300 x 300 DPI)



Figure S4. (a) A lamivudine-colloidal silica particle obtained from emulsions containing 400 mg/mL of colloidal silica and 80 mg/mL of lamivudine. The inset shows a higher magnification of the particle surface marked with a white box, revealing the close packing of silica. (b) A cross-section of a particle with cluster of lamivudine crystals with reduced aspect ratio (marked with a white circle). 182x62mm (300 x 300 DPI)


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Spherical Crystalline Anti-Retroviral Drug Particles with Tunable

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