Nanocrystallization confined to porous matrices with and ...

Nanocrystallization confined to porous matrices with and without

surface functionalization effects

By

Leia M. Dwyer

Bachelor of Science in Engineering in Chemical Engineering

University of Connecticut, 2013

Master of Science in Chemical Engineering Practice

Massachusetts Institute of Technology, 2015

Submitted to the Department of Chemical Engineering in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy at the

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

June 2018 © Massachusetts Institute of Technology 2018. All rights reserved.

The author hereby grants to MIT permission to reproduce

and to distribute publicly paper and electronic

copies of this thesis document in whole or in part

in any medium now known or hereafter created.

Signature of Author: __________________________________________

Department of Chemical Engineering

May 4th, 2018

Certified by: ______________________________________________

Allan S. Myerson

Professor of the Practice of Chemical Engineering

Thesis Supervisor

Accepted by: ________________________________________________

Patrick S. Doyle

Robert T. Haslam Professor of Chemical Engineering

Chairman, Committee for Graduate Students

1

Nanocrystallization confined to porous matrices with and without

surface functionalization effects

By

Leia M. Dwyer

Submitted to the Chemical Engineering Department on May 4th, 2018, in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

ABSTRACT

Poorly water-soluble active pharmaceutical ingredients (APIs), which represent a major fraction

of the molecules in drug discovery and development, are a challenge to the pharmaceutical

industry given their low bioavailability. One way to address this issue is to generate nanocrystals

of these APIs. Nanocrystals have a significantly increased surface area to volume ratio as

compared to standard micron-sized crystals, which results in improved solubility and dissolution

rates. There already exist some industrially relevant techniques for producing pharmaceutical

nanocrystals, which typically exploit contact forces and high pressures to bring crystals of a

normal micron range down to the nanocrystal scale. However, these techniques are often plagued

with challenges such as low production rates, high energy input, and issues with stabilization and

control over the final crystalline form produced. Because of this, techniques which produce

nanocrystals in the desired size range from the start are gaining interest.

In this work, crystallization in confinement is used to produce stable pharmaceutical nanocrystals

of a well-controlled size. Rigid, nanoporous silica matrices were used to confine crystallization

volumes to the nanoscale, resulting in the formation of nanocrystals within these pores. The

technique was demonstrated across a wide range of pore sizes, and using several poorly water-

soluble APIs. When the principles were extended to a two-stage continuous crystallizer setup,

the loadings of API in these porous matrices were improved to over 50 weight percent. When

these drug loaded porous silicas were tested in a dissolution rate apparatus, the resulting

dissolution profiles showed dramatic improvements as compared to the dissolution of bulk

micron-sized crystals. In the later stages of this research, porous silica with surface

functionalization was used rather than bare porous matrices. Herein, it was demonstrated that, at

the small pore volumes present in these systems, the surface functionalization from the media

may contribute enough functional group interaction to the solvent-solute system for the solubility

of a dissolved API within these pores to change. Thus, through the combination of surface

functionalization and confinement effects, this work demonstrated nanocrystallization from

undersaturated API solutions using functionalized nanoporous matrices.

Thesis Supervisor:

Allan S. Myerson

Title: Professor of the Practice

2

ACKNOWLEDGEMENTS

I would like to first thank my thesis advisor, Professor Allan S. Myerson. His guidance and

direction on my thesis work as well as personal development has been indispensable. He is kind

and generous with his time and support and I am extremely grateful to have been mentored by

him for my doctoral studies. My other thesis committee members, Professor Bernhardt L. Trout

and Professor William A. Tisdale, deserve my thanks as well. They brought support and helpful

questioning to my work. I have appreciated their input greatly.

Next, I would like to thank all of the members of the Myerson research group who have helped

me and made for a good working environment. In particular, I’d like to thank Dr. Marcus

O’Mahoney who assisted me when I joined the lab and my undergraduate student Luzdary and

collaborator Dr. Lucrèce Nicoud. Next, I thank Jennifer for her friendship as both a lab-mate and

Communication Lab fellow, and for braving conference travel with me. Of course, my

experience in the lab has been critically shaped for the better by the support of my office-mate

Carlos Pons Siepermann, who has been on this journey with me from joining the lab to now

graduating. Not every lab-mate becomes a true friend, but I am lucky to have gone through this

experience with such a supportive, energetic, enthusiastic, and caring individual as Carlos.

I would also like to thank members of the department and MIT at large for helping me establish a

community where I have enjoyed spending my time. The many intramural participants who

joined me on various teams have given me a great sense of camaraderie and fun memories. I’d

like to thank my fellow ChemE Communication Lab members for starting something new

together during this past inaugural year. The MIT Student Art Association ceramics studio has

been a critical component of feeding my creativity and maintaining my sanity during my doctoral

work. I thank Darrell for his mentorship, positive attitude, and constant teaching moments as

well as Jay for keeping the studio lively and fun. I also thank the MIT Recreation community, in

particular Anna, James, and Fen, who have helped me strengthen my mind and body.

I am ever grateful for the support of the many friends I am lucky to have made here. I thank

everyone who joined me on one of my many hiking or camping adventures to get a dose of

nature away from Cambridge, as well as those who adventured in Cambridge during board game

nights. A special thanks goes to Kristen for prioritizing a lunch break every day and for always

being there for me through any problem. Of course, I thank my dear friends who have also been

my roommates for their never-ending support: Brinda, Kathryn, Nikunja, and Sepideh.

Of course I thank my family, my rocks, for their support, realism, delightful eccentricity, and

unconditional love. To Cara, the Carl to my Jimmy. To Garth, finally an older brother to nerd out

with. To my sister Laurel, my first best friend, who will always know my mind like no other. To

my father, Robert, who has made the opportunities I’ve had in life possible and who I know I

need never worry about disappointing. And finally, to my mother, Ellen, my first teacher, for

whom I strive every day to face the world with childlike wonder and cherish life in all its

richness. Without you all, the journey would mean nothing, and I have been lucky.

Leia M. Dwyer

3

TABLE OF CONTENTS

Chapter 1 : Introduction ................................................................................................................ 15

1.1 Introduction to crystallization in confinement ............................................................... 15

1.2 Introduction to surface functionalization effects in crystallization ................................ 19

1.3 Thesis outline ................................................................................................................. 22

Chapter 2 : Confinement effects on properties of pharmaceutical nanocrystals .......................... 26

2.1 Introduction ......................................................................................................................... 26

2.2 Experimental ....................................................................................................................... 28

Materials ............................................................................................................................... 28

Experimental Apparatus........................................................................................................ 28

X-Ray Powder Diffraction Analysis ..................................................................................... 29

Differential Scanning Calorimetry Analysis ......................................................................... 30

Thermogravimetric Analysis ................................................................................................ 30

Solid-state Nuclear Magnetic Resonance Spectroscopy ....................................................... 30

Dissolution testing ................................................................................................................ 31

2.3 Results and discussion ........................................................................................................ 32

Crystal form identification with XRPD ................................................................................ 35

Crystal form identification with ssNMR ............................................................................... 37

Analysis of melting point depression of nanocrystals by DSC ............................................. 38

4

Enhanced dissolution profile for nanocrystalline fenofibrate ............................................... 42

Extension to Aeroperl® porous matrix ................................................................................. 44

2.4 Pore size and shape effects on dissolution rates ................................................................. 46

Understanding dissolution profile factors ............................................................................. 46

Dissolution Profile Modeling ................................................................................................ 49

Experiments to understand transport from a porous matrix.................................................. 52

2.5 Conclusions ......................................................................................................................... 55

2.6 Acknowledgements ............................................................................................................. 56

2.7 Appendix ............................................................................................................................. 57

Chapter 3 : Continuous crystallization in confinement to improve matrix loading ...................... 72

3.1 Introduction ......................................................................................................................... 72

3.2 Experimental ....................................................................................................................... 75

Materials ............................................................................................................................... 75

Experimental apparatus ......................................................................................................... 75

Analytical techniques ............................................................................................................ 79

3.3 Results and discussion ........................................................................................................ 82

Selection of MSMPR operating parameters ......................................................................... 82

Analysis of FEN loading in MSMPR experiments ............................................................... 83

Dissolution profile enhancement .......................................................................................... 86

5

Melting point depression analysis of nanocrystals with DSC .............................................. 87

Crystal form identification with X-Ray powder diffraction (XRPD) ................................... 89

Extension of principle to poorly soluble compounds ........................................................... 90

3.4 Conclusions ......................................................................................................................... 92

3.5 Acknowledgements ............................................................................................................. 93

Chapter 4 : Understanding nanoscale surface functionalization’s role on supersaturation in

crystallizing solutions ................................................................................................................... 94

4.1 Introduction ......................................................................................................................... 94

4.2 Experiments and results ...................................................................................................... 96

Materials for solvent/solute systems ..................................................................................... 96

Production and size control of iron oxide nanoparticles ....................................................... 96

Functionalization of silica-coated nanoparticles ................................................................. 102

Continued testing of functionalized nanoparticles in relevant systems .............................. 105

Initial results indicating effects on heterogeneous nucleation ............................................ 106

Aggregation of nanoparticles and its role in the failure of the intended mechanism ......... 111

4.3 Conclusions ....................................................................................................................... 113

4.4 Acknowledgements ........................................................................................................... 114

Chapter 5 : Surface functionalization in combination with confinement for crystallization from

undersaturated solutions.............................................................................................................. 115

5.1 Introduction ....................................................................................................................... 115

6

5.2 Experimental ..................................................................................................................... 118

Materials ............................................................................................................................. 118

DSC and TGA analysis ....................................................................................................... 118

XRPD analysis .................................................................................................................... 119

Submerged loading experiment .......................................................................................... 119

Column loading experiment ................................................................................................ 120

Capillary crystallization experiment ................................................................................... 122

5.3 Results and discussion ...................................................................................................... 123

Submerged loading experiment .......................................................................................... 123

Column loading experiment ................................................................................................ 126

Capillary crystallization experiment ................................................................................... 135

5.4 Summary and application of the work .............................................................................. 137

Submerged loading experiments ......................................................................................... 137

Column loading experiments .............................................................................................. 138

Capillary crystallization experiments ................................................................................. 138

Proposed extensions of the technology ............................................................................... 139

5.5 Acknowledgements ........................................................................................................... 142

Chapter 6 : Conclusions and recommendations .......................................................................... 143

6.1 Outlook for use of nanoconfinement for crystallization ................................................... 143

7

6.2 Future work on using surface functionalized materials for crystallization in confinement

................................................................................................................................................. 145

Chapter 7 : References ................................................................................................................ 147

8

LIST OF FIGURES

Fig. 1: Organic molecule of interest, fenofibrate (FEN) for API loading in porous silica used for

this study ....................................................................................................................................... 28

Fig. 2: Loading procedure to impregnate porous silica particles with API solution .................... 29

Fig. 3: XRD patterns of bulk fenofibrate presented in (a), fenofibrate loaded on 53 nm CPG in 3

distinct trials in (b), and fenofibrate loaded on three representative pore sizes of CPG in (c) ..... 36

Fig. 4: 13C CP MAS NMR spectra of fenofibrate: molecule (top), bulk drug (middle) and loaded

in 20 CPG (bottom) ....................................................................................................................... 38

Fig. 5: DSC scans of all CPG pore sizes showing single peaks (no surface crystals) with

increasing melting point temperatures. Peaks are separated by color and correspond left to right

to increasing pore sizes 20 to 300 nm. .......................................................................................... 39

Fig. 6: Constant enthalpy-constant surface interaction energy Gibbs-Thomson equation fit to

melting points of fenofibrate nanocrystals confined to porous silica. .......................................... 41

Fig. 7: Enthalpy of fusion of fenofibrate nanocrystals showing a linear relationship with 1/d

across varying CPG pore sizes. ..................................................................................................... 42

Fig. 8 Average dissolution profiles of all CPG-confined fenofibrate nanocrystals showing

enhanced dissolution rates compared to crushed and uncrushed bulk fenofibrate, also shown ... 43

Fig. 9: Comparison of the most enhanced dissolution profile of nanocrystalline fenofibrate in 70

nm CPG compared to bulk crushed fenofibrate ............................................................................ 44

9

Fig. 10: XRPD scans of fenofibrate confined to CPG versus Aeroperl(R) show that it is the same

crystalline form ............................................................................................................................. 45

Fig. 11: Dissolution enhancement profiles showing Aeroperl® outperforming all other porous

matrices ......................................................................................................................................... 46

Fig. 12: Mass transport schematic diagram of nanocrystal dissolution and diffusion from porous

beads ............................................................................................................................................. 48

Fig. 13: Schematic representation of diffusion path length and geometry for basic transport

model of nanocrystal diffusion from within a bead. ..................................................................... 50

Fig. 14: Tortuosity model compared with experimental results for dissolution profiles .............. 51

Fig. 15: Porous supports used for fenofibrate loading: anodic alumnimum oxide (left) and porous

silica (right) ................................................................................................................................... 53

Fig. 16: The results of the dissolution testing of FEN loaded on AAO indicate that the loading

was not confined to the pores, nanocrystals were not produced, and the dissolution profile was

not improved over the bulk form of the drug ................................................................................ 54

Fig. 17: Crystallizer design schematic showing the (a) single-stage design and (b) two-stage

design for improved drug loading ................................................................................................. 78

Fig. 18: Comparison of the MSZW of FEN in ethyl acetate showing a decrease in the width with

the addition of porous silica .......................................................................................................... 83

10

Fig. 19: FEN confined to 35 nm Aeroperl® showing dramatically enhanced dissolution profiles

when compared to the bulk crystals .............................................................................................. 87

Fig. 20: Constant-enthalpy surface interaction energy Gibbs-Thomson equation fit to melting

points of confined nanocrystalline FEN averaged from all MSMPR runs ................................... 89

Fig. 21: XRPD scans of FEN nanocrystals from various runs show crystallinity and consistent

formation of form I ....................................................................................................................... 90

Fig. 22: DSC scans of GSF in porous silica compared to bulk crystals show that the two-pass

loading technique was able to produce confined nanocrystals with no significant surface crystals

....................................................................................................................................................... 92

Fig. 23: Proposed schematic of the technique in the case where the nanoparticles are acting as

antisolvent ..................................................................................................................................... 95


solvent ........................................................................................................................................... 95

Fig. 25: Size description of synthetic IONPs as measured by DLS............................................ 100

Fig. 26: TEM picture of the iron oxide nanoparticles from Spherotech ..................................... 101

Fig. 27: Scheme of the mechanism of silane deposition on the silica surface ............................ 102

Fig. 28: FTIR spectrum of the silica coated iron oxide nanoparticles functionalized with

hexyltriethoxysilane. As a comparison, the FTIR spectra of the plain nanoparticles and of the

11

silane are also shown. The spectra on the right-hand side of the figure are similar to those on the

left-hand side but with a different scale ...................................................................................... 104

Fig. 29: TGA analysis of the silica coated iron oxide nanoparticles functionalized with hexane

(silane SIH6167.5). ..................................................................................................................... 105

Fig. 30: Experimental data of the solubility of DPH as a function of the volumetric percentage of

heptane (symbols). The black line represents the dilution line upon heptane addition. ............. 108

Fig. 31: Crystallization likelihood at various concentrations of heptane and bare nanoparticles.

Five samples were prepared for each condition 103 ..................................................................... 109

Fig. 32: Crystallization of DPH at 33 % heptane and various concentrations of bare nanoparticles

followed by FBRM. (left) Total number of counts as a function of time. (Right) Chord length

distribution at 1 hour ................................................................................................................... 110

Fig. 33: Pictures of DPH crystals formed at 43 % heptane with 0.25 g/L (left) bare and (right)

decane functionalized nanoparticles 103 ...................................................................................... 111

Fig. 34: FBRM indicates that the nanoparticles are around 20 microns in size, indicating

substantial aggregation 103........................................................................................................... 112

Fig. 35: Images taken with a particle vision ParticleView V19, Mettler Toledo show

nanoparticles before crystallization (left) and in the presence of DPH crystals (right) 103 ......... 112

Fig. 36: Schematic of the column setup for porous matrix loading experiments ....................... 121

12

Fig. 37: DSC scans of porous glass material loaded with a submerged basket from an

undersaturated solution of FEN in ethyl acetate (550 mg/mL). The Zorbax® material shows two

distinct melting points indicating the presence of crystallization in the pores. .......................... 125

Fig. 38: Average FEN loading on functionalized Zorbax® chromatographic media versus parent

solution concentration ................................................................................................................. 126

Fig. 39: Flowthrough concentration of DPH collected as the column was run with CPG shows

little change ................................................................................................................................. 127

Fig. 40: Trace DPH with bulk melting temperature only seen in the CPG powder collected from

the column runs ........................................................................................................................... 127

Fig. 41: Flowthrough concentration of DPH in isopropyl alcohol on runs where the column was

packed with Zorbax® show a significant decrease indicating DPH retention on the column.... 129

Fig. 42: DSC scan of the Zorbax® material collected from the column run showing DPH

confined to the pores of the material........................................................................................... 129

Fig. 43: Flowthrough concentration of solids collected as the column was run with CPG and

impurity shows little change ....................................................................................................... 131

Fig. 44: Flowthrough concentration of solids in isopropyl alcohol on runs where the feed also

contained impurity. The column was packed with Zorbax® and shows a significant decrease in

flowthrough solids concentration indicating DPH retention on the column ............................... 132

13

Fig. 45: DSC scan of the Zorbax® material collected from the column run with impurity in the

feed solution showing DPH confined to the pores of the material ............................................. 132

Fig. 46: XRPD of loaded Zorbax® collected from the column runs in which an impurity was

added to the feed solution. Neither column run's collected Zorbax® shows any trace of the

impurity, showing crystalline DPH only .................................................................................... 134

Fig. 47: XRPD of the solids collected from the flowthrough material on the column from runs in

which an impurity was added to the feed solution. The flowthrough is a clear mix of both DPH

and benzophenone. ...................................................................................................................... 134

Fig. 48: Time series XRPD scans of the API solutions loaded in capillary tubes filled with

Zorbax® media showing crystallization within 1 hour .............................................................. 137

14

LIST OF TABLES

Table 1: Pore sizes, surface areas, and volumes of porous silica used in the study as provided by

producer ........................................................................................................................................ 32

Table 2: Fenofibrate loaded in porous silica particles .................................................................. 33

Table 3: Comparison of bead pore and grain sizes used in API release studies ........................... 49

Table 4: Modeling expressions for release of dissolved API from straight and tortuous pores ... 50

Table 5: Room temperature (25 °C) MSMPR loading summary of FEN wt% in porous silica ... 84

Table 6: Loading results for FEN wt% in porous silica in single-stage MSMPR as a function of

filter temperature ........................................................................................................................... 85

Table 7: Loading results of multi-step procedure for nucleation then growth of crystals to

increase the weight percent loading .............................................................................................. 91

Table 8: Summary of the commercial nanoparticles under investigation ................................... 101

15

Chapter 1 : Introduction

1.1 Introduction to crystallization in confinement

Traditional crystallization of active pharmaceutical ingredients (APIs) in both batch and

continuous crystallization is the main separation and purification process for these ingredients.

The resulting crystal population must be controlled for purity, crystal form, crystal shape, and

particle size, among other properties 1. These parameters have implications for other downstream

processing techniques, such as filtering and drying, as well as the effectiveness of the drug itself

as a medicine 2. Various methods to supply the driving force for crystallization, including

cooling, evaporation, and antisolvent addition, can result in differences in the resulting crystal

population and are therefore controlled to meet target product profiles 3.

A major issue facing the pharmaceutical industry is the processing and development of APIs

which are poorly water-soluble. It has been estimated that up to 40% of pharmaceuticals in the

development phase and up to 70% in the discovery phase are poorly water-soluble molecules 4.

This poses a challenge in the body for uptake of these pharmaceuticals, given that the

bioavailability is directly related to the dissolution rates as well as permeability within the body,

composed of aqueous environments 5.

Nanocrystals of APIs offer a simple physical solution to this issue. Because of their high surface

area to volume ratios, the dissolution behavior of pharmaceutical nanocrystals is much improved

over their bulk crystal counterparts 6. Furthermore, while the saturation solubility of bulk crystals

is fixed for a given molecule in a given solvent at a temperature and pressure, below the micron

range saturation solubility is additionally a function of particle size 7. Pharmaceutical

16

nanocrystals have now become an established topic of interest in the industry, and many

techniques already exist for producing them, some of which are actively used in the processes for

APIs currently on the market.

Broadly, the techniques for producing pharmaceutical nanocrystals may be described as top-

down or bottom-up approaches. Top-down approaches take bulk crystals produced at a larger

size, typically in the micron range, and process them down to the nanoscale range. Methods of

this type include high pressure homogenization and various forms of milling 8. High pressure

homogenization standardly applies a very high pressure to mixture of crystals in solvent 9. Often,

the suspension is passed through a valve or membrane with extremely small slits to result in

shear forces in addition to high pressure. Alternatively, a high pressure stream may be aimed at

physical elements such as blades or plates to produce impact forces, or take advantage of

cavitation within a system 10. High pressure homogenization has been employed in many

different pharmaceutical systems, and typically produces crystals in the range of 50-500 nm.

However, the high pressure in the system may cause changes in the crystal structure in addition

to the crystal size, including a transformation to the amorphous form of the drug. These present

serious issues for a finely controlled pharmaceutical product 5.

Milling is the other approach commonly used to bring crystals from the micron range down to

nanocrystal size, typically resulting in particles not smaller than about 500 nm. In milling

procedures, compression, shear, and impact stresses are imparted to the crystals, typically in a

liquid suspension, through a milling media. This media is a hard and dense material, typically

present in the form of beads 11. Due to the straightforward nature of these technologies, the top-

down approaches are those most commonly used today for pharmaceutical nanocrystal products

17

on the market 12. The most prevalent issue with milling is contamination of the pharmaceutical

product by the milling media or apparatus 8.

Bottom-up approaches instead produce crystals in the desired nanoscale range from the onset at a

molecular level. Some of these techniques are based on solvent evaporation to provide the

driving force for nucleation, including spray-drying, electrospraying, and cryogenic spray

processes 6. In these techniques, the volume of evaporating solution is limited, and thereby

controls the resulting crystal size. This often results in low production volumes for these

techniques 6. Furthermore, the high pressures or low temperatures required in these techniques

renders them quite energy intensive 8. Others use the driving force of antisolvent addition,

namely liquid antisolvent addition and supercritical antisolvent addition. These processes

produce nanocrystals in a similar size range to the top-down approaches, typically around 500

nm, but often result in very broad particle size distributions which is undesirable 13.

An issue with all of the methods discussed above is the stabilization of nanoparticle suspensions.

In all of these methods, the nanocrystals are typically produced and held in a liquid suspension,

often to which surfactants or polymers are added to stabilize the nanocrystals and prevent

aggregation to keep them in the desired size range. This results in another additive to the drug

formulation with potential adverse side effects for some patients 5.

Thus, nanocrystallization in confinement has emerged as a viable method for producing

pharmaceutical nanocrystals from a bottom-up approach that doesn’t require subsequent

stabilization of a nanocrystal suspension. Here, various matrices which can control the

crystallization environment are used. Typically porous in nature, the crystals are made in and

thereby confined to these matrices. Materials which have been used for confinement matrices

18

include ordered mesoporous silica 14–21, controlled pore glass (CPG) 22,23, porous

polycyclohexylethylene and polystyrene (p-PCHE) 22,24, nanostructured lipid carriers 25, fumed

silica 26, and electrospun materials 27. While some of these materials have also been used to

confine the amorphous form of APIs, keeping the API in its crystalline state with associated

polymorphism is more desirable for long term stability of the formulated products 28,29. The

resulting product of a crystal confined to a porous matrix stabilized by the matrix itself has the

advantage of not needing any additional surfactants or polymers to maintain the desired size

distribution. Control over the size of the pores present in the matrices lends itself to control over

the size of the nanocrystalline pharmaceuticals produced.

19

1.2 Introduction to surface functionalization effects in crystallization

Heterogeneous nucleation, the formation of a crystal at a surface, plays a large role in

crystallization processes. This is particularly true in industrial processes where large-scale

crystallizers involve many surfaces such as vessel walls, stirring blades and shafts, and baffles,

making heterogeneous nucleation the major contributor in these processes. However, even in

small-scale research situations, the heterogeneous nucleation of crystals on surfaces such as vial

walls, stir bars, or undesirable particulates is an important factor with respect to the end

crystalline product.

For research purposes, often surface modifications are made to a heterogeneous surface to study

the effects on nucleation and crystal growth. In particular, self-assembled monolayers (SAMs)

have been broadly used to examine the effects of functionalization on promoting or obstructing

crystallization at surfaces. These are surfaces to which surface functional groups have been

chemically added, resulting in an outward-facing functionality or functionalities30. When these

SAMs have been used in crystallization studies, typically the functionalities have been selected

to either promote or suppress crystallization though interactions with the functional groups of

crystallizing molecules, or preferential facial interactions with different crystalline faces of a

particular form 31. Previous studies have shown the use of SAMs as crystallization templates to

have effects on crystal polymorphism, size, morphology, and nucleation rate kinetics 23,32–36. In

these systems, the interaction between the solute, either in molecular or crystalline form, and the

SAM functionality is the basis for selective crystallization.

20

Antisolvent crystallization is one of the most relevant industrial crystallization techniques. In this

method, a solvent in which a solute is poorly soluble, an antisolvent, is added to a solution of

dissolved solute in a highly soluble solvent. The addition of the antisolvent changes the solvent

makeup, and the solubility of the solute in the new solvent system decreases. Thus, the driving

force for nucleation is supplied and crystallization may occur 3,37.

A theory was proposed in our lab and demonstrated in proof-of-concept work that perhaps the

solvent makeup of a system could be changed by the introduction of functional groups through

the addition of surface functionalized nanoparticles 38, building upon well-established SAM

crystallization principles. However this work is different from most studies involving SAMs, in

that here the surface functionality is being used to modify the solution phase of the system, as

opposed to interact with the solute. The motivating thought process behind this work was that the

surface area of an addition of nanoparticles could be so high that the addition could contribute

reasonably to the surface functionality present in the solvent phase of a system, thus changing a

dissolved solute’s saturation solubility. This work was tested in normal, bulk crystallization

experiments with some success, however it was desired to understand how it might extend to

crystallization in confinement as well.

Due to difficulties knowing the exact structure of crystals confined to porous environments as

discussed above, it is unclear how much of a role the surface of the porous confinement media

plays in the crystallization process in confinement. It is likely that crystals form preferentially in

the larger volumes at intersections of pore channels; however, it is also possible that adherence to

the pore walls plays a role in the crystallization in confinement. It may be conjectured that the

heterogeneous effect is potentially quite important, given the small pore sizes, the relative

21

surface area of the walls of the confinement media to the pore volume in a given pore is high.

Understanding how concepts of surface functionalization which apply to crystal nucleation in

these previous studies with nanoparticle addition and SAM surfaces might translate to

crystallization in confinement is an important motivator for some of the investigations carried

out in this thesis.

22

1.3 Thesis outline

This thesis began with the objective of understanding how nanocrystals may be produced in

confinement in a stable, reproducible way and how this may affect their physical properties. We

were particularly interested in how the dissolution rate behavior would change, as this is the

metric most closely aligned with how the drug will behave in the body. As we then gained an

understanding of the role of nanoscale surface functionalization in crystallizing solutions, we

began to investigate how surface functionalization effects could work in combination with

crystallization in confinement.

In Chapter 2, we present a study to crystallize a single pharmaceutical molecule, fenofibrate, in a

range of different pore sizes in porous silica matrices. We found that something fundamentally

lacking from other studies of crystallization in confinement was a systematic investigation across

a broad range of pore sizes. Thus, we obtained and used porous silica of pore sizes from 300 to

12 nm. A basic benchtop procedure was developed for loading parent API solution into these

matrices, and evaporation was used to provide the driving force for crystallization. The resulting

drug-loaded porous silica was subjected to a variety of characterization techniques. It was found

that we achieved reasonable loadings of API in the porous matrices, at about 30 weight percent.

The thermal techniques showed that the melting point behavior of the crystals behaved well

according to a Gibbs-Thomson relationship. The dissolution profiles of the drugs confined to

porous silica had dramatic improvements relative to the bulk crystal dissolution profiles. In

particular, the fenofibrate contained to Aeroperl®, a fumed silica used in the study, had greater

than 80 percent dissolution in just 22 minutes. Differences in the dissolution profiles led to a

further brief investigation into how the dissolution profiles are influenced not just by nanocrystal

23

dissolution but also by transport from the porous matrices, dependent upon pore tortuosity and

grain size of the matrices.

In Chapter 3, we look at the advantages of using a continuous crystallization setup to produce

nanocrystals confined to porous matrices and improve the loadings. Continuous crystallization is

a technique well used in both our lab and industry, as it has advantages in product consistency

and scalability. We designed a single-stage mixed suspension mixed product removal (MSMPR)

crystallizer to load the porous silica matrices used in our previous work with API. We then

extended the principle to a two-stage design, in which the first stage was intended to load the

crystals into the pores. Then, a second stage was added to grow the crystals within the pores

ideally without nucleating additional crystals on the surfaces of the matrices or in solution. We

demonstrated that the two-stage design can improve the loading of API in these matrices

significantly, up to greater than 50 weight percent. The concept to have a second growth stage

after crystals were already nucleated within the pores in a first stage led us to extend the

principle in a benchtop procedure to other poorly water-soluble drugs, griseofulvin and

indomethacin. Ultimately, the poor solubility of these two candidate drugs in many solvents led

to poor loadings in the porous matrices.

Next, we changed the theme of investigation slightly to look at surface functionalized

nanoparticles as additives to crystallization systems. Building on previous results from within the

lab, it was speculated that the addition of surface-functionalized nanoparticles to a solvent

system would add so much resulting functionality due to the high surface area of these particles

that it could substantially change the functional group makeup of the solvent system. Thus, the

solubility of a dissolved solute may change in these systems. Several types of surface-

24

functionalized nanoparticles were investigated in this study: pure gold particles, iron oxide

particles, and silica-coated iron oxide particles. We aimed to achieve a balance of particle size,

permanent surface functionality, and easy separation due to the magnetic nature of the iron oxide

used. Ultimately, however, we found that the achievable size of nanoparticles that had the

desired magnetic and surface functional properties was just too large to be feasible for the

proposed applications. Aggregation of the nanoparticles and an inability to produce them below

200 nm resulted in the required quantity of added nanoparticles to achieve reasonable surface

functionalization addition to the solvent makeup being far too high to be practical.

However, this led us to wonder how we may combine surface functionalization with

crystallization in confinement, to tie together the two somewhat separate avenues of the work

that had been conducted thus far. A chromatographic material, Zorbax® was investigated which

is a porous silica of about 7 nm pore diameter. It has a surface functionalization resulting in C8-

like functionality. We therefore proposed that APIs which showed poor solubility in alkane-type

solvents may be good candidates for testing with this material. We proposed that the addition of

the Zorbax® media would provide the driving force for crystallization because of the unique

combination of the physical confinement in a nanoscale volume with the additional of surface

functional groups. A benchtop, column, and capillary crystallization procedure were investigated

for the crystallization of APIs from undersaturated solutions using Zorbax® functionalized

media. We demonstrate that Zorbax®, through the combined effects of functionalization and

nanoconfinement, was able to provide the required driving force for crystallization in these

systems, resulting in pharmaceutical nanocrystals confined to Zorbax® media.

25

Finally, in Chapter 6, the thesis is concluded with remarks on the research conducted and

recommendations for future studies that build upon the foundations of this work.

26

Chapter 2 : Confinement effects on properties of pharmaceutical

nanocrystals

2.1 Introduction

Pharmaceutical nanocrystals have been targeted as a solution for improving the bioavailability of

poorly water soluble pharmaceuticals 5,39 . Nanocrystals have an increased surface area to

volume ratio compared to their bulk crystal counterparts and can increase dissolution rate

according to the Noyes-Whitney equation 40 and enhance permeability 4. It has also been shown

that the solubility increases with decreasing particle size below a cut-off size of 1-2 μm 41. It is

desirable to produce nanocrystals directly without resorting to other processing steps such as

milling to achieve nanocrystal size while also controlling the polymorphism of the crystal 42.

Several bottom-up methods to produce nanocrystals of a given size in reproducible polymorphs

exist. These include the “hydrosol” method 41, freeze-drying 42, supercritical fluid methods 43,44,

cryogenic spray processes 45, and evaporative precipitation into aqueous solution 46,47. These

processes often produce amorphous material or an undesired polymorphic form. In addition,

control of size distribution and can be difficult to scale up. They can be plagued with low

production rates and typically do not achieve particle sizes below 100 nm 5,8,42,48.

Alternative approaches for producing stable pharmaceutical nanocrystals employ the bottom-up

approach of conducting the crystallization in confinement. Ordered mesoporous silica 14–21,

controlled pore glass (CPG) 22,23, porous polycyclohexylethylene and polystyrene (p-PCHE) 22,24,

nanostructured lipid carriers 25, fumed silica 26, and solutions of active pharmaceutical ingredients

27

(APIs) in electrospun materials 27 have all been used to confine APIs to small volumes 49.

Confining crystals to porous matrices of known size addresses the issue of particle size

distributions, and has been proposed to lead to higher polymorph control through regulation of

nucleation 50 as well as the stabilization of otherwise metastable polymorphs 51. Producing

confined nanocrystals has also been proposed as a way to better understand fundamentals of

polymorph formation, due to the unique surface energy effects of the confined systems 29.

Ha et al. 22 have found a Gibbs-Thomson like relationship between melting point depression and

crystal size in crystals confined to CPG and p-PCHE. They found size-dependent polymorphism

and the potential for polymorph discovery with varying pore size, making particular note of the

interplay between surface energy and volume free energy at the nanoscale. Three pore sizes of

CPG and one size of p-PCHE monolith were studied.

This work aims to explore the crystallization of APIs in rigid nanoporous media over a broad

range of pore sizes, which is lacking in existing studies, allowing a fundamental understanding of

the relationship between pore size, crystallinity and bioavailability. The API fenofibrate (shown

in Fig. 1), which is known in two polymorphic forms, was crystallized over a range of pore sizes

(10 different pore sizes between 12 and 300 nm) of CPG and a porous fumed silica, Aeroperl®.

The drug loadings were determined with thermogravimetric analysis (TGA) and the nanocrystal

melting points and enthalpies of fusion were studied with differential scanning calorimetry

(DSC). Crystallinity was assessed with X-ray powder diffraction (XRPD), while both

polymorphism and degree of crystallinity was studied using solid-state nuclear magnetic

resonance (ssNMR). It is the intent that the porous matrices used be biocompatible excipient

28

media, such that a formulation of the nanocrystalline API could conceivably be as simple as

encapsulated API-loaded matrix.

Fig. 1: Organic molecule of interest, fenofibrate (FEN) for API loading in porous silica used for this

study

2.2 Experimental

Materials

Fenofibrate (FEN) was obtained from Xian Shunyi Bio-chemical Technology Company. Silicon

dioxide (silica) particles of varying pore sizes were obtained from three sources. Controlled pore

glass (CPG) was obtained from Millipore in pore sizes of 300 nm and 70 nm. CPG was also

obtained from Prime Synthesis in pore sizes of 191.4, 151.5, 105.5, 53.7, 38.3, 30.7, 20.2, and

12.7 nm. Colloidal fumed silica which fulfills requirements of Ph. Eur. as well as the USP/NF

(AEROPERL ® 300 Pharma) were obtained from Evonik USA. AEROPERL ® consists of

bead-like mesoporous granules with a pore size of ~35 nm 26.

Experimental Apparatus

Experimental setup is shown in Fig. 2: (1) A small amount (~ 0.25 g) of CPG was placed in a 20

mL scintillation vial, resulting in a CPG bed height of about 0.3 cm and a top surface area of ~

29

3.1 cm2. For this study, the preparation of 0.25 g of CPG to be loaded with drug was plenty for

analytical purposes. (2) The pore volume present in the entire CPG sample was then calculated

based on the given pore volume/gram CPG. A 60% weight/volume solution of fenofibrate in

ethyl acetate was prepared. API solution in equal amount to the pore volume present in the CPG

was then micropipetted over the surface of the CPG in the scintillation vial as uniformly as

possible. (3) Immediately after pipetting, a metal spatula was used to stir the mixture, to wet as

much of the CPG as possible, ceasing only when the mixture appeared dry. The drug-loaded

CPG was then left in a fume hood for an additional 24 hours to continue evaporation of excess

solvent. It is noteworthy that no wash step was required in this method. Samples were prepared

in triplicate for each pore size.

Fig. 2: Loading procedure to impregnate porous silica particles with API solution

X-Ray Powder Diffraction Analysis

X-Ray powder diffraction (XRPD) was performed on all samples using a PANalytical X’Pert

PRO diffractometer at 45 kV with an anode current of 40 mA. The instrument has a PW3050/60

standard resolution goniometer and a PW3373/10 Cu LFF DK241245 X-ray tube. Samples were

placed on a spinner stage in reflection mode. Settings on the incident beam path included: soller

30

slit 0.04 rad, mask fixed 10 mm, programmable divergence slit and fixed 1º anti-scatter slit.

Settings on the diffracted beam path include: soller slit 0.04 rad and programmable anti-scatter

slit. The scan was set as a continuous scan: 2θ angle between 4 and 40 º, step size .0167113 º and

a time per step of 31.115 s.

Differential Scanning Calorimetry Analysis

A Q2000 instrument from TA instruments was utilized for the differential scanning calorimetry

(DSC) analysis. Inert atmosphere environment was maintained in the sample chamber using a

nitrogen gas cylinder set to a flow rate of 50 ml/min. An extra refrigerated cooling system (RCS

40, TA instruments) was used to broaden the available temperature range between -40 and 400

ºC. Tzero ® pans and lids were used with ~5 mg of sample. A heating rate of 10 ºC/min was

applied and the samples were scanned from -20 to 180 ºC. When determining the enthalpy of

fusion for a given sample, the DSC curve was integrated for 30 ºC centered on the melting

temperature of each pore size to capture the entire melting event.

Thermogravimetric Analysis

Thermogravimetric analysis (TGA) was performed on a Q500 instrument from TA instruments

connected with a nitrogen gas cylinder to maintain a flow rate of 25 mL/min to keep the sample

chamber under an inert gas environment. Between 5 and 10 mg of sample were loaded on

platinum sample pans from TA instruments. The samples were allowed to equilibrate at 30 ºC

and then heated at 10 ºC/min to 300 ºC.

Solid-state Nuclear Magnetic Resonance Spectroscopy

Solid-state nuclear magnetic resonance experiments (ssNMR) were conducted on a homebuilt

500 MHz spectrometer (D. Ruben, Francis Bitter Magnet Laboratory (FBML), Massachusetts

31

Institute of Technology). Prepared samples were packed into Revolution NMR (Fort Collins,

USA) 4 mm o.d. (60 μl fill volume) ZrO2 rotors, equipped with Vespel drive and top caps.

Spectra were acquired on a 4 mm Chemagnetics triple resonance (1H/13C/15N) magic-angle

spinning (MAS) probe. 13C natural abundant spectra were acquired using cross-polarization (CP)

52, a recycle delay of 3 seconds, between 16,384 and 65,536 co-added transients and a spinning

frequency of 9,000 ± 3 Hz. The Hartman-Hahn match condition was optimized by setting 1H to

50 kHz (ƔB1/2π), a positive ramp contact pulse for 13C (centered at 58 kHz) and a contact time of

1.5 ms. All data were acquired using two-pulse phase-modulated, TPPM 53 1H decoupling (100

kHz, 1H ƔB1/2π). The magic-angle was adjusted using potassium bromide (KBr) at a spinning

frequency of 5 kHz, (rotational echoes > 11.5 ms). 13C spectra were referenced (and shimmed,

FWHM = 4 Hz) using solid adamantane to 40.49 ppm (high frequency resonance) with respect to

DSS (0 ppm).

Dissolution testing

The dissolution tests were designed following USP standards. Analysis of the percentage of

dissolved API was done using built-in ultraviolet-visible spectroscopy at 286 nm. The

dissolution buffer used was .025 M sodium dodecyl sulfate solution (7.21 grams of powdered

SDS (Sigma Aldrich) was dissolved and brought up to 1000 mL in water). The dissolution

profile of the sample was determined using USP Dissolution Apparatus 2 at 37 ºC. The apparatus

operated at 75 RPM. 900 mL of the buffer solution was allowed to reach the equilibrium

temperature before sample was placed in the apparatus. Enough sample of API-loaded CPG was

added such that the targeted concentration of fenofibrate in solution was 15 μg/mL, within the

32

expected linear range 54. Samples of both uncrushed and crushed bulk fenofibrate were analyzed

as comparison. Samples were acquired for about 29 hours.

2.3 Results and discussion

Fenofibrate was selected as a model API to work with in preliminary studies. It is poorly water

soluble, < 1 mg/mL at 37 ºC 55 , and has two known polymorphs, crystalline form I with a

melting point around 80 ºC and a metastable form II with a melting point around 73 ºC 56,57. The

metastable form has been collected in a sample of amorphous fenofibrate that was heated to

around 40ºC 56. Fenofibrate was chosen for initial studies due to its lack of multiple stable

polymorphs; it is advantageous to first study how a single polymorph changes with varying

crystal size. Table 1 summarizes the sizes of porous media used and the pore volumes as

provided by the supplier.

Table 1: Pore sizes, surface areas, and volumes of porous silica used in the study as provided by

producer

Pore Size (nm) Pore Volume (cc/gram) Producer

300 >1 Millipore

191.4 1.5 Prime Synthesis



70 >1 Millipore






Aeroperl (~35) 1.6 Evonik

33

Table 2: Fenofibrate loaded in porous silica particles

Pore size

(nm)

FEN mass loaded

(wt %)

Melting point by

DSC (ºC)

Polymorph by

XRPD

Polymorph

by ssNMR

300 29.4±1.2 79.9±0.1 Form I Form I

191.4 40.0±2.0 79.8±0.5 Form I Form I

151.5 31.5±1.0 79.0±0.2 Form I Form I

105.5 35.7±0.5 78.7±0.2 Form I Form I

70 28.1±0.4 77.7±0.2 Form I Form I

53.7 33.4±0.4 75.2±0.6 Form I Form I

38.3 29.4±0.4 71.8±0.5 Form I Form I

35 28.0±1.7 70.3±0.8 Form I Form I

30.7 29.4±0.7 71.2±0.1 Form I Form I

20.2 26.2±0.8 64.2±0.4 Form I Form I

12.7 16.3±0.6 N/A Amorphous Amorphous

/Form I

All loading data, melting points, and polymorph observations via XRPD and ssNMR are

summarized in Table 2. High drug loadings were achieved via the method of applying the pore

volume of drug solution. In the XRPD samples, there is a large amorphous feature which

disrupts the baseline (to be subtracted) due to the amorphous silica matrix which makes up the

bulk of the sample. NMR is isotope selective and invariant to the substrate that the API is placed

upon offering an approach to probe the degree of crystallinity and identify polymorphs easily

using 13C CP MAS NMR. Overall drug loading is reasonably well correlated to both pore

volume and mean pore size but appears more closely dependent on the nominal pore size.

Fenofibrate in 20 to 300 nm CPG illustrated clean 13C spectra with high crystalline API

formation. DSC and XRPD data indicated an inability to crystallize fenofibrate in the 12 nm

CPG, suggesting an amorphous form (vide infra). In examining the literature, it has been

reported that the pore diameter should be at least 20 times the molecular diameter for

crystallization in confined spaces 58. Fenofibrate has an estimated molecular size of 0.98-1.27 nm

34

54. It is hypothesized that this is the reason why the 12 nm CPG showed no crystalline fenofibrate

in the powder x-ray diffraction results, as it is less than 20 times the diameter of fenofibrate. Ha

et al. found an similar size limit to crystalline versus amorphous stability in porous matrices,

noting that crystallization of the compound ROY was suppressed in 20 nm pores as compared to

30 nm pores when carried out either by evaporation or by melting/cooling 23. We postulate that

under slow crystallization conditions, crystals could be formed in pore sizes under 20 times the

molecular diameter, which would explain the combination of broadened (i.e., amorphous phase)

and narrow (i.e., crystalline) 13C resonance observed in the 12 nm sample (Appendix 2: Figure

IVa).

A major challenge in this work was to produce crystals which are successfully loaded in the

porous matrix, rather than on the external surfaces. DSC was used to determine if surface

crystals were present; in cases where both nanocrystals and surface crystals were formed, there

are two obvious peaks present on the DSC scans corresponding to melting points of the confined

crystals and the surface crystals (Appendix 2: Figure II). Each trial was deemed successful in

producing confined crystals with surface crystals when there was no measurable second peak on

the DSC scans. This conclusion is supported by the work of O’Mahony et al., where SEM

imaging of the surface of drug-loaded nanoporous substrate was used to confirm that crystals

were confined to the pores and that no significant amount of bulk crystals were present on the

surface of the substrate 59. In this study, there was no occurrence of surface crystals that were

nanosized rather than bulk-sized.

35

Crystal form identification with XRPD

With the exception of fenofibrate in 12 nm CPG which showed no crystallinity, all samples

showed the same XRPD peak pattern, both within trials of the same size CPG and across

different sizes of CPG. Fig. 3 (a) is a scan of bulk fenofibrate and Fig. 3 (b) shows the XRPD

scans of a single representative size of 53 nm CPG, across all three trials. It is evident that the

crystal pattern is consistent throughout trials of a given pore size, which was also seen in all

other pore sizes. Fig. 3 (c) shows an overlay of scans from three representative CPG sizes (191,

53, and 70 nm). Crystalline fenofibrate form I has reported theoretical diffractogram main peaks

at 12º (2θ), 14.5º (2θ), 16.2º (2θ), 16.8º (2θ), and 22.4º (2θ) 56. The identity of all samples of

nanocrystalline fenofibrate as form I can be confirmed by matching peaks and the absence of

other peak positions.

36

Fig. 3: XRD patterns of bulk fenofibrate presented in (a), fenofibrate loaded on 53 nm CPG in 3

distinct trials in (b), and fenofibrate loaded on three representative pore sizes of CPG in (c)

37

Crystal form identification with ssNMR

13C CP MAS NMR spectra for all fenofibrate loaded porous silica particles were used to identify

amorphous or crystalline fenofibrate and identify whether the crystalline phase present were

form I or II. All 13C MAS NMR spectra illustrate highly crystalline fenofibrate (form I), with line

widths between 60 and 85 Hz (Appendix 2: Figures IVa-i). Isotropic chemical shift data for silica

particles with pore sizes ranging between 20 and 300 nm revealed identical spectra with no

evidence of structural disorder. The slight decrease in resolution (13C line broadening from 300

to 20 nm) is due to the increase of surface disorder as the nanocrystals become increasingly

smaller (i.e., surface vs nanocrystalline core).

The 13C MAS NMR spectrum of the 12 nm CPG sample is slightly more complex; although the

three well-resolved high frequency 13C resonances indicate form I of fenofibrate, all 13C

resonances were broadened with the aromatic region being most affected (Appendix 2: Figure

IVa). We attribute this broadening to the small pore size causing a high degree of structural

disorder, which often occurs when API’s begin to form an amorphous phase or very small

nanocrystalline formation. This observation agrees with the powder x-ray diffraction data which

exhibits a single broad featureless lump, consistent with the lack of long-range periodic order.

Finally, the poor fenofibrate loading on the 12 nm CPG pore size as determined by the TGA is

reflected by a rather poor signal-to-noise after considerable averaging and comparable sample

mass when ssNMR was performed on this material. The small pore size could retard the ability

of fenofibrate to form nanocrystals as discussed above.

38

Fig. 4: 13C CP MAS NMR spectra of fenofibrate: molecule (top), bulk drug (middle) and loaded in

20 CPG (bottom)

Analysis of melting point depression of nanocrystals by DSC

The melting point of bulk fenofibrate crystals was measured and found to be 81.6 ± 0.2 ºC. Fig.

5 shows an overlay of the DSC scans for representative trials of fenofibrate crystallized in each

CPG pore size. Individual, sharp peaks can be found at decreasing melting point temperatures,

moving left as the CPG pore size decreases. Double peaks were not seen in the trials, indicating

the preparation method was successful inhibiting the formation of any surface crystals.

39

Fig. 5: DSC scans of all CPG pore sizes showing single peaks (no surface crystals) with increasing

melting point temperatures. Peaks are separated by color and correspond left to right to increasing

pore sizes 20 to 300 nm.

It is well known that the Gibbs-Thomson equation can be used to describe the melting point

depression seen in nanocrystals 3. The complete Gibbs-Thomson equation for nanocrystals

confined to pores is as follows:

4( ) cos( )

solid liquid m

m m m

fus solid

MTT T T d

d H

Eq. 1

where 𝑇𝑚 is the bulk melting temperature, 𝑇𝑚(𝑑) is the melting temperature of a confined

crystal with diameter d assumed equal to the pore diameter, M is the molecular mass, 𝜌𝑠𝑜𝑙𝑖𝑑 is

the density of the solid,𝛾𝑠𝑜𝑙𝑖𝑑−𝑙𝑖𝑞𝑢𝑖𝑑 is the surface free energy of the solid-liquid interface, 𝛥𝐻𝑓𝑢𝑠

is the molar enthalpy of fusion, and θ is the contact angle between the wall and crystal. If a

standard contact angle of 180º is assumed, the equation reduces to

40

4( )

solid liquid m

m m m

fus solid

MTT T T d

d H

Eq. 2

At this point, it is evident that if the surface energy interaction term and the enthalpy of fusion,

𝐻𝑓𝑢𝑠, remains constant, there is an expected linear relationship between the melting point and

1/d. Fig. 6 is the plot of 1/d for the fenofibrate in the given range of pore diameters versus the

melting point temperature, taken from the DSC peaks. The data fits a linear trend. If the linear fit

is extrapolated to 1/d = 0, it predicts the melting point of an infinite diameter particle which is

the bulk melting point. The fit predicts a bulk melting point of 81.6 ºC, equal to the measured

bulk melting temperature of 81.6 ºC.

It is clear that the linear Gibbs-Thomson equation wherein the enthalpy of fusion and surface

interaction energies are assumed constant accurately predicts the bulk melting temperature.

However, the enthalpy of fusion was also measured in the DSC experiments, and was determined

to be non-constant. It, too, can be plotted against 1/d, as seen in Fig. 7. It is shown to decrease

linearly with 1/d.

In accordance with a study done by Ha et al. 22 the Young equation to describe the equilibrium

contact angle allows the substitution of (𝛾𝑠𝑜𝑙𝑖𝑑−𝑙𝑖𝑞𝑢𝑖𝑑 cosθ) in the original Eq. 1

with(𝛾𝑠𝑜𝑙𝑖𝑑−𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 − 𝛾𝑙𝑖𝑞𝑢𝑖𝑑−𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒). This yields the modified equation:

4( )( )

solid substrate liquid substrate m

m m

fus solid

MTT T d

d H

Eq. 3

As nanocrystals size decreases, it is expected that the surface energy of the solid approaches that

of its corresponding liquid. The difference term, (𝛾𝑠𝑜𝑙𝑖𝑑−𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 − 𝛾𝑙𝑖𝑞𝑢𝑖𝑑−𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒), should

41

therefore decrease and approach zero. The decrease in this term, found in the numerator, likely

offsets the decrease in the enthalpy of fusion term found in the denominator. This would produce

the apparent linear Gibbs-Thomson relationship reflected in the data despite the known change in

enthalpy.

Fig. 6: Constant enthalpy-constant surface interaction energy Gibbs-Thomson equation fit to

melting points of fenofibrate nanocrystals confined to porous silica.

42

Fig. 7: Enthalpy of fusion of fenofibrate nanocrystals showing a linear relationship with 1/d across

varying CPG pore sizes.

Enhanced dissolution profile for nanocrystalline fenofibrate

Dissolution profiles were tested and shown in Fig. 8. The nanocrystalline fenofibrate with the

most enhanced dissolution profile from controlled pore glass confinement occurred in the 70 nm

CPG matrix, shown in detail in Fig. 9. These fenofibrate nanocrystals showed a roughly 7 fold

increase in dissolution rate compared with crushed bulk fenofibrate. They reached >80%

dissolution in 42.5 minutes where crushed bulk fenofibrate reached >80% dissolution in 295.5

minutes. Fenofibrate nanocrystals confined to 20 and 30 nm CPG had profiles which aligned

closely with the crushed bulk profile indicating that, at small pore sizes, diffusional resistance

likely matters to enhancing dissolution rate. Nanocrystals in CPG above 30 nm showed improved

dissolution over the bulk crushed and uncrushed fenofibrate crystals at all time points of the

study. The dissolution profiles can be clustered into two groups based on manufacturer. The 70

nm and 300 nm (Millipore CPG) confined fenofibrate nanocrystals are the most enhanced

43

profiles and show the expected faster dissolution with smaller pore/crystal size. The fenofibrate

nanocrystals confined to the other pore sizes (Prime Synthesis CPG) all have very similar, still

improved, dissolution profiles with no discernible trend by pore size. It is likely that the

differences in pore geometry and tortuosity of the two types of CPG contribute to the differences

in improvement in dissolution rate seen in the study

Fig. 8 Average dissolution profiles of all CPG-confined fenofibrate nanocrystals showing enhanced

dissolution rates compared to crushed and uncrushed bulk fenofibrate, also shown

44

Fig. 9: Comparison of the most enhanced dissolution profile of nanocrystalline fenofibrate in 70 nm

CPG compared to bulk crushed fenofibrate

Extension to Aeroperl® porous matrix

The fumed silica matrix Aeroperl® behaved similarly to the CPG in terms of loading achieved

and depressed melting point. The melting point of the fenofibrate confined to Aeroperl® was

70.3 ± 0.8 °C, compared to the bulk melting temperature of 81.6 ± 0.2 ºC. It had an average

enthalpy of fusion of 55.2 ± 2.5 J/g compared to the bulk enthalpy of fusion of about 71.4 J/g.

While the depressed melting point followed the linear regression extrapolated from all of the data

points collected across different controlled pore glasses, the measured enthalpy of melting for the

fenofibrate confined to Aeroperl® does not lie along the same trend line computed for the

controlled pore glass samples. This is likely due to differences in the surface or shape of the

Aeroperl ® which could affect the enthalpy of fusion of the crystals confined to it. The

45

fenofibrate crystals confined to Aeroperl® showed the same polymorphism and XRPD patterns

as those confined to the CPG media as shown in Fig. 10. The underlying amorphous baseline for

the XRPD scan of the Aeroperl® material is different than that of the CPG simply due to its

difference in grain size and pore structure.

Fig. 10: XRPD scans of fenofibrate confined to CPG versus Aeroperl(R) show that it is the same

crystalline form

Fenofibrate confined to the Aeroperl® behaved dramatically better in dissolution testing than its

counterparts in controlled pore glass. Again, this was likely attributable to differences in the

shape and structure of the pores for Aeroperl®. The nanocrystalline fenofibrate with the fastest

dissolution profile occurred in the AEROPERL ® matrix. It reached ~50% dissolution in ~10

minutes where a control of crushed bulk fenofibrate reached ~50% dissolution in ~100 minutes,

as shown in Fig. 11. The same CPG curves previously discussed are shown as well for

comparison.

46

Fig. 11: Dissolution enhancement profiles showing Aeroperl® outperforming all other porous

matrices

2.4 Pore size and shape effects on dissolution rates

Understanding dissolution profile factors

An outstanding question from previous work was how the shape and material of the matrix

affects the dissolution behavior and possible interactions between the matrix and the confined

nanocrystals. Upon initial examination of the dissolution profiles, no clear trend was observed

between pore size and improvement to dissolution rate. One might assume that the smallest

nanocrystals (20 nm or 30 nm) would dissolve more rapidly than the 300 nm crystals if purely

surface area based dissolution mechanisms were at play. Upon closer look, however, a

generalization can be drawn from the above data that, besides Aeroperl®, the nanocrystals

confined to silica provided by Millipore (300 nm and 70 nm) showed more enhanced dissolution

profiles than those confined to silica obtained from Prime Synthesis (all others). Understanding

47

the contribution of the porosity of the material and pore shape and how this might affect

transport properties of the system was a natural extension of understanding the improvements to

dissolution profiles.

Fig. 12 shows a schematic representation of the combination of dissolution and diffusion effects

at play when considering the final dissolution profile observed for an API released from silica

beads. The dissolution of the nanocrystal embedded in the pores of the bead is actually thought

to be quite fast in terms of the overall time measurements in the system, as the length scale

predicted here is on the 10-1000 nm scale, related to the pore diameter. However the path length

for diffusion is much longer, related to the diameter of the porous bead. The CPG beads used in

this study tended to have grain sizes between 75 and 125 μm. Aeroperl® grain size was even

smaller, at nominally around 30 μm. Thus, the diffusion length from the pore would be expected

to matter greatly. Indeed, a study done on mesoporous silica matrices and the controlled release

of ibuprofen found that “The release process is found to be mainly diffusion controlled, but clear

differences were observed between the studied materials, which we mainly ascribe to differences

in the pore connectivity and pore geometry of the materials, and the aqueous stability of the

matrix.” 60

48

Fig. 12: Mass transport schematic diagram of nanocrystal dissolution and diffusion from porous

beads

Work done in the Doyle Group at MIT has been investigating the effect of bead size in similar

work, looking at the release of APIs from core-shell hydrogels that have a porous network and a

defined grain size 61. They found that lag time release and overall dissolution time was

minimized in smaller hydrogel beads. They found that >80% dissolution in 30 minutes could be

achieved with their hydrogels of about 400 μm in grain size. This is comparable to the best

release profiles seen from our rigid matrices in which >80% dissolution was achieved in 22.5

minutes. While this study’s alginate beads obviously have different pore structures and surface

properties that account for some differences in the release times, it points towards the size and

shape properties of the physical matrix that the nanocrystals are confined to as having a very

important role on the final dissolution profiles. A summary of the pore and grain sizes discussed

here is found in Table 3.

49

Table 3: Comparison of bead pore and grain sizes used in API release studies

Matrix material

(pore size)

Producer Surface area

(m2/gram)

Pore volume

(cc/gram)

Grain size

(diameter)

Alginate bead

(1-2 μm)

Doyle Lab

(MIT)

-- -- 430 μm

Aeroperl

(~35 nm)

Evonik 281 1.6 D50 = 33 μm

CPG

(70 nm)

Millipore 43 1.2 75-125 μm

CPG

(53 nm)

Prime

Synthesis

94 1.3 75-125 μm

Dissolution Profile Modeling

A very basic transport model was made to determine the contribution of tortuosity to the

dissolution rates. A 1987 paper “Hindered transport of large molecules in liquid-filled pores”

was consulted and used to form a simple diffusion model 62. In one case, the pore was modeled

as a straight channel, and in another it was given a sinusoidal path length. A visual of the

proposed path geometries is provided in Fig. 13. Csat indicates the expected saturation

concentration of the API in solution at the nanocrystal interface and CL the concentration at a

given length, L, away from the center of the bead. Csat was found to be 8*10-4 mg/mL for

fenofibrate in the water/SDS mixture which was used for dissolution rate testing, which makes

sense given its extremely poor water solubility 63. The radius of the pore is given by rpore and is

defined by the nominal pore size of the matrices and rsolute is the radius of the solute molecule.

For this case, fenofibrate was used as a model compound for comparison to our experimental

data and the radius was estimated from literature 64.

50

Fig. 13: Schematic representation of diffusion path length and geometry for basic transport model

of nanocrystal diffusion from within a bead.

The estimated diffusivity of fenofibrate in water at room temperature was taken from literature.

A hindrance factor, H, was adapted from this paper to give an effective diffusivity for fenofibrate

in water, to match dissolution rate data. The parameters and expressions used for the model are

summarized in Table 4.

Table 4: Modeling expressions for release of dissolved API from straight and tortuous pores

Straight Pore Tortuous Pore

Deff

= Dwater, 298 K

Deff

= H*Dwater, 298 K

No hindrance factor 𝐻 = 0.984 (1 −𝑟𝑠𝑜𝑙𝑢𝑡𝑒𝑟𝑝𝑜𝑟𝑒

)

92

Path length = L Path length = 𝑎𝑟𝑐𝑙𝑒𝑛𝑔𝑡ℎ(3 sin𝑥) |0

𝐿

𝐿

The solution to the diffusion problem where 𝐶(𝑥, 𝑡) solving for the concentration at a given point

in the x direction away from the surface of the bead at a given time, t, is in the form of an error

function:

51

Eq. 4

This is plotted for fenofibrate in a dissolving water system at a given distance x outside the pore

to compare results with experimental data generated and discussed in the previous section. The

path length of the straight pore L was set equal to the expected radius of the grain size of the

matrix bead. Results are shown in Fig. 14. The distance x was set at 10 μm to be sufficiently far

from the surface of the bead such that a concentration profile would develop that would be

similar to that sampled by the UV spectrometer in the dissolution rate apparatus.

Fig. 14: Tortuosity model compared with experimental results for dissolution profiles

The straight pore model captures the behavior of the Aeroperl® confined fenofibrate dissolution

profile reasonably well, with a similar shape and similar end concentration at about 2 hours. We

know that Aeroperl® had the best dissolution profile of the porous matrices examined, and the

highest surface area. It may be reasonable to assume that its pores are not particularly tortuous.

𝐶(𝑥, 𝑡) = 𝐶𝑠𝑎𝑡 (1 − erf (𝑥

2√𝐷𝑒𝑓𝑓𝑡))

52

However, the model slightly underpredicts in the time between 1000 and 6000 seconds. The

model assumes that the drug is at the center of the bead providing a constant source of solute to

the external system, and must diffuse the entire length out. In fact, some API may be present

throughout various interior points of the bead to the surface and actually have less far to travel.

This may account for the true dissolution profile from Aeroperl® showing slightly higher values

in this range.

The model continues to underpredict for the tortuous pore as compared to the 30 nm pore real

dissolution data. Here, too, the initial behavior is not captured well. There is a lag time for the

tortuous pore model which is not seen for the real data. This is likely explained by the same

phenomenon previously discussed, where some API is present throughout the interior of the

bead, and in fact is likely quite close to the surface at some points. This initial transport of this

material probably prevents a concentration lag from being recorded in the real dissolution

profiles. Overall, the remainder of the curve is underpredicted but the shape is a reasonable

match.

Continued modeling with more advanced parameters could have been performed to better predict

the dissolution profiles seen experimentally. A better model would account for pore wetting,

dissolution of a crystal of solute within the pore as well as diffusion of solute from the pore, and

a distribution of crystals throughout the interior of the bead. A better defined geometry of pores

could improve a model as well, accounting for a pore network with intersections which are at a

juncture of two pores. Further modeling was not continued in this work.

Experiments to understand transport from a porous matrix

As a final experimental step, anodic aluminum oxide (AAO) membranes were purchased from

ACS Material. These membranes possess highly ordered straight pores with a pore aperture of

53

80-100 nm, and a hole depth of 60±10 μm. The pore-to-pore distance is 100-120 nm. This

membrane was wetted with a 60% weight per volume solution of fenofibrate in ethyl acetate and

allowed to evaporate in a similar method to the loading procedure for the porous silica outlined

in this chapter. For comparison, porous CPG of 105.5 nm was loaded as well. A visual of the two

porous supports is shown in Fig. 15. After loading with API, the membranes were subjected to

the standard analytical techniques performed on the porous glass material, of TGA, DSC, and

dissolution testing. The dissolution test in particular was of interest. The straight pores of the

AAO were hypothesized to yield faster dissolution profiles than the tortuous porous network of

CPG.

Fig. 15: Porous supports used for fenofibrate loading: anodic alumnimum oxide (left) and porous

silica (right)

The following graph shows the dissolution profile of fenofibrate confined to porous CPG, in its

bulk form, and the results of the AAO loading indicated above (Fig. 16).

54

Fig. 16: The results of the dissolution testing of FEN loaded on AAO indicate that the loading was

not confined to the pores, nanocrystals were not produced, and the dissolution profile was not

improved over the bulk form of the drug

It is clear from the profile that there is no improvement between the dissolution profile of

fenofibrate loaded on the AAO with the method described above and the bulk fenofibrate. This is

in sharp contrast to the very different and much improved dissolution profile seen for fenofibrate

confined to the nanopores of the CPG. We believe that this is simply a result of not actually

having loaded the fenofibrate into the pores of the AAO. As mentioned previously, the AAO

membrane had pore diameters and lengths comparable to the controlled pore glass used.

However, a critical difference was that the AAO membrane’s pores were not open at both ends.

Thus, we believe the simple drop method used did not wet or penetrate the pores of AAO, and

the fenofibrate crystals formed only at the surface. This would make sense given that their

dissolution essentially behaves as the bulk crystals. In the future, to properly load FEN in an

55

AAO pore, we would likely have to perform the loading under vacuum, to drive the API solution

into the pore since capillary action alone can’t work for the close-ended pore.

We also planned to investigate silica of different grain size but similar pore sizes, however cost

of procurement has proven a hindrance to this investigation. The grain size is not typically finely

controlled in the process of producing the porous glass matrices, so to order or produce sieved

material of different grain sizes would be very costly. Developing a solution to procuring porous

silica in a variety of grain sizes with a single pore size would be an extremely valuable addition

to this work

2.5 Conclusions

We successfully obtained nanocrystalline fenofibrate over a broad range of sizes by using rigid

matrices of porous silica. The results of the study indicate a decreasing melting point and

enthalpy of fusion with decreasing pore size in nanocrystals. The decrease in the enthalpy of

fusion is offset by a simultaneous decrease in the difference between the surface interaction of

the solid-substrate and melt-substrate. This produces seemingly linear behavior in melting point

depression as predicted by the Gibbs-Thomson equation. The dissolution testing showed

enhanced dissolution profiles for the nanocrystalline materials confined to different porous

matrices, with fenofibrate confined in 70 nm CPG from Millipore showing the greatest

improvement in dissolution. Further investigation remains to be done to understand the effect of

pore size, shape, and geometry on the dissolution profiles of APIs confined to porous matrices.

The controlled pore glass matrices used in this study are not currently accepted materials for oral

drug delivery as they do not fulfil requirements of the European Pharmacopeia or the United

States Pharmacopeia and the National Formulary; however, the ability to form nanocrystals

56

across a range of pore sizes in this material suggests the potential to use drug-loaded rigid

matrices of biocompatible materials to serve as an oral dosage forms with enhanced dissolution

ability for poorly water soluble APIs. As a rigid porous material which does meet the USP/NF

guidelines, AEROPERL ® is an attractive matrix for use in simply preparing nanocrystals of

poorly water soluble APIs which could be formulated into capsules with no additional

formulation.

2.6 Acknowledgements

This chapter has been adapted and published as a peer-reviewed journal article. Dwyer LM,

Michaelis VK, O’Mahony M, Griffin RG, Myerson AS. Confined crystallization of fenofibrate

in nanoporous silica. CrystEngComm / RSC. 2015;17(41):7922-7929. doi:10.1039/C5CE01148E.

We thank the Novartis-MIT Center for Continuous Manufacturing for financial support and use

of instrumentation. R.G.G thanks the NIBIB through the National Institutes of Health grant EB-

002026. V.K.M. is grateful to the Natural Sciences and Engineering Research Council of Canada

and the Government of Canada for a Banting Postdoctoral Fellowship. L.M.D. is grateful to Dr.

Samir A. Kulkarni for valuable assistance and discussions.

57

2.7 Appendix

I: Individual DSC Scans: one trial of each pore size shown as a representative

a. 12 nm CPG

b. 20 nm CPG

58

c. 30 nm CPG

d. 38 nm CPG

59

e. 53 nm CPG

f. 70 nm CPG

60

g. 105 nm CPG

h. 151 nm CPG

61

i. 191 nm CPG

j. 300 nm CPG

62

II. DSC Scan of Poor Quality Preparation: scan of a trial wherein bulk and nanosized crystals

were produced in a sample with CPG of 38.3 nm pore size clearly showing two distinct peaks

III. Individual XRPD Scans: one trial of each pore size shown as a representative

a. 12 nm CPG

63

b. 20 nm CPG

c. 30 nm CPG

64

d. 38 nm CPG

e. 53 nm CPG

65

f. 70 nm CPG

g. 105 nm CPG

66

h. 151 nm CPG

i. 191 nm CPG

67

j. 300 nm CPG

IV. Individual ssNMR Spectra: one trial of each pore size shown as a representative

a. 12 nm CPG

68

b. 20 nm CPG

c. 38 nm CPG

69

d. 53 nm CPG

e. 70 nm CPG

70

f. 105 nm CPG

g. 151 nm CPG

71

h. 191 nm CPG

i. 300 nm CPG

72

Chapter 3 : Continuous crystallization in confinement to improve

matrix loading

3.1 Introduction

The previous work demonstrated that porous matrices could be used effectively for confining an

API and demonstrating a degree of control over the size of nanocrystal formed for improved

release profiles. However, improving the loading of API in a porous matrix was a necessary next

step for allowing this to be a viable candidate for a dosage form to achieve the correct dosing

amounts of drug with a reasonable additional volume of porous matrix for confinement.

The low bioavailability of poorly water soluble active pharmaceutical ingredients (APIs) is a

challenge for API formulation and even selection in the drug discovery phase 65,66. However, as

nearly 90% of drugs in the discovery pipeline have low aqueous solubility 67, finding solutions to

improve the physiochemical properties of these drugs is of high importance 68. Forming

nanocrystals of these APIs is a simple and promising solution to this problem 5. Nanocrystalline

APIs (<1000 nm) have improved surface area to volume ratios compared to bulk crystals,

increasing dissolution rates 40,69, improving solubility 39,41,70, and enhancing permeability 4.

Numerous methodologies exist for producing pharmaceutical nanocrystals in both “top-down”

approaches that control the nanosizing of larger crystals or “bottom-up” technologies which

control the size of the crystal formed directly. These methods include milling 5, high pressure

homogenization 71, hydrosol methods 5,72, freeze-drying 73, supercritical fluid methods 44,45,73, and

evaporative or antisolvent precipitation 46,47,74–76. These methods have associated problems with

73

contamination, high surfactant requirements, complex and energy-intensive procedures,

difficulties controlling particle size distribution and polymorphism, and low production rates 8.

Many of these challenges are addressed with the confined crystallization approach in which

crystallization of the API is restricted to a micro- or nanoporous environment to form

nanocrystals. Of particular interest and widely studied for drug delivery applications are rigid

silica matrices including ordered mesoporous silica with and without surface modifications and

grain size control 14,15,20,77–80, controlled pore glass 19,22,23,81,82, and fumed silica 26 due to the high

degree of control over pore size and inert nature leading to nucleation control and polymorph

stabilization 29,49,51,83 .

While many studies have used porous silica matrices with extremely small pores (<10 nm) to

confine high loadings of the amorphous forms of poorly water soluble drugs to the effect of

dramatically enhanced dissolution profiles 28,84–87 , this study aimed to retain crystallinity of the

drug loaded in porous matrices due to the long-term stability requirements for formulated

pharmaceuticals 41,88. In a previous study, a flow column-based loading process has been

developed to first load the poorly water soluble API ibuprofen into nanoporous silica and then

crystallize the material in the pores through evaporation 59. In this process, a maximum loading

of about 33 wt% ibuprofen in the silica matrix was achieved. The researchers found that high

API solution concentration and high solvent viscosity (within common solvents) were able to

improve the loading achieved in the pores. Rinse volume was also a significant process

parameter, which needed to be adjusted to be low enough to maintain drug loading but high

enough to remove bulk micron sized crystals on the surface of the silica. This important work is

74

a starting point for the development of continuous flow procedures for loading API into porous

silica matrices.

Based on successful multi-stage continuous crystallizer designs in which the second stage and

beyond aim to grow crystals which have nucleated in a first stage 89–92, a two-stage mixed

suspension mixed product removal (MSMPR) crystallizer was proposed to address the challenge

of increasing the loading of API in porous silica. A single-stage MSMPR was employed for the

continuous wetting of porous silica in a variety of pore sizes with a subsequent filtration step

which accomplished the crystallization of fenofibrate (FEN) within the confined pores by either

evaporation or cooling. This was then coupled to a second stage in which the drug-loaded silica

was submerged in a slightly supersaturated FEN solution, allowing for growth of the

nanocrystals within the pores.

Fenofibrate is a BCS Class II drug (low solubility, high permeability) used for lowering

cholesterol and was chosen for its low aqueous solubility of 0.8 μg/mL 93 . It has a molecular

weight of 360.8 g/mol in a structure with an aromatic portion of the molecule containing two

benzyl rings adjacent to an aliphatic region 56. The principle was then applied to a bench-scale

multiple-step impregnation procedure using an even less soluble compound, griseofulvin (GSF),

to demonstrate improved loadings in this system. Samples were analyzed with thermogravimetric

analysis (TGA) to determine loadings, differential scanning calorimetry (DSC) to study their

nanocrystalline nature, and X-ray powder diffraction (XRPD) for form identification. This

methodology was shown to improve drug loading of poorly water soluble compounds, making

drug-loaded biocompatible porous silica a viable dosage form.

75

3.2 Experimental

Materials

Fenofibrate (FEN), griseofulvin (GSF), and indomethacin (IMC) were obtained from Xian

Shunyi Bio-chemical Technology Company. Silicon dioxide (silica) particles of varying pore

sizes were obtained from three sources. Controlled pore glass (CPG) was obtained from

Millipore in a pore size of 300 nm. More than 90% of the particle mass by weight fell within a

120/200 mesh size (75-125 μm). CPG was also obtained from Prime Synthesis in pore sizes of

191.4, 151.5, 105.5, 53.7, and 38.3 nm. All had 100% particle mass with a grain size within a

120/200 mesh. Both the Millipore and Prime Synthesis material were produced from borosilicate

glass. At least 80% of the pores were within 10% of the mean pore diameter in pore size. Finally,

Aeroperl® 300 Pharma, a fumed silica, was obtained from Evonik with a pore size of about 35

nm. These particles had an average grain size of 30 μm. Solvents were purchased at ACS grade

or higher purity from Fisher Scientific.

Experimental apparatus

Single-stage MSMPR

A single-stage MSMPR was used to crystallize fenofibrate in porous silica. A 50 mL round-

bottomed water jacketed reaction vessel with standard dimensions was used with magnetic stir

bar and temperature separately controlled by an external water circulation controller (Thermo

Scientific NESLAB RTE). A solids content within the reactor of 10 mg porous silica/mL API

solution was chosen; the empty reactor was primed with 500 mg silica to meet this value. The

volume contribution of the silica content was deemed negligible. Peristaltic pumps (Masterflex

76

P/S, Thermo Scientific) with Viton tubing (Cole-Parmer) were used for solution and slurry

transfer. For the single-stage MSMPR, an undersaturated feed solution of 600 mg/mL fenofibrate

in ethyl acetate was fed to the reactor at a flow rate of 1.7 mL/min to maintain a residence time

of 30 min in the reactor. Slurry was removed intermittently so that every 3 minutes, 5 mL of

slurry (10% of the vessel volume) was removed rapidly near the pump maximum flow rate of

about 170 mL/min. After the liquid level again dropped below the outlet dip tube, the tube was

pumped with air to clear remaining slurry. At this time, a replacement quantity of 50 mg of silica

was added to the reactor volume to maintain the solids density at 10 mg/mL. The temperature of

the feed and MSMPR were maintained at 25°C. The pore size of the porous silica was varied to

study the effect of confinement and subsequent crystal size on crystal properties and loading.

Fig. 17 (a) shows a schematic of the apparatus.

The slurry removed containing API-solution loaded porous silica was fed to a water jacketed

glass frit filter connected to an external water circulation temperature control (Thermo Scientific

NESLAB RTE) with qualitative Grade 1 filter paper (Whatman). The filter was maintained at

25°C. The first ten volumes of recovered slurry were discarded to allow the system a full

residence time turnover. The system was then run for 90 minutes to capture 3 full residence

times. This resulted in the capture of 150 mL of slurry containing a total of about 1500 mg

porous silica. The final filter cake was dried in air and washed with 5 mL cold (chilled on ice

0−5 °C) ethanol to wash off residual solvent or surface API crystals from the outside of the

porous silica beads. In a second study, the temperature of the filter was varied below 25°C at a

fixed pore volume to induce crystallization while minimizing solvent evaporation. The powder

was then dried in the filter by flowing air. All experiments were carried out in triplicate.

77

Two-stage MSMPR

For the two-stage MSMPR, two 50 mL water jacketed reaction vessels were used. The same

peristaltic pumps, tubing, and external water temperature circulators were employed. The first

stage was essentially a replica of the single-stage experiment, with a 600 mg/mL fenofibrate in

ethyl acetate feed with continuously supplied porous silica. The reactor vessel and filter were

held at 25°C with intermittent slurry withdrawals every 3 minutes of 10% of the volume of the

reactor and unloaded porous silica replaced to the vessel at this time. Due to the inability to

continuously harvest the filter cake of API loaded porous silica from the first stage for supply to

the second stage, the second stage was time-decoupled from the first to allow for the buildup of

enough filtered, loaded material from stage one to accumulate to be fed to stage two.

The feed to the second stage was a supersaturated solution of fenofibrate in ethyl acetate at 1080

mg/mL and 1.7 mL/min. The second reactor vessel was primed with 500 mg of stage one API-

loaded silica. The same residence time of 30 min was maintained and the reactor was held at

25°C. The same intermittent withdrawal scheme of 5 mL every 3 minutes was employed. The

silica added to the reactor in 50 mg intervals every three minutes was API loaded silica from

stage one. The slurry was sent to a water jacketed glass frit filter with qualitative Grade 1 filter

paper (Whatman) maintained at 25°C with an external water circulation controller (Thermo

Scientific NESLAB RTE). The samples were washed at each withdrawal with 1 mL cold (chilled

on ice 0−5 °C) ethanol to wash off residual solution and surface crystals. 6 (b) shows a schematic

of the setup. The samples were then dried in the filter by flowing air. Again, the first ten slurry

volumes were discarded. The crystallizer was run for two complete residence times to utilize all

78

of the recovered loaded silica from stage one, resulting in approximately 1000 mg of captured

material at the end of stage two. Experiments were carried out in triplicate.

Fig. 17: Crystallizer design schematic showing the (a) single-stage design and (b) two-stage design

for improved drug loading

Extension of principle to poorly water soluble compounds

Griseofulvin and indomethacin were selected for a bench scale extension of the multiple-stage

MSMPR principle of a first stage to load the pore with an initial crystalline material and a second

stage to grow the crystals in pores. Single, double, and triple-pass loading methods were

employed for each system. In the single loading method, a previously established technique 59

was employed of submerging the particles in a large volume (>250 mL) of undersaturated API

solution using a fine mesh basket. About 1 gram of porous silica particles were immersed in a

79

solution of 30 mg/mL GSF in acetone using a fine mesh basket uncontrolled at room

temperature. The solution was left to wet and enter the silica pores for 20 min before removing

and wicking away excess solution using a paper towel. After the silica particles were removed

from the solution, the particles were rinsed with 2.5 mL of cold ethanol (chilled on ice 0−5 °C).

The rinse was wicked away using a paper towel and the porous silica was removed to dry at

room temperature for approximately 15 hours.

In the double-pass loading method, prepared loaded samples from a single-pass experiment were

then submerged using the fine mesh basket in a large volume very slightly supersaturated

solution of 45 mg/mL GSF in acetone. The samples were held for 5 days held at 25 °C in a

recirculated water bath (Thermo Scientific NESLAB RTE) at 25 °C. The samples were then

removed, solution wicked away, and rinsed with cold ethanol as above before being separated

from any visible bulk crystals and dried at room temperature for about 15 hours. Finally, for the

triple-pass loaded systems, a third submersion step in a slightly more supersaturated GSF

solution of 50 mg/mL was completed for another 5 days, with subsequent identical wash and dry

procedures. The experiments were repeated using IMC solutions in acetone at the same

concentrations for the one-pass, two-pass, and three-pass loading methods. All experiments were

carried out in triplicate.

Analytical techniques

X-Ray powder diffraction

XRPD was performed on all samples using a PANalytical X'Pert PRO diffractometer at 45 kV

with an anode current of 40 mA. The instrument has a PW3050/60 standard resolution

80

goniometer and a PW3373/10 Cu LFF DK241245 X-ray tube. Samples were placed on a spinner

stage in reflection mode. Settings on the incident beam path included: soller slit 0.04 rad, mask

fixed 10 mm, programmable divergence slit and fixed 1° anti-scatter slit. Settings on the

diffracted beam path include: soller slit 0.04 rad and programmable anti-scatter slit. The scan

was set as a continuous scan: 2θ angle between 4 and 40°, step size .0167113° and a time per

step of 31.115 s.

Thermogravimetric analysis

TGA was performed on a Q500 instrument from TA instruments connected with a nitrogen gas

cylinder to maintain a flow rate of 25 mL/min to maintain an inert gas environment in the sample

chamber. Between 5 and 10 mg of sample were loaded on platinum sample pans from TA

Instruments. The samples were allowed to equilibrate at 30 °C and then heated at 10 °C/min to

300 °C.

Differential scanning calorimetry

A Q2000 instrument from TA instruments was used for the DSC analysis. Inert atmosphere

environment was maintained in the sample chamber using a nitrogen gas cylinder set to a flow

rate of 50 ml/min. An extra refrigerated cooling system (RCS 40, TA Instruments) was used to

widen the available temperature range to between −40 and 400 °C. Tzero® pans and lids were

used with ~5 mg of sample. A heating rate of 10 °C/min was applied and the samples were

scanned from −20 to 250 °C. When determining the enthalpy of fusion for a given sample, the

DSC curve was integrated for 30 °C centered on the melting temperature of each pore size to

capture the entire melting event. An exception to this rule was made in samples with a secondary

81

peak present from surface crystals in which case the integration limit was set to exclude the

secondary melting event.

Solubility measurements

The saturation solubility for FEN was measured in ethyl acetate at different concentrations by

adding a known amount of FEN and 1 mL of solvent, respectively, to a 1.5 mL glass vial. The

vials were then placed in a Crystal16 (Avantium), and the heating and cooling rates were set to

0.3 °C/min. The samples were stirred with a controlled stirring speed of 700 rpm using magnetic

stirring bars. The samples were heated with a heating rate of 0.3 °C/min from 5 to 50 °C. The

temperature at which the suspension turned into a clear solution was recorded and assumed to be

the saturation temperature. The clear points generated the solubility curve. After a waiting time

of 30 min at 50°C, the clear solution was cooled to 5 °C with a cooling rate of 0.3 °C/min to

recrystallize the FEN. The cloud point curve was generated as the metastable zone limit (see

Section 3 for discussion).

Dissolution testing

A dissolution test meeting the USP standard for fenofibrate was performed using a USP

Dissolution Apparatus 2 at 37 °C. Built-in ultraviolet-visible spectroscopy was used to determine

percentage of dissolved FEN at 286 nm. The dissolution buffer used was .025 M sodium dodecyl

sulfate solution (Sigma Aldrich). The dissolution apparatus operated at 75 RPM using 900 mL

buffer solution which was allowed to equilibrate at temperature before the addition of drug-

loaded silica. Enough sample of FEN-loaded silica was added such that the targeted

82

concentration of fenofibrate in solution was 15 μg/mL, within the expected linear range. Samples

were acquired for about 29 hours.


Poorly water soluble compounds are of interest for confined crystallization work to produce

small crystals with better dissolution ability. In previous studies, FEN nanocrystals confined to

rigid porous silica matrices have been shown to have well-behaved thermal properties and

enhanced dissolution rates 28,40. A single polymorph has been shown to crystallize in pore sizes

from 20-300 nm which is ideal for studying melting point behavior in a simple system 81.

Selection of MSMPR operating parameters

Experiments were conducted on a Crystal 16 apparatus to determine the saturation solubility and

metastable zone width (MSZW) of the FEN systems. The solubility was measured of FEN in

ethyl acetate alone, and with porous silica also present in the vials. Figure 1 clearly shows the

decrease in the metastable zone width with the addition of porous silica. The need to understand

the working MSZW in a confined crystallization procedure is critical in the selection of a

supersaturation level. The feed to stage two of the crystallizer was carefully selected to lie within

the narrowed MSZW resulting from the presence of silica. This feed was designed to be

supersaturated such that the feed could allow growth of the FEN crystals in the crystallizer while

not dissolving the crystals already loaded in the pores. It also needed to be stable enough so as to

not precipitate on its own. Indeed, some crystallizer runs were discarded due to issues with bulk

crystal formation in the second stage. The saturation solubility of FEN in ethyl acetate at room

temperature (25 °C) was found to be approximately 750 mg/mL. The supersaturation selected for

83

the second stage feed was about 1.4, a concentration of 1080 mg/mL. This value was selected to

be well within the MSZW window.

Fig. 18: Comparison of the MSZW of FEN in ethyl acetate showing a decrease in the width with the

addition of porous silica

The experiments were designed to maintain 10% w/v silica in the crystallizers. It is likely that

the crystallizers could be run at much higher than 10% solids density with similar growth and

loading. The low value of 10% was selected to be conservative in the “infinite solution”

approximation for the second stage wherein the mechanics of crystal growth and supersaturation

were being carefully monitored.

Analysis of FEN loading in MSMPR experiments

Table 5 summarizes the loading results from the single- and two-stage MSMPR experiments

held at 25 °C as determined by thermogravimetric analysis (TGA). The theoretical maximum

loading from the first stage of the MSMPR was calculated assuming that all of the fenofibrate

84

contained in the original 600 mg/mL (60% w/v) solution crystallized in the pore and that the pore

volume of the silica was completely filled with solution. The theoretical “filled pore maximum”

theoretical loading was also calculated based on the density of fenofibrate assuming that the

entirely pore volume was filled with crystal 94. This basis is a maximum for comparison;

however, complete filling of the tortuous porous network of the silica would be unlikely.

Table 5: Room temperature (25 °C) MSMPR loading summary of FEN wt% in porous silica

Only runs which did not show bulk crystallization in the vessels were selected for loading

analysis. However, differential scanning calorimetry (DSC) scans did detect the presence of a

small (<10 wt%) amount of surface crystals present on the silica powder in the 38 and 53 nm

samples. This skews the loading data to suggest slightly higher possible loadings for these two

pore sizes. In all pore sizes, the single-stage MSMPR experiments were able to achieve >75% of

the theoretical maximum loading based on the amount of FEN solution expected to have wetted

the pores. The loadings from the two-stage MSMPR were higher than their one-stage counterpart

in all cases, indicating that the crystals confined to the pores in the first stage were then able to

grow within the pores in the second stage. High weight percentage loadings were achieved in all

Pore

size

(nm)

Specific

surface

area

(m2/g)

Pore

vol.

(mL/g)

Theoretical single-

stage max. loading

from FEN soln.

(wt%)

Single-stage

loading

(wt%)

Theoretical

filled pore

max. FEN

loading (wt%)

Two-stage

loading

(wt%)

300 10 1.0 37.5 31.4 ± 1.7 54.1 41.6 ± 1.0

191 30 1.5 47.4 36 ± 1.9 63.9 50.2 ± 1.8

151 31 1.2 41.9 36.1 ± 1.7 58.6 40.2 ± 2.5

105 52 1.4 45.7 36.5 ± 4.2 62.3 54.8 ± 3.7

53 94 1.3 43.8 39.1 ± 1.0 60.5 55.6 ± 2.2

38 138 1.3 43.8 40.3 ± 2.3 60.5 56.1 ± 3.5

35 300 1.6 49.0 - 65.4 56.7 ± 1.6

85

cases. Most notably, due to its large pore volume, the porous silica Aeroperl® was able to be

loaded with FEN to more than 50 wt% loading. While the samples with 38 and 53 nm pores did

show some surface crystals, the relative contribution of surface to confined crystals indicated that

there was still growth of the crystals confined to the pores in the two-stage MSMPR.

The single-stage MSMPR was also run at varying filter temperatures for a single pore size of 191

nm. Rather than allowing the crystallization to occur as a result of evaporation of the solvent and

potentially losing more crystals in the wash step, cooling the solution in the crystal pores was

hypothesized to allow for higher loadings even in the single-stage MSMPR. Table 6 summarizes

the loadings determined by TGA and a description of the DSC scans for the varied temperature

experiment.

Table 6: Loading results for FEN wt% in porous silica in single-stage MSMPR as a function of

filter temperature

Temp

(°C)

Loading (wt%)

Notes from DSC thermogram Trial

1

Trial

2

Trial

3 Avg.

25 36.0 42.3 37.2 38.5 ± 3.5 Single peak, confined crystals




15 64.6 61.0 65.7 63.8 ± 2.5 Two peaks, confined and surface

crystals

For all temperatures but 15°C, the DSC scans showed single peaks at a melting point depression

indicating that the crystals formed were confined to the pores of the silica matrix. There is a

trend of higher loadings with cooler filter temperature. The samples crystallized at 15°C were

shown to have two peaks in the DSC scans corresponding to both confined crystals and crystals

86

on the surface of the porous supports. Previous calculations based on the pore volume and

density of FEN indicated that the theoretical filled pore maximum for FEN entirely occupying

the pore volume for this pore size is 63.9 wt%. The loading values in Trials 1 and 3 at 15°C

further support the interpretation of DSC thermograms indicating that both confined and surface

crystals were present as the measured loading values exceed the theoretical maximum.

Dissolution profile enhancement

The FEN-loaded silica generated from the MSMPR crystallizers was studied in a dissolution

apparatus to generate the dissolution profiles. Fig. 19 shows the dissolution profile of FEN

loaded Aeroperl® from the two-stage MSMPR in contrast with bulk FEN crystals from the

manufacturer. The Aeroperl® dissolution profiles are the most dramatically enhanced, due to a

combination of the small crystal size (35 nm) and also likely diffusion effects from differences in

the tortuosity and porosity between the different silicas used. The nanocrystalline FEN loaded in

Aeroperl® was able to achieve more than 80% dissolution in 22.5 minutes, whereas the bulk

FEN took 656 minutes for the same. The release time for FEN confined to Aeroperl® is the

fastest seen of this API confined to any other form of porous silica 81.

87

Fig. 19: FEN confined to 35 nm Aeroperl® showing dramatically enhanced dissolution profiles

when compared to the bulk crystals

Melting point depression analysis of nanocrystals with DSC

DSC scans were performed on each sample both to confirm that the crystals formed were

confined to the pores and to study the effect of confinement on melting point depression. For the

majority of the silica supports, the DSC scans showed a single, sharp peak at a melting point

below the melting point of bulk FEN (81.6 °C ± 0.2 °C.). This can be interpreted as indicating

the presence of API crystals confined to the pores, without the presence of significant surface

crystals of bulk size on the surface of the porous silica 59,81. A few samples from pore sizes 38

and 53 nm showed two peaks on the DSC scans: a large peak at a temperature below 81.6 °C and

a secondary peak at about 81.6 °C accounting for less than 10% of the total peak area. While this

is interpreted as the presence of some crystals on the surface of the porous silica, the small

relative quantity was accepted and these samples were still used for loading analysis.

88

A basic simplified Gibbs-Thomson analysis was employed to study the effect of confinement on

melting point depression of the nanocrystals produced via the MSMPR methods 24. Eq. 4 relates

the melting point depression to the nanocrystal size:

4( )

solid liquid m

m m m

fus solid

MTT T T d

d H

Eq. 5

where 𝑇𝑚 is the bulk melting temperature, 𝑇𝑚(𝑑) is the melting temperature of a confined crystal

with diameter d assumed equal to the pore diameter, M is the molecular mass, 𝜌𝑠𝑜𝑙𝑖𝑑 is the

density of the solid ,𝛾𝑠𝑜𝑙𝑖𝑑−𝑙𝑖𝑞𝑢𝑖𝑑 is the surface free energy of the solid-liquid interface, and

𝛥𝐻𝑓𝑢𝑠 is the molar enthalpy of fusion.

In previous studies, Eq. 4 has been used to fit melting point data to show that the confined

nanocrystals have the expected predictable linear-fit Gibbs-Thomson melting point depressions,

indicating that a decrease in the surface interaction energy of the nanocrystals with the substrate,

found in the numerator, is balanced by a simultaneous decrease in the enthalpy of fusion of the

nanocrystals, found in the denominator. The melting point data from both the single-stage and

two-stage MSMPR were plotted against the inverse of the pore diameter. Fig. 20 shows that the

confined crystals produced by MSMPRs have the expected melting point depression behavior

with minimal deviation from the theory and minimal error in melting point temperature within a

single pore size. The fit predicts a bulk melting point of 81.9 ºC, very close to the measured bulk

melting temperature of 81.6 ºC.

89

Fig. 20: Constant-enthalpy surface interaction energy Gibbs-Thomson equation fit to melting

points of confined nanocrystalline FEN averaged from all MSMPR runs

Crystal form identification with X-Ray powder diffraction (XRPD)

All samples showed the same XRPD peak pattern, both within trials of the same size porous

silica and across different pore sizes. Furthermore, the crystal structure was the same regardless

of the number of stages used in the MSMPR setup or the temperature of the filter. Fig. 21 shows

a representative scan of each type of experiment conducted (with the baseline silica amorphous

feature present and scaled or shifted for clarity of comparison) showing identical peak patterns.

Crystalline fenofibrate form I has reported theoretical diffractogram main peaks at 12° (2θ),

14.5° (2θ), 16.2° (2θ), 16.8° (2θ), and 22.4° (2θ) 56. The identity of all samples of nanocrystalline

fenofibrate as form I can be confirmed by matching peaks and the absence of other peak

positions. The high loading of the single-stage MSMPR run with a cold (17°C) filter for the

loading step is evidenced in the relatively lower amorphous silica underlying baseline

contribution to this scan.

90

Fig. 21: XRPD scans of FEN nanocrystals from various runs show crystallinity and consistent

formation of form I

Extension of principle to poorly soluble compounds

Griseofulvin (GSF) and indomethacin (IMC) were chosen as compounds that are not only poorly

water soluble but which also have low solubilities in most of the FDA Class III solvents as

challenging systems in which to achieve high pore loadings in a nanoporous silica matrix.

Saturation solubility estimates exist for both APIs of about 40 mg/mL in acetone at 25°C 95,96.The

theoretical maximum loading from the single-pass loading method was calculated assuming that

all of the GSF or IMC contained in the original undersaturated solution of 30 mg/mL crystallized

in the pore and that the pore volume of the silica was completely filled with solution. The

theoretical “filled pore maximum” theoretical loading was also calculated assuming that the

entirely pore volume was filled with crystals based on the density of GSF, 1.4 mg/mL 97, and

assumed density of 1.0 mg/mL for a conservative estimate for IMC due to lack of available data.

The experiments were all conducted in Aeroperl® of pore size 35 nm to have the highest pore

volume silica (1.6 mL/g). A previous study 98 used IMC as a model compound to test similar

91

loading procedures, however this study used silica with smaller pore sizes and did not produce

the crystalline form of drug which was targeted in this work.

Table 7: Loading results of multi-step procedure for nucleation then growth of crystals to increase

the weight percent loading

API

Theoret. one-

pass max. load

from 30 mg/mL

soln. (wt%)

Theoret. filled

pore max. load

(wt%)

One-pass

loading

(wt%)

Two-pass

loading (wt%)

Three-pass

loading (wt%)

GSF

4.6 69.1

3.0 ± 0.6 16.9 ± 1.7 26.1 ± 2.0

DSC

notes

No surface

crystals

No surface

crystals

Some surface

crystals

IMC

4.6 61.5

- 10.9 ± 2.2 13.2 ± 3.9

DSC

notes Undetected

Mostly surface

crystals

Mostly surface

crystals

Table 7 summarizes the results of the experiments. Extremely low loadings were seen for the

one-pass loading method, attributable to the very low concentration of API in solution. GSF was

loaded to around 3.0 ± 0.6 wt% and the IMC was undetectable in these samples, likely indicating

that what IMC had crystallized from the acetone solution was only on the surface of the porous

silica and was washed off. In the two pass loading system, the loading of GSF was increased to

an appreciable value greater than 15 wt% and in the three-pass system the loading was above 25

wt%. The DSC thermograms of the loaded samples shown in Fig. 22 show that in the two-pass

system, all of the crystalline GSF produced was confined to the pores and showed the

characteristic depressed melting point. However, in the three-pass system significant surface

crystals added to the higher loading as evidenced by the second peak at the bulk melting

92

temperature. With the two and three-pass methods, IMC was detectable in the samples but the

DSC thermograms indicated the majority of the contribution was from bulk crystals on the

surface of the silica or amorphous content and the methodology was unable to produce IMC

nanocrystals confined to the silica pores.

Fig. 22: DSC scans of GSF in porous silica compared to bulk crystals show that the two-pass

loading technique was able to produce confined nanocrystals with no significant surface crystals

3.4 Conclusions

Fenofibrate was successfully loaded into a range of nanoporous silica with varying pore sizes

using a continuous MSMPR methodology. A single-stage MSMPR was shown to reliably wet

the pores for subsequent crystallization by evaporation to yield average nanocrystalline FEN

loadings across pore sizes of 37 wt% at 25 °C. Cooling crystallization was also employed on

wetted porous silica from a single-stage MSMPR to achieve average FEN loadings of 48 wt%.

When the single-stage design was coupled to a second stage crystallizer in which a

supersaturated solution was allowed to grow the nanocrystals which had already been loaded in

93

the pores, loadings increased without substantial contributions from crystals on the surface of the

porous silica. The average two-stage MSMPR loading across pore sizes was 51 wt% at 25 °C.

This was an important improvement in methodology and loading from the previous chapter’s

work. The nanocrystalline FEN produced was the same polymorph across all studies and showed

the expected melting point depression behavior expected from a Gibbs-Thomson relationship. A

benchtop extension of the principle was shown to improve the loading of griseofulvin in porous

silica from just 3.0 wt% to over 15 wt% with no surface crystals, but was unable to load the

poorly water soluble drug indomethacin with appreciable quantities in the pores; indomethacin

showed a strong propensity for crystallization on the surface of the silica related to its nucleation

and growth kinetics. Using a multi-stage MSMPR apparatus to load poorly soluble drugs in silica

matrices has the ability to decouple nanocrystal formation from growth and allow for high drug

loadings for these compounds. In particular, a material used in this study, Aeroperl® 300

Pharma, which not only has the highest pore volume but also meets the USP/NF guidelines, is an

attractive matrix for further pursuit for oral dosage forms.


This chapter has been adapted and published as a peer-reviewed journal article. Dwyer, L.;

Kulkarni, S.; Ruelas, L.; Myerson, A. Two-Stage Crystallizer Design for High Loading of Poorly

Water-Soluble Pharmaceuticals in Porous Silica Matrices. Crystals 2017, 7, 131. We would like

to thank the Novartis-MIT Center for Continuous Manufacturing for financial support and

instrumentation use. L.M.D. is grateful to Carlos Pons Siepermann, Jennifer Moffitt Schall, and

Dr. Marcus O’Mahony for valuable assistance and discussions

94

Chapter 4 : Understanding nanoscale surface functionalization’s

role on supersaturation in crystallizing solutions

4.1 Introduction

Heterogeneous nucleation is the formation of a crystal at a surface, at which the free energy

barrier for nucleation is lower due to the effective surface area energy being lower 3. In industrial

crystallization, surfaces are common, examples including vessel walls, mechanical stirrers, and

even undesirable particulates 99.

Many experimental studies have aimed to look at the effects of surfaces on crystal nucleation, in

particular surfaces which have modifications which may further induce or suppress nucleation

beyond the lowering of the surface energy barrier. In particular, self-assembled monolayers

(SAMs) have been used extensively including in this research group, as surfaces for

heterogeneous nucleation 31–33. SAMs are highly ordered molecular assemblies formed when a

surfactant adsorbs to a solid surface, creating a layer of functional groups oriented outward from

a typically inert surface 34. Using SAMs as surfaces for crystallization has been shown to have

effects on polymorphism35, enantiopurity 36, crystal size 100, and crystal morphology 101.

Within our group, an idea was proposed to use the concept of the surface functionality of SAMs

to act in a different way. It was theorized that if nanoparticles were surface functionalized, they

may provide enough surface area that the interaction of these surface functional groups with the

solvent and solute system in a crystallization would act like an additional solvent or antisolvent,

thereby promoting crystallization or dissolution. In the case of promoting crystallization, for

95

example, the addition of the functionalized nanoparticles would cause the system to become

supersaturated, and thus provide the driving force for crystallization. The types of nanoparticles

used could be exploited for desirable properties including magnetism for easy separation or core-

shell coatings for ease of surface functionalization. A similar concept has been shown in protein

crystallization systems 102. Initial promising results were developed by our group 38 which

prompted a continued study of this methodology presented here. This work was done in close

collaboration with Lucrèce H. Nicoud. Fig. 23 and Fig. 24 summarize the proposed schemes of

how the nanoparticles were thought to interact with solvent/solute systems.


antisolvent


solvent

96

4.2 Experiments and results

Materials for solvent/solute systems

All solvents used in this study were purchased at ACS grade or higher purity from Fisher

Scientific. All chemical precursors for iron oxide particle synthesis were purchased at reagent

grade from Sigma-Aldrich. Fenofibrate (FEN) was obtained from Xian Shunyi Bio-chemical

Technology Company. Diphenhydramine hydrochloride (DPH) was purchased from BeanTown

Chemical at a purity ≥ 99%. Silica coated magnetic nanoparticles with a total diameter of 200

nm were purchased from Spherotech Inc (catalog number SIM025-10H). N-decyltriethoxysilane

was purchased from Gelest (catalog number SID2665.0) to perform functionalization of the

silica surface of the nanoparticles with decane.

Production and size control of iron oxide nanoparticles

Over the course of a year, we attempted to continue to investigate the work previously conducted

by Kulkarni in our lab 38. In this work, gold (procured from Spherotech, Inc., Lake Forest, IL) and

iron oxide nanoparticles (synthesized in lab) ranging from 1.8 nm in diameter to >10 nm in

diameter were functionalized with different surface chemistries to behave as antisolvent-like

molecules for a given system. It was later proposed that, similarly, the surfaces could be

functionalized to act as solvents in a system to improve the solubility of a compound of interest.

It was shown that D-mannitol could be crystallized from water using 1.8 nm gold particles

functionalized with 2-mercaptoethanol (Spherotech, Inc.). Fenofibrate was similarly crystallized.

A saturated solution of fenofibrate in ethyl acetate was prepared, and decanoic acid

functionalized iron oxide particles reported to be about 5 nm in diameter were added to solution.

The spontaneous crystallization of fenofibrate was observed within minutes. The iron oxide

97

particles were easily separated from solution with a magnet. It was proposed that the high surface

area of the nanoparticles allowed their functional groups to behave as a traditional liquid addition

to a mixed-solvent system.

These initial successes prompted us to continue to investigate this mode of action, as well as the

reverse mode of action, with solvent-like chemistry to act to improve the solubility of a

compound. This would be most applicable in a recrystallization, where a slurry of API in

antisolvent is made to go into solution again, followed by a subsequent recrystallization, to

remove impurities. However, for both directions of the system, we believed that significant effort

on producing small, well-dispersed nanoparticles of the iron oxide would contribute to the

success of the system, because the gold system with true nanoparticles of <10 nm had the most

promising initial results. We therefore began investigation of a matrix of synthesis conditions to

assess their effect on the nanoparticles produced as well as investigating various procured iron

oxide nanoparticles. We also determined that the thiol functionalization used in the gold

nanoparticles with limited reversibility was likely a good contributor to the success of the

functionalized particles. We therefore investigated silica-coated iron-oxide so that we could do

silane functionalization for a more permanent functionalization than weak surface interactions

directly to the iron oxide surface.

Focusing on size, the previous method employed to produce the iron oxide nanoparticles was

successful at producing the correct form of iron oxide to be magnetic (Fe3O4), however the

particle size was much larger than desired. As measured by dynamic light scattering (DLS), these

particles were in the range of 1000+ nm. The method is summarized here:

98

IONPs were synthesized by chemical co-precipitation method under alkaline

condition and molar ratio between Fe2+ salt and Fe3+ salt was maintained at 1:2. In

order to synthesize 1 g of Fe3O4 particles, 0.86 g of FeCl2_4H2O and 2.35 g of

FeCl3_6H2O were dissolved in 40 mL ultrapure water under N2 atmosphere with

vigorous stirring at speed of 1000 rpm. As the solution was heated to 80 °C, 5 mL

of NH4OH solution was added and the reaction was continued for another 30 min.

The resulting suspension was cooled down to room temperature and washed with

ultrapure water. The product of bare magnetic nanoparticles (IONP) was isolated

from the solvent by magnetic decantation. The IONP’s further washed with water

5 times and separation by magnetic decantation.

In consultation with the literature and members of the Roman lab at MIT, we determined that to

produce the correct size of nanoparticles we would employ a microemulsion method to confine

the reaction volume to micro droplets of aqueous solution in heptane, with a surfactant to

stabilize the emulsion. Several important factors in this synthesis were highlighted and are being

systematically tested to determine a procedure to produce nanoparticles in the size range we

desire (~10 nm), consistently in the correct form (Fe3O4 as opposed to FeO and Fe2O3). These

factors include:

Headspace and atmospheric conditions (air or pure nitrogen)

Vessel size

Heptane/water ratio

Surfactant concentration and type

Iron salt precursor concentration

pH upon addition of base

Heating

Wash, dry, recovery steps

99

The following method was been shown to produce stable magnetic iron oxide nanoparticles at a

size range nearer to the desired size particles, up to about 200 nm. Variations have been

completed wherein the ratio of water to heptane was decreased and the headspace was increased,

both of which produced the wrong form of iron oxide. We also aimed to develop a method in

which a silica shell may be grown on the iron oxide particles, for use in functionalization by

silanes.

A 20 mL scintillation vial is filled with 15 mL heptane and 2 mL Brij, a

commercial surfactant. This is stirred vigorously at 1000 RPM at room

temperature. In a separate vial, 107.4 mg of FeCl2_4H2O and 293.7 mg of

FeCl3_6H2O are dissolved in 5 mL ultrapure water. This is then added to the Brij

and heptane mixture and allowed to stir vigorously to form a reverse

microemulsion with salt contained in the aqueous phase within the larger heptane

phase. After this has stirred for 15 minutes at minimum, 1 mL concentrated

NH4OH was added to the solution. A black precipitate formed immediately,

however the solution was allowed to continue stirring for another 15 minutes.

In the case where we desired to produce bare IONP, the emulsion was broken with the addition

of about 20 mL methanol. The iron oxide nanoparticles were sized directly, after collecting and

washing with water, and after drying and re-suspending. In the case where we desired to coat the

particles with silica, 100 μL Tetraethyl orthosilicate (TEOS) was added. The TEOS partitions

into the water phase and decomposes to silica in the presence of water. This solution was

allowed to stir for 24 hours, at which point the nanoparticles were sampled for sizing.

100

The following diagram summarizes the size of IONP produced with the second method above.

These results are from dynamic light scattering experiments performed on a DynaPro NanoStar

by Wyatt Corporation available at the MIT BioInstrumentation facility. The particles synthesized

by the reverse microemulsion method are nearer to the desired size range but work remains to

produce smaller particles of the correct form and understand the operating parameter effects on

the synthesis.

Fig. 25: Size description of synthetic IONPs as measured by DLS

Besides working on the synthesis of nanoparticles, we also looked for commercial suppliers.

Table 8 summarizes the commercial nanoparticles that were ordered.

101

Table 8: Summary of the commercial nanoparticles under investigation

Particle type Particle size Supplier Comments

Silica nanopowder 5-15 nm Sigma

$ 2/g

We did not manage to properly disperse

those nanoparticles (using vortexing,

sonication, and different solvents)

There are also safety issues related to

such nanopowders.

Iron oxide core

+ silica shell

dispersed in hexane

14 nm

(core 8 nm)

Particle

works

$ 2’000/g

We could not separate these nanoparticles

with a magnet.

Iron oxide core

+ silica shell

dispersed in water

200 nm

(core 160

nm)

Spherotech

$ 600/g

These particles require several minutes to

separate them with a magnet from a 1 mL

solution.

We are currently working with these

nanoparticles to develop our

functionalization protocol.

Iron oxide core

+ silica shell

dispersed in water

40 to 100 nm

Creative

diagnostics

$ 20’000/g

We received samples of these particles.

DLS analysis revealed that the

nanoparticles are larger than expected

(140 to 300 nm), so these nanoparticles

were not used anymore.

Fig. 26 shows a TEM picture of the nanoparticles from Spherotech. The silica shell appears with

a lighter grey than the iron oxide core.

Fig. 26: TEM picture of the iron oxide nanoparticles from Spherotech

102

Functionalization of silica-coated nanoparticles

We aimed at functionalizing silica coated iron oxide nanoparticles with silanes. Fig. 27 shows a

scheme of the functionalization reaction. Several silanes were purchased from Gelest, including

hexyltriethoxysilane, 2-cyanoethyltriethoxyehtylsilane, acetoxyethyltrietoxysilane.

Fig. 27: Scheme of the mechanism of silane deposition on the silica surface

103

We then focused on the functionalization of the commercial 200 nm silica coated iron oxide

nanoparticles with hexyltriethoxysilane. The current protocol goes as follows:

Wash 5 mg of nanoparticles with water and then with anhydrous ethanol. Remove

the solvent with magnetic separation. Prepare a solution with: 95 % w/w

anhydrous ethanol + 5 % w/w water acidified at pH ~4 with acetic acid. Add the

silane to reach a 2 % w/w concentration. Sonicate for 2 min in the bath sonicator.

Add 1 mL of this reaction mixture to the washed nanoparticles and vortex.

Perform the reaction at 25 °C under shaking at 500 rpm for 24 hours. Dry the

nanoparticles for 2 hours in an oven at 90 °C. Wash the nanoparticles with

ethanol. To do so, use the tip sonicator to properly redisperse the nanoparticles.

Remove the solvent with magnetic separation.

The functionalized nanoparticles were analyzed by Fourier-transform infrared spectroscopy

(FTIR) and the results are shown in Fig. 28 with an orange line. As comparison, the FTIR spectra

of the non-functionalized nanoparticles and of the silane are also shown. The small peak small at

around 3000 cm-1 corresponds to the stretching of the C-H bond, which suggests that the

functionalization was successful.

104

Fig. 28: FTIR spectrum of the silica coated iron oxide nanoparticles functionalized with

hexyltriethoxysilane. As a comparison, the FTIR spectra of the plain nanoparticles and of the silane

are also shown. The spectra on the right-hand side of the figure are similar to those on the left-hand

side but with a different scale

The nanoparticles were also analyzed by thermogravimetric analysis (TGA) and the results are

shown in Fig. 29. For both the non-functionalized and functionalized nanoparticles, the mass

decreases when the temperature is increased. Nevertheless, the two signals differ at around

500 °C. These results suggest that the grafted silane represent around 1 % of the mass of the

nanoparticles. However, quantifying accurately the degree of functionalization with FTIR and

TGA reveals quite challenging due to the small mass of grafted silane with respect to the mass of

the nanoparticle.

105

Fig. 29: TGA analysis of the silica coated iron oxide nanoparticles functionalized with hexane

(silane SIH6167.5).

The protocol described above was also applied to 2-cyanoethyltriethoxyehtylsilane,

acetoxyethyltrietoxysilane.

Continued testing of functionalized nanoparticles in relevant systems

The bulk of the results of trying to replicate the initial successes with both the gold nanoparticles

and iron oxide nanoparticles indicate that because we are working with such fine degrees of

changing thermodynamic equilibrium, temperature obviously plays a large role in the

experiments. Through trying to repeat experiments with mercaptoethanol functionalized gold

nanoparticles of 1.8 and 5 nm diameters, whose surface areas should have been quite large

enough to contribute the originally understood effect, we were unable to produce crystallization

from solutions of saturated mannitol dissolved in water, even with the addition of nearly 1:1

parts water and gold nanoparticle slurry. We held these solutions at a constant temperature of

25 °C rather than relying on room temperature to stay constant over the entire laboratory, as it

was done in the preliminary experiments. These solutions never crystallized when held in this

temperature regulated environment. Unfortunately, similar results were shown with the

106

functionalized iron oxide nanoparticles and silica-coated and functionalized iron oxide, which is

unsurprising because the concentration of these particles would allow for even less surface area

than their nanometer gold counterparts. When temperature was properly controlled for, it was

determined that the functionalized particles could not induce crystallization from saturated

solutions.

Initial results indicating effects on heterogeneous nucleation

Cooling crystallization

However, there was some indication that the functionalized nanoparticles promoted faster

nucleation than their unfunctionalized counterparts, or than control experiments in which no

nanoparticles were added. The effect on heterogeneous nucleation was also studied in

diphenhydramine hydrochloride (DPH). We prepared a solution of DPH in isopropanol at 30

mg/mL by heating to about 30°C. All of the DPH dissolved. We determined the saturation

solubility of our samples of DPH in IPA at bath conditions of about 23°C to be around 26

mg/mL. We removed our 30 mg/mL solution from heat and brought it to bath temperature of 23°

at which point we saw no crystallization. We then added around 1.2 mg/mL of iron oxide

nanoparticles functionalized with decanoic acid synthesized in the lab, of about 200 nm in

diameter, and saw crystallization within a minute. We understand that the effect of temperature

would lead to crystallization in this scenario after some time, so we aimed to then compare the

effect of the nanoparticles on nucleation in comparison with cooling crystallization. A solution

of 250 mg/mL DPH in isopropanol was made at 60°C in multiple vials. To one set of vials, 1

mg/mL synthesized iron oxide nanoparticles functionalized with decanoic acid were added, in

another set iron oxide particles with no functionalization were added, and all sets of vials were

107

cooled at 1°C/min from 60°C to 5°C. The vials with the addition of the functionalized iron oxide

nanoparticles crystallized in under 15 minutes, those with non-functionalized particles took

slightly longer averaging 20 minutes, and those with cooling alone took approximately 1 hour. A

magnet easily separated the nanoparticles from the solution leaving the crystals undisturbed at

the bottom of the vial.

Anti-solvent crystallization

After examining the impact of nanoparticles on heterogeneous nucleation in cooling

crystallization experiments, we investigated their effect during anti-solvent crystallization. To do

so, diphenhydramine (DPH) was dissolved in ethanol at a concentration of 300 mg/mL and

heptane was added to reach different levels of supersaturation. The experiments were performed

at 25 °C either in the absence of nanoparticles or in the presence of commercial core-shell

magnetic nanoparticles having a 160 nm iron oxide core and a 20 nm thick silica shell.

Fig. 30 shows experimental data of the solubility of DPH as a function of the volumetric

percentage of heptane in the absence of nanoparticles. The black line represents the dilution line

upon heptane addition starting from a solution at 300 mg/mLEtOH = 277 mg/gTOT. This graph

shows that crystallization is expected to occur at heptane concentrations above approximately

10 % v/vTOT.

108

Fig. 30: Experimental data of the solubility of DPH as a function of the volumetric percentage of

heptane (symbols). The black line represents the dilution line upon heptane addition.

In a first set of experiments, crystallization was investigated at the milliliter scale at various

heptane concentrations above 10 % v/vTOT. Five samples were prepared for each condition and it

was checked after 24 hours of shaking whether crystallization occurred or not. The results are

summarized in Fig. 31. It is seen that in the absence of nanoparticles, most of the samples did not

crystallize after 1 day. In particular, none of the samples below 20 % heptane crystallized. At

heptane concentrations between 25 and 55 %, only some of the samples crystallized and it was

necessary to increase the heptane concentration up to 60 % to observe crystallization in the 5

samples. This shows that kinetic limitations hinder crystal formation at low heptane

concentrations, i.e. in the metastable zone. Interestingly, it was observed that the presence of

bare nanoparticles promotes crystal formation at low supersaturation. Indeed, all the samples

above 33 % heptane crystallized in the presence of nanoparticles.

109

Fig. 31: Crystallization likelihood at various concentrations of heptane and bare nanoparticles. Five

samples were prepared for each condition 103

In a second set of experiments, crystallization was performed in a 100 mL reaction vessel and

was followed by focused beam reflectance measurements (FBRM). The heptane concentration

was set to 33 %, while bare nanoparticles were added in various concentrations varying from 0 to

0.25 g/L. Fig. 32 (left) shows the total number of counts recorded as a function of time. It is seen

that in the absence of nanoparticles, there is a lag time during which no crystals are detected.

After about 30 min, crystallization starts and the number of counts per second rises. When

nanoparticles are added, this lag time disappears and the total number of counts increases, which

suggests that more nanoparticles are formed. Fig. 32 (right) shows the chord length distribution

recorded at 1 hour. In the absence of nanoparticles, the chord length distribution is monomodal

and is centered around 80 μm. When nanoparticles are added, a second peak arises at around 20

μm and its relative contribution increases when increasing the concentration of nanoparticles.

110

Overall, these data show that the presence of nanoparticles promote crystal nucleation and leads

to smaller crystal sizes.

Fig. 32: Crystallization of DPH at 33 % heptane and various concentrations of bare nanoparticles

followed by FBRM. (left) Total number of counts as a function of time. (Right) Chord length

distribution at 1 hour

All the experiments presented above were performed with bare silica coated magnetic

nanoparticles. In a last set of experiments, the impact of surface functionalization was

investigated. To do so, the nanoparticles were functionalized with decane using N-

decyltriethoxysilane. As in the case of the bare nanoparticles, it was found that decane

functionalized nanoparticles promote crystal formation at low heptane concentration. However, it

was observed that the color of the crystals formed in the presence of decane functionalized

nanoparticles is lighter than those formed in the presence of bare nanoparticles, as shown in Fig.

33. This suggests that less nanoparticles are incorporated in the crystal lattice when the

nanoparticles are functionalized with decane.

111

Fig. 33: Pictures of DPH crystals formed at 43 % heptane with 0.25 g/L (left) bare and (right)

decane functionalized nanoparticles 103

To summarize, the major findings of this study are that: a) nanoparticles promote nucleation at

low antisolvent concentrations, i.e. in the metastable zone, b) nanoparticles lead to smaller

crystal sizes, and c) nanoparticle incorporation in the crystal lattice depends on the surface

functionalization.

Aggregation of nanoparticles and its role in the failure of the intended mechanism

Over the course of these experiments and the subsequent work performed by Lucrèce Nicoud in

an EasyMax crystallizer system, it became evident that the nanoparticles were not well dispersed

when used in crystallization systems. In these experiments, non-functionalized silica coated iron

oxide particles from Spherotech of diameter 200 nm were used. The following Fig. 34 shows an

FBRM chord length distribution measurement of one of the crystallizations performed in the

EasyMax, indicating that the nanoparticles were significantly larger than expected. A similar

phenomenon was seen using an imaging probe, a particle vision ParticleView V19 by Mettler-

Toledo which again provided evidence that the nanoparticle aggregates were significant in size,

as seen in Fig. 35.

112

Fig. 34: FBRM indicates that the nanoparticles are around 20 microns in size, indicating

substantial aggregation 103

Fig. 35: Images taken with a particle vision ParticleView V19, Mettler Toledo show nanoparticles

before crystallization (left) and in the presence of DPH crystals (right) 103

113

A simple calculation may be done taking the iron oxide particles which we had worked with

early on as an example. It was calculated that 1 gram of 5 nm IONPs has the same surface area

as 50 grams of 200 nm IONPs. At the nanoscale, even a small difference in diameter translates to

large differences in available surface area. This concept which we initially hoped to exploit as

the basis for why surface functionalization may have allowed the particles to behave as a solvent

addition to the system was inaccessible due to the particle aggregation. Addition of surfactants to

the system to separate the nanoparticles was considered. However the attraction of the system at

the start included the simplicity of the addition of nanoparticles only for ease of separation, as

well as the minimization of additional liquid solvent addition steps.

4.3 Conclusions

From an applicability standpoint, iron oxide particles and their magnetic separation ability are a

better target as a crystallization technology than gold nanoparticles due to the rather prohibitive

cost of gold nanoparticles. However, as a result of this work we discovered that due to synthesis

and aggregation, iron oxide nanoparticles are too large and aggregate too much to behave as a

pure antisolvent. We determined that nanoparticles at reasonable concentrations in the available

size range will not substantially affect solubility. Similar aggregation problems were seen even in

commercial particles with silica shells. However, larger particles that are still in the micron range

represent good targets for studying the effects of functionalized surfaces on heterogeneous

nucleation. In this line of reasoning, it was found that nanoparticles change the heterogeneous

nucleation kinetics and thus impact the crystal size and that functionalized nanoparticles

incorporate differently into the crystal lattice than non-functionalized nanoparticles indicating

that there is an effect on the nucleation behavior.

114

This work prompted continued investigation of the effects that the coated nanoparticles had on

heterogeneous nucleation primarily conducted by Lucrèce Nicoud. This work was later published

as “Heterogeneous Nucleation of Small Molecule Crystals in the Presence of Nanoparticles” in

AIChE Proceedings, 2017 103. Additional investigation into how surface modification might be

used at the nanoscale in conjunction with our knowledge of confinement at the nanoscale is

discussed in the next chapter of this work.


We would like to thank the Novartis-MIT Center for Continuous Manufacturing for financial

support and instrumentation use as well as the DARPA ASKCOS project at MIT for support and

funding. L.M.D. is grateful in particular to Lucrèce Nicoud without whom this work would not

have taken place. We would also like to thank Aaron Garg of the Roman Group at MIT for his

assistance with synthesis.

115

Chapter 5 : Surface functionalization in combination with

confinement for crystallization from undersaturated solutions

5.1 Introduction

This chapter pertains to the crystallization of organic compounds, focusing on active

pharmaceutical ingredients (APIs), from undersaturated solutions through a new combination of

confinement and surface functionalization effects using Zorbax® functionalized

chromatographic media. The technique is demonstrated in several experimental methods, the

most conclusive of which confines the media and API solution to a sealed capillary tube chamber

wherein crystallization is monitored in real time. Discussions of the extension of these principals

to alternative porous matrices and functionalizations may be found in the applications portion of

the chapter.

Crystallization is an important separation and purification technique, especially in the

pharmaceutical industry. The tight regulations on product quality including crystal form, particle

size distribution, crystal shape, purity, and yield require fine control of the crystallization process

104. The crystallization process includes both nucleation and growth, both of which have

implications for the end product of the process. For nucleation to occur, the solution must be

supersaturated, meaning the concentration of solute is greater than the equilibrium concentration

at that given temperature and solvent condition 3. Supersaturated conditions are generated most

often by changing the solvent with the addition of an antisolvent or by cooling the solution. The

addition of heterogeneous surfaces to the crystallization process also changes the dynamics of

nucleation 105.

116

Antisolvent crystallization is particularly common in pharmaceutical processes when the solute

or active pharmaceutical ingredient (API) may be sensitive to changes in temperature 3. A solute

molecule is dissolved in one solvent, typically at ambient temperature. The addition of an

antisolvent to the solution generates supersaturation because the solute is less soluble in this new

anti-solvent. The choice of antisolvent and composition of the solvent/antisolvent mixture can

have implications on crystal size, shape, form, and yield 106–108.

Heterogeneous surfaces can be used to facilitate the formation of crystal nuclei from

supersaturated solutions. These surfaces may include vessel walls or stirring mechanisms, or

seeds of the desired crystal product 99. The activation energy of nucleation is lowered in these

instances due to the addition of the surface site, corresponding to a decrease in the surface area of

the nuclei in classical nucleation theory. Templates whose surface chemistries or geometries

(such as self-assembled monolayers, SAMs) are selected to be favorable for certain solute

crystallizations have been shown to promote nucleation and growth 31,33,35,101. The selection of a

surface chemistry can orient solute molecules in addition to providing a surface site, and thus

lower even further the energy barrier to nucleation.

Crystallization in confinement has been demonstrated as a way to produce stable pharmaceutical

nanocrystals of a controlled size 49,59,81. In this approach, crystallization of the API is restricted to

a micro- or nanoporous environment to form nanocrystals, with contributions to nucleation both

from the confinement of the crystallization volume and heterogeneous surfaces of the pore. This

study combines the effects of crystallization in confinement as well as the principles of

heterogeneous nucleation and surface functionalization to produce nanosized crystals from

undersaturated solutions. Crystallization in pores has been carried out before, however there the

117

driving force was always present due to antisolvent addition, evaporation, or cooling

crystallization as opposed to this unique combined effect of surface functionalization and

confinement.

Zorbax® chromatographic media with C8-like surface groups was used, to mimic the functional

group interaction of alkanes, which tend to be poor solvents for the organic APIs chosen for this

study. We postulate that in the confined volumes of the pores of the matrix, the addition of these

surface groups rendered a change in the solubility of API in solution in these nanoscale volumes,

providing the driving force for nucleation and crystallization. This has been applied to the

crystallization of several small molecule organic compounds. Given that the parent solutions are

undersaturated, when held at a fixed temperature left alone or in the presence of non-

functionalized control silica media which mimics the surface area of the Zorbax® media, the

solutions will not crystallize because there is no driving force for nucleation. However, we

demonstrate that, in several experimental setups, the addition of the functionalized Zorbax®

media induces crystallization. The confined nanoscale volumes of the pores have high surface

areas, and thus the expected contribution of the surface functionalization interaction with the

solvent in this environment is high. We believe the combined surface functionalization and

confinement effect allows for the Zorbax® media to act as an antisolvent, reducing the solubility

of the APIs and causing nucleation and crystallization.

118

5.2 Experimental

Materials

Zorbax® functionalized chromatographic media was obtained from Agilent in bulk packing

form. This is porous silica functionalized with a C8-like group (n-octyldimethylsilane). The

average listed grain size of the beads is 7 μm and the nominal pore size is 7 nm in diameter with

a nominal surface area of 160-180 m2/gram. As a control, unfunctionalized controlled pore glass

(CPG) was purchased from Prime Synthesis (Aston, PA, USA). This is a fumed silica with

controlled pore size of approximately 12 nm. The grain size of this material is about 100 μm.

Acetaminophen (APAP) was purchased from Sigma-Aldrich (BioXtra ≥99.0%, Lot

#SLBR2060V). Aspirin (ASA) and nicotinamide (NIC) were purchased from Sigma Life

Science (USP grade, Lot #MKBQ8444V and ≥99.5%, Lot #BCBF9698V, respectively).

Diphenhydramine hydrochloride (DPH) was purchased from BeanTown Chemical (>99.0%).

Fenofibrate (FEN) was obtained from Xian Shunyi Bio-chemical Technology Company

(Shaanxi, China). Solvents were purchased at ACS grade or higher purity from Fisher Scientific

(Waltham, MA, USA). Benzophenone was purchased from Sigma-Aldrich in ReagentPlus form

at purity ≥99%.

DSC and TGA analysis

Thermogravimetric analysis (TGA) was performed on a Q500 instrument from TA instruments

(Newcastle, DE, USA) connected with a nitrogen gas cylinder to maintain a flow rate of 25

mL/min to maintain an inert gas environment in the sample chamber. Between 5 and 10 mg of

119

sample were loaded on platinum sample pans from TA Instruments. The samples were allowed

to equilibrate at 30 °C and then heated at 10 °C/min to 300 °C.

A Q2000 instrument from TA instruments was used for the differential scanning calorimetry

(DSC) analysis. Inert atmosphere environment was maintained in the sample chamber using a

nitrogen gas cylinder set to a flow rate of 50 mL/min. An extra refrigerated cooling system (RCS

40, TA Instruments) was used to widen the available temperature range to between −40 and

400 °C. Tzero® pans and lids were used with ~5 mg of sample. A heating rate of 10 °C/min was

applied and the samples were scanned from −20 to 200°C.

XRPD analysis

XRPD was performed in a capillary setup using a PANalytical X’Pert PRO (Almelo, the

Netherlands) diffractometer at 45 kV with an anode current of 40 mA. The instrument has a

PW3050/60 standard resolution goniometer and a PW3373/10 Cu LFF DK241245 X-ray tube.

Samples were loaded in capillary tubes (see later section) and aligned on a goniometer capillary

holder in stage capillary spinner mode with a focused point incident beam using a Cu Si focusing

mirror. Settings on the incident beam path included: soller slit 0.04 rad, mask fixed 10 mm,

programmable divergence slit and fixed ½° anti-scatter slit. Settings on the diffracted beam path

include: soller slit 0.04 rad and 1/8°anti-scatter slit. The scan was set as a continuous scan: 2θ

angle between 2° and 90°, step size .0167113° and a time per step of 15.240 seconds.

Submerged loading experiment

In initial experiments, an undersaturated solution of fenofibrate in ethyl acetate was made at

25°C. Fenofibrate is highly soluble in ethyl acetate with a saturation solubility of about 650

120

mg/mL at 25°C, as determined by gravimetric analysis of the filtrate of a stirred slurry. The

undersaturated solution was prepared at 550 mg/mL. One gram of Zorbax® was added to a 20

mL scintillation vial, and 10 mL of the fenofibrate solution was pipetted on top. The vial was

sealed and held for 6 hours at 25°C. A second vial was made in the same way using the control

CPG instead of the Zorbax® material. After the 6 hours, the vials were poured over a paper filter

with vacuum and immediately filtered and rinsed with 5 mL cold ethanol to remove any residual

API solution. The resulting powder was dried and analyzed with DSC and TGA. The

experiments were repeated for initial fenofibrate concentrations in ethyl acetate of 450, 350, and

250 mg/mL. All experiments were repeated in triplicate.

Column loading experiment

A low-pressure glass column was purchased from Bio-Rad Laboratories (Econo-Column®) that

was 1.0 cm in diameter and 10 cm in length with a fill volume of about 8 mL. The column

contained polypropylene end fittings and a porous polymer bed support at the bottom to retain

fine particles. A feed solution bottle was placed in a water bath maintained at 25°C (Thermo

Scientific NESLAB RTE, Waltham, MA, USA). Peristaltic pumps (Masterflex P/S, Thermo

Scientific) with Viton tubing (Cole-Parmer, Vernon Hills, IL, USA) were used for solution

transfer. The column was compatible with isopropyl alcohol at room temperature. A feed

solution of was prepared of diphenhydramine hydrochloride in isopropyl alcohol 25°C at 30

mg/mL. The column was packed with 5 grams of Zorbax®, or CPG for the control case. This

solution was pumped to the column at 1.0 mL/min. The column was run for 60 minutes and the

flowthrough solution was collected. Two mL of solution was collected at time points 0, 20, and

40 minutes, and the total collected volume was sampled at the end of the run at 60 minutes.

121

Gravimetric analysis was used to determine the concentration of DPH in solution. At the end of

the run, the column was flushed with 10 mL of isopropyl alcohol at 0°C followed by air.

Samples of the dried column packing were collected and analyzed with TGA and DSC. Two runs

each of both the Zorbax® and the CPG packing were performed. A schematic of the apparatus is

shown in Fig. 36.

Fig. 36: Schematic of the column setup for porous matrix loading experiments

Additionally, a final experiment was performed in which an impurity was added to the feed

solution sent to the column. All details of the column setup remained identical to those above.

Benzophenone is a known impurity of diphenhydramine hydrochloride. The USP monograph on

the purity of diphenhydramine hydrochloride lists that the known impurity benzophenone must

be present below at an impurity concentration of .0025 mg/mL or no more than 0.4% of DPH. 109

122

It was expected that the column would selectively crystallize the desired crystal, DPH, and the

impurity, benzophenone, would remain in solution and enter the flowthrough.

A feed solution of was prepared of diphenhydramine hydrochloride in isopropyl alcohol 25°C at

(29.5 mg DPH and 0.5 mg benzophenone)/mL. The column was packed with 5 grams of

Zorbax®, or CPG for the control case. This solution was pumped to the column at 1.0 mL/min.

The column was run for 60 minutes and the flowthrough solution was collected. Two mL of

solution was collected at time points 0, 20, and 40 minutes, and the total collected volume was

sampled at the end of the run at 60 minutes. Gravimetric analysis was used to determine the

concentration of solids in solution. At the end of the run, the column was flushed with 10 mL of

isopropyl alcohol at 0°C followed by air. Samples of the dried column packing were collected

and analyzed with TGA and DSC. Two runs each of both the Zorbax® and the CPG packing

were performed.

Capillary crystallization experiment

Slightly undersaturated solutions of several APIs were prepared at 25°C. The saturation

solubilities of these compounds in the chosen solvents were first found by stirring a slurry of the

API in solvent at 25°C for several days, filtering the solution, and performing gravimetric

analysis. These experiments were repeated in triplicate. The saturation solubility of

acetaminophen in water was found to be 15.5 mg/mL. An undersaturated solution of 12.0 mg/mL

was prepared. The saturation solubility of aspirin in isopropyl alcohol was determined to be 90.0

mg/mL. An undersaturated solution of 75.0 mg/mL was prepared. The saturation solubility of

diphenhydramine hydrochloride in isopropyl alcohol was found to be 38.0 mg/mL and an

undersaturated solution of 30.0 mg/mL was prepared. Finally, the saturation solubility of

123

nicotinamide in isopropyl alcohol was found to be 46.5 mg/mL and a solution of 38.0 mg/mL

was prepared.

Glass capillary tubes were purchased from Hampton Research (Aliso Viejo, CA). The tubes were

made of glass #50, with a 1.0 mm OD, wall thickness of 0.01 mm and length of 80 mm. The

capillary tubes were packed with Zorbax® or CPG material by scooping powder into the tip,

placing the capillary in a larger diameter glass pipette, and tapping to allow gravity to act on the

powder to settle. Once packed, the tubes were suctioned at the open end with a 1-mL plastic

syringe, using a small piece of rubber tubing to adapt the fit. The filled capillary was then

immediately dipped into the undersaturated API solutions to allow the solution to fill the tube.

Molten was used to plug the end of the capillary, and the tip was then heated in a Bunsen burner

flame to seal. Additional capillaries were filled with liquid API solution alone.


Submerged loading experiment

In previous studies, it was shown that wetting the pores of CPG with an API solution and then

allowing for evaporative crystallization can produce crystals confined to the pores of the silica

matrix. The driving force for crystallization in these instances was the increase in concentration

of the parent solution by evaporation of the solvent. The API solutions which was loaded into

pores in these studies was near saturation, and quickly became supersaturated as evaporation

occurred.

This experiment differed by use of an undersaturated API solution that was never allowed to

evaporate, and thus should have maintained the same undersaturated condition under which no

124

crystal nucleation should have occurred. Any driving force for crystallization would have to stem

from the dynamics of confinement and surface functionalization resulting from the addition of

the porous silica matrices. Additionally, some driving force for crystallization could be expected

in the handling of the material at the end of the experiment. The wash and dry steps were

intended to minimize any crystallization from evaporation of remaining solution. However some

crystallization of the parent solution could have occurred in this step. Indeed, in the control

experiment using unfunctionalized CPG, a trace amount of FEN was detected in the resulting

powder, measuring less than 5 wt% by TGA analysis.

Fig. 37 shows the DSC scan of samples loaded from the 550 mg/mL solution. The samples

loaded onto the Zorbax® material have two distinct melting peaks, one at a depressed melting

point indicative of smaller sized crystals confined to the pores of the material, and one at the bulk

melting temperature. The DSC scan of the control material showed a very small peak at the bulk

FEN melting point, indicating that the crystals formed were of normal micron size and likely

formed on the surface of the porous glass material as a result of the wash and dry steps.

125

Fig. 37: DSC scans of porous glass material loaded with a submerged basket from an

undersaturated solution of FEN in ethyl acetate (550 mg/mL). The Zorbax® material shows two

distinct melting points indicating the presence of crystallization in the pores.

When Zorbax® was used as opposed to unfunctionalized CPG, the average loading as

determined by TGA was significantly higher at an average of 22.4 ± 3.8 wt% for the samples

loaded from solutions of 550 mg/mL FEN in ethyl acetate. The concentration of FEN in the

loading solution was varied over several more undersaturated values, shown in Fig. 38. The

resultant loading of fenofibrate on the collected powders seems to decrease with decreasing

initial API concentration, indicating that this is correlated with the driving force for

crystallization. In combination with the two melting peaks seen in the DSC, this indicated that it

was likely crystallization was occurring as a result of the presence of the functionalized

nanoporous Zorbax® material as opposed to just during the wash and dry steps of the process.

126

Fig. 38: Average FEN loading on functionalized Zorbax® chromatographic media versus parent

solution concentration

Column loading experiment

The flowthrough collected from the two runs of the column setup performed using

unfunctionalized controlled pore glass and a feed solution of DPH only showed little change in

the concentration of API in solution over the duration of the experiment. The solution fed to the

column was undersaturated DPH in isopropyl alcohol, so it is therefore not surprising that in the

well-controlled environment of the column no evaporation or temperature changes occurred to

provide the driving force for nucleation. Fig. 39 shows the flowthrough concentrations in each

run of the CPG material. The concentrations of DPH in isopropyl alcohol vary little with time

from the original 30 mg/mL value, average 28.2 and 29.1 mg/mL at the end of the two runs. The

powder was also extracted at the end of the column run. This material showed less than 2 wt%

DPH present by TGA. The DSC scan showed only the slightest presence of bulk form DPH,

127

likely crystallized on the surface of the porous glass or column during the wash and dry steps

(Fig. 40).

Fig. 39: Flowthrough concentration of DPH collected as the column was run with CPG shows little

change

Fig. 40: Trace DPH with bulk melting temperature only seen in the CPG powder collected from the

column runs

128

When the column runs were performed with Zorbax® chromatographic media, the flowthrough

concentration of DPH in solution changed dramatically over the course of the experiment. Fig.

41 shows the decrease in concentration in the flowthrough, from an initial concentration of 30.0

mg/mL to an average end concentration of 15.9 and 16.0 mg/mL in the two runs, as measured in

the collected pool of flowthrough at the end of the run. The powder was extracted from the

column and analyzed with TGA and DSC. The DSC (Fig. 42) shows a single large peak at a

decreased melting temperature, indicating that the majority of crystals formed in the column are

confined to the pores. The TGA indicated loadings of 14.7 wt% and 15.4 wt% in the two runs. A

significant amount of the DPH in solution was crystallized in the column due to the presence of

the functionalized porous silica media as opposed to the control unfunctionalized silica. A mass

balance may be performed on each of these runs. Of the original 30 mg/mL solution flowed at 1

mL/minute for 60 minutes, the total original API present may be calculated (1800 mg). The

average TGA determined loading for the runs correspond to a yield of 41% and 43%

respectively. We can look at the flowthrough to account for the remainder of the material, and

find that for Run 1 the mass balance summing the loaded and flowthrough API accounts for 94%

of the total material and in Run 2, 96% is accounted for. Considering the fact that the sampling

of the loaded Zorbax® is an average over the entire column and that some API material may

have been trapped in the inlet and outlet apertures, these mass balances are very successfully

closed. While the <50% yields may seem poor at first glance, the flowthrough material may be

easily recycled and passed through another column or the same in a recycle loop, thereby

increasing the expected yield. Furthermore, we believe a primary application here, as indicated

earlier, would be in the purification of APIs. If the API that may interact with the selected

functional groups preferentially crystallizes on the column, we may enrich the flowthrough

129

solution in impurity, and collect purified API in the column. We may then elute the column with

fresh solvent and achieve a high purity API solution dissolved off the column.

Fig. 41: Flowthrough concentration of DPH in isopropyl alcohol on runs where the column was

packed with Zorbax® show a significant decrease indicating DPH retention on the column

Fig. 42: DSC scan of the Zorbax® material collected from the column run showing DPH confined

to the pores of the material

130

The experiments were repeated with an impurity present in the original solution. The feed

solution to the column was formulated to be (29.5 mg DPH and 1 mg benzophenone)/mL in

isopropanol. The flowthrough collected from the two runs of this column setup with impurity

present that were performed using unfunctionalized controlled pore glass showed little change in

the concentration of API in solution over the duration of the experiment. As before, the solution

fed to the column was undersaturated, so it is therefore not surprising that in the well-controlled

environment of the column no evaporation or temperature changes occurred to provide the

driving force for nucleation. Fig. 43 shows the solids flowthrough concentrations in each run of

the CPG material. Because gravimetry alone was used, this concentration is a total solids

concentration as opposed to having information about specifically the composition of DPH and

benzophenone. In the future, HPLC should be used to assess the concentrations of each in the

flowthrough. The concentrations of solids in isopropyl alcohol vary little with time from the

original 30 mg/mL value, average 29.6 and 28.7 mg/mL at the end of the two runs. The powder

was also extracted at the end of the column run. This material showed less than 1.5 wt% solids

present by TGA. Due to time constraints and the lack of change in the flowthrough

concentrations, DSC was not performed on these samples.

131

Fig. 43: Flowthrough concentration of solids collected as the column was run with CPG and

impurity shows little change

When the column runs were performed with Zorbax® chromatographic media and the impurity

solution feed, the flowthrough concentration of solids in solution changed dramatically over the

course of the experiment. Error! Reference source not found. shows the decrease in

oncentration in the flowthrough, from an initial concentration of 30.0 mg/mL solids to an

average end concentration of 17.3 and 13.2 mg/mL solids in the two runs, as measured in the

collected pool of flowthrough at the end of the run. The powder was extracted from the column

and analyzed with TGA and DSC. The DSC (Fig. 44) shows a single large peak at a decreased

melting temperature, indicating that the majority of crystals formed in the column are confined to

the pores. Furthermore, this depressed melting temperature point corresponds well to the point

found in the previous experiment with no impurity present, thereby indicating that the crystals in

the column are DPH only. The TGA indicated loadings of 13.4 wt% and 18.1 wt% in the two

runs.

132

Fig. 44: Flowthrough concentration of solids in isopropyl alcohol on runs where the feed also

contained impurity. The column was packed with Zorbax® and shows a significant decrease in

flowthrough solids concentration indicating DPH retention on the column

Fig. 45: DSC scan of the Zorbax® material collected from the column run with impurity in the feed

solution showing DPH confined to the pores of the material

A significant amount of the DPH in solution was crystallized in the column due to the presence

of the functionalized porous silica media as opposed to the control unfunctionalized silica. A

133

mass balance may be performed on each of these runs. Of the original (29 mg DPH and 1 mg

benzophenone)/mL solution flowed at 1 mL/minute for 60 minutes, the total original API present

may be calculated (1740 mg). The average TGA determined loading for the runs correspond to a

yield of 38% and 51% respectively. The second run is over a 50% yield, which was even better

than the impurity-free runs. The DPH is preferentially crystallized in the porous media over the

impurity, which can even result in this boost in yield.

We can look at the flowthrough to account for the remainder of the material, and find that for

Run 1 the mass balance summing the loaded and flowthrough API accounts for 95% of the total

material and in Run 2, 94% is accounted for. Again, if we consider the fact that the sampling of

the loaded Zorbax®is an average over the entire column and that some crystalline material may

have been trapped in the inlet and outlet apertures, these mass balances are very successfully

closed. To further confirm that the column loaded material contained DPH alone and no

benzophenone, XRPD was performed on the samples. XRD was also performed on the solids

collected from the flowthrough to confirm that this was a mix of DPH and benzophenone.

134

Fig. 46: XRPD of loaded Zorbax® collected from the column runs in which an impurity was added

to the feed solution. Neither column run's collected Zorbax® shows any trace of the impurity,

showing crystalline DPH only

Fig. 47: XRPD of the solids collected from the flowthrough material on the column from runs in

which an impurity was added to the feed solution. The flowthrough is a clear mix of both DPH and

benzophenone.

135

These column runs successfully demonstrated that we were able to selectively crystallize DPH

present in a parent solution with impurity on the Zorbax® packed column. We saw the formation

of DPH crystals in the column material without the formation of benzophenone crystals. This

demonstrates what we believe to be a primary application of this technology. If the API that may

interact with the selected functional groups preferentially crystallizes on the column, we may

enrich the flowthrough solution in impurity, and collect purified API in the column. We may

then elute the column with fresh solvent and achieve a high purity API solution dissolved off the

column.

Capillary crystallization experiment

The results of the previous experiments indicated that the presence of the functionalized

Zorbax® material caused crystallization where unfunctionalized CPG did not. However, in both

the submerged loading and column experiments, there was a wash and dry step which would

have potentially caused crystallization of the API from solution due to cooling, evaporative, or

antisolvent crystallization. Indeed we saw trace amounts of API that were attributable to this

cause in the control experiments. To eliminate the question of whether the crystallization seen in

the Zorbax® samples was attributable to the Zorbax® itself, we desired to isolate the system

from these other possible causes of driving force for crystallization and monitor the crystallinity

in a non-invasive way. We isolated the Zorbax® material to a glass capillary tube and loaded it

with the API solution and sealed the tube. The crystallinity was then monitored with XRD. When

the same procedure was carried out with unfunctionalized CPG, no changes were seen in the

XRD over time, with just an amorphous background signal from the silica. This was true for all

136

four API solutions tested. Furthermore, capillaries that were filled with API solution only also

did not crystallize.

In the Zorbax® experiments the four API’s tested all crystallized within the sealed capillary after

loading while being monitored in the XRD machine. All API solutions tested in capillary tubes

filled with Zorbax® functionalized media showed no crystallinity at the initial XRD scan at 0

minutes. In the case of aspirin and nicotinamide, the second XRD run at 20 minutes in to the

experiment showed crystalline form of the compound. The diphenhydramine hydrochloride scan

at 40 minutes showed the first crystallinity and the acetaminophen scan at 60 minutes showed

crystallinity. Samples were held for another 20 minutes after first showing crystallinity to see

that they maintained crystalline API within the sealed capillary. The time series of XRD scans

for each of the compounds discussed is shown in Fig. 48. An artificial offset was placed in the y-

axis so that each scan was visible.

137

Fig. 48: Time series XRPD scans of the API solutions loaded in capillary tubes filled with Zorbax®

media showing crystallization within 1 hour

5.4 Summary and application of the work

The following experiments demonstrated formation of nanoscale crystals from undersaturated

solutions in the presence of Zorbax® chromatographic media.

Submerged loading experiments

1) Zorbax® media is added to an undersaturated API solution and held at constant temperature with no

evaporation of solvent. The media is held for several hours.

2) The media is recovered through filtration followed by a fast 10 mL cold solvent wash, dried, and

analyzed.

138

3) The Zorbax® media is shown to have both surface crystals as well as confined nanocrystals which

were not present in control experiments performed with unfunctionalized silica media, suggesting

that the Zorbax® media allowed for the formation of the nanosized crystals.

Column loading experiments

1) Zorbax® media (5 grams) is held in a 10 mL column and temperature of the solutions is controlled

at 25 °C.

2) Undersaturated API solution is flowed slowly over the column (1 mL/hour) and flowthrough is

collected.

3) Flowthrough analysis demonstrates that in the control experiments performed with unfunctionalized

media, little API is retained on the column. However when the Zorbax® media is used, the

concentration of API in flowthrough solution decreases dramatically.

4) The Zorbax® in the column is analyzed and nanoscale crystals are formed. No nanoscale crystals

are formed when control unfunctionalized media was used.

Capillary crystallization experiments

1) Zorbax® media is packed into 1 mm OD glass capillaries for XRPD.

2) Undersaturated solutions of APIs are prepared. The capillaries are placed under

slight vacuum and filled with API solution.

3) Continuous XRPD is performed on these capillaries to determine crystallinity in live

time.

4) Capillaries packed with functionalized Zorbax® media showed crystallization from

four different undersaturated API solutions within 1 hour, whereas capillaries packed

with control media or API solution alone did not.

139

Proposed extensions of the technology

Crystallization schemes using Zorbax® media

This approach demonstrates a unique combination of confinement and surface functionalization

to cause crystallization in undersaturated solutions. It may be applied to the following

crystallization schemes:

1) Crystallization of APIs: Screening of API crystallization from various solvents with this approach

may be used as an alternative to antisolvent crystallization. It may elucidate differences in nucleation

induction time, polymorphs, or crystal morphologies.

2) Purification of API solutions: The column setup could be particularly advantageous for the

purification of API from solution containing impurities. A solution of this type could pass through a

column loaded with Zorbax® media. The API would crystallize in the column and a solution of

impurities could pass through the column. The API could be harvested with the Zorbax® media, re-

dissolved, and crystallized again with greater purity.

3) Protein crystallization: Protein and large biologic molecule crystallization is typically challenging.

There is a lack of generalized methods, and protein solutions may be dilute. This technique could be

used for crystallization of undersaturated protein solutions for discovery and better understanding of

crystal structure and interaction.

Alternative media

We have demonstrated the combination of the principles of crystallization through surface

functionalization and nanoconfinement. The selection of a functionalized nanoporous media was

critical to the success of these studies. The small pore size contributes to the generation of

140

nanovolumes for crystallization and the surface functionalization is able to alter these

environments due to their small volumes such that the crystallization may occur from

undersaturated parent solutions. Thus, looking at the extension of the technique to other

materials and functionalizations, these critical factors must be considered.

The matrix material itself must be inert to the solvents used in crystallization. Porous silica

represents a natural choice due to its chemically inert nature and ease of production of a variety

of porous silicas. These include controlled pore glass, Vycor ®, Zorbax ®. Other nanoporous

materials that may be inert to solvents used in typical crystallizations include anodic aluminum,

nanoporous zeolites, and nanoporous carbon. The material must be easily functionalized, which

glass may be through a straightforward silane reaction. Optimizing the cost of these materials

versus the surface coverage of functionalization is of key importance. The Zorbax ® material

used in the present study is expensive. Finding cheaper silica alternatives which still possess

good surface coverage but also are more cost effective would be an ideal route forward.

Nanopore size

The ratio of surface functional groups to nanovolume within a pore is critical to the successful

application of these methods. Zorbax® demonstrated success with a pore volume of about 7 nm.

Nanoporous materials typically have best controlled pore volumes at above 1 nm. It has been

demonstrated before that evaporative crystallization in nanopores is successful down to about 20

nm. Thus, for this specific technique of crystallization from undersaturated solutions using the

combined effects of surface functionalization and confinement, we suggest a pore range in

materials between 1 nm and 20 nm.

141

Surface functionalizations

The C8 functionalization was chosen specifically for these experiments because a wide range of

APIs are poorly soluble in alkane solvents such as hexane, heptane, or decane. The

functionalization on the Zorbax® mimicked this poor solubility, and decreased the solubility of

the API in solution in the confinement volumes such that crystallization could occur from an

undersaturated solution. Alternative functionalizations, such as phenyl groups, carboxyl, or –CN

groups already exist for chromatographic media and would be natural choices for a system in

which the desired crystal product is poorly soluble in solvents with functional groups of the

same. For example, a poorly water-soluble compound such as ibuprofen may demonstrate

successful crystallization if a carboxyl functional group on a nanoporous surface were used to

mimic the polar –OH groups present in an aqueous system. Surface functionalizations to

represent the range of solvent types typically available in pharmaceutical solubility studies can

be achieved through easy silane reactions on the surfaces of glass. Available surface groups that

could be made to project from the surface of porous glass matrices through organosilane

chemistry amine, carboxyl, vinyl, or thiol groups effectively spanning a variety of hydrophobic

and hydrophilic functional group types.

The technology discussed here would be highly applicable in the pharmaceutical industry, as fine

control over crystallization of commodity APIs is of interest and these APIs may be both heat

and solvent sensitive. This would also be applicable to chemical products crystallization;

however, the driving force available from the functionalized porous matrices is small and cannot

achieve the yields of a traditional cooling or antisolvent crystallization. Additionally the matrices

142

are costly and would therefore be better used in a fine chemicals setting. The technology is also

applicable in the crystallization of proteins and macromolecules.


We would like to thank the Novartis-MIT Center for Continuous Manufacturing for financial

support and instrumentation use as well as the DARPA ASKCOS project at MIT for support and

funding. L.M.D. is grateful in particular to Lucrèce Nicoud for initial direction with this project.

This material is currently under preparation for publication as “Crystallization from

undersaturated solutions using nanoconfinement matrices with functionalization.”

143

Chapter 6 : Conclusions and recommendations

6.1 Outlook for use of nanoconfinement for crystallization

The work carried out in this thesis as well as the body of work which it built upon has

successfully demonstrated that rigid porous media is a viable confinement tool for

nanocrystallization. A variety of APIs were successfully loaded into porous matrices in a range

of pore sizes, producing pharmaceutical nanocrystals from 20 nm up through 300 nm. These

nanocrystals showed marked improvements in dissolution profiles as compared to profiles from

standard micron-sized pharmaceutical crystals. The loading of API in these rigid matrices was

further improved from a benchtop procedure to successful loading in a continuous crystallizer, as

well as a consecutive two-stage crystallizer setup for loading and subsequent growth of crystals

within the pores.

These results offer a potentially simple method of drug formulation, wherein a poorly water-

soluble API is loaded in a rigid nanporous matrix and the resulting loaded matrix powder is

encapsulated. This is a much simpler method with fewer stabilizing chemicals than the methods

currently available on the market to produce formulations of nanocrystals relying on top-down

approaches to nanocrystal formation.

Further work to elucidate the size and shape of crystals confined to porous matrices is an

interesting avenue of further research. This work and others assume that the nanocrystals

confined to the matrices are nominally spheres of the same size as the nominal pore size

provided by the matrix. While thermal analysis of melting point depressions do indicate that

crystals of different sizes have formed in matrices with pores of different sizes, the spherical

144

assumption is likely invalid. It neglects standard crystal morphology, where a crystal may be

prompted to grow at a particular face if the energy barrier to do so is lower. Perhaps with crystals

confined to a pore, the energy barrier for growth is lowest if the crystal grows along the length of

the pore, but alternatively it could be more common for larger crystals to form at pore junctions.

Answering questions about the size and shape of confined crystals is an extremely interesting but

challenging topic that deserves further exploration.

Investigations into the grain size of the silica beads could provide information about the

contribution of the transport element of the dissolution enhancement. Conducting dissolution

measurements of silica with the same pore size with different grain sizes could help indicate

whether transport through the bead is causing significant delays in dissolution rate. Improving

modeling capabilities to account for different pore geometries and bead sizes could help explain

experimental results seen in dissolution profiles as well. Advanced models which take into

account dissolution of nanocrystals within pores and subsequent diffusion of the solute to the

surface of the beads are a valuable next step to accompany much of the experimental work which

has been done with nanocrystals in confinement. Such models could also provide insight into

features about these systems which are difficult to know, such as the location of nanocrystals

within porous beads, and the size and shape of these crystals therein.

Ultimately, a technique which would allow for the imaging of nanocrystals confined to these

pores would provide extremely valuable information about the size and shape of crystals formed

by these techniques. Surface microscopy techniques will unfortunately not give the full picture of

crystals confined to the pores. Cross sectioning a porous matrix bead that has been loaded with

API nanocrystals would be ideal for imaging; however, the small grain sizes of the matrices used

makes this less practical. Also the force of cross sectioning a bead may likely damage crystals at

145

the cutting interface. Focused ion beam technology is a potential route forward for this kind of

precise cross sectioning that would not disrupt the embedded crystals.

6.2 Future work on using surface functionalized materials for crystallization

in confinement

The work which resulted from the combination of our investigations with surface functionalized

nanoparticles for addition to bulk solutions for crystallization and the previous work on

crystallization in confinement has been an exciting demonstration of a new concept. The ability

to crystallize within very small pores from undersaturated drug solutions due to the presence of a

surface functionalization in these pores is a previously undemonstrated phenomenon. The surface

functionalization on the porous media support is thought to provide enough interactions in the

small nanovolumes within pores that it may change the saturation solubility of dissolved API.

Due to the relatively high cost of the functionalized media, we believe that this material is best

used in a setup where the confined crystals may then be redissolved, and the functionalized

porous media captured, cleaned, and recycled for use again. The column setup that we showed to

be successful where an impurity was spiked into the parent feed solution and shown to be

enriched in the flowthrough off the column without crystallizing the impurity in the column

demonstrates a useful application of this principle.

Furthermore, it is possible that through a silane surface functionalization, we could functionalize

a more inexpensive rigid porous matrix. It remains to be investigated at what point the pore size

would become too large, such that the surface functionalization could no longer contribute

significantly to the functional groups that the solute molecule would interact with in solution. It

146

is expected through our failed studies using functionalized nanoparticles as additives to solutions

that the tipping point of the pore size needed such that a surface functionalization would

contribute significantly to the solvent makeup actually occurs at a quite small pore size.

Finally, there exists great room for exploration into how this concept may be applied for a

variety of crystallizations. Different surface functionalizations which mimic different

solvent/antisolvent-like groups could be used to crystallize APIs of differing solubilities. In this

work, only one surface functionalization that behaved like an alkane-type solvent was

investigated. Other porous matrices made of materials other than glass may be used provided

they can meet both the nanoconfinement and surface functionalization criteria.

147

Chapter 7 : References

(1) Abu Bakar, M. R.; Nagy, Z. K.; Saleemi, A. N.; Rielly, C. D. The Impact of Direct

Nucleation Control on Crystal Size Distribution in Pharmaceutical Crystallization

Processes. Cryst. Growth Des. 2009, 9 (3), 1378–1384.

(2) Fujiwara, M.; Nagy, Z. K.; Chew, J. W.; Braatz, R. D. First-Principles and Direct Design

Approaches for the Control of Pharmaceutical Crystallization. J. Process Control 2005, 15

(5), 493–504.

(3) Myerson, A. Handbook of Industrial Crystallization; Butterworth-Heinemann, 2002.

(4) Hu, J.; Johnston, D. K. P.; III, D. R. O. W. Nanoparticle Engineering Processes for

Enhancing the Dissolution Rates of Poorly Water Soluble Drugs. Drug Dev. Ind. Pharm.

2004, 30 (3), 233–245.

(5) Shegokar, R.; Müller, R. H. Nanocrystals: Industrially Feasible Multifunctional

Formulation Technology for Poorly Soluble Actives. Int. J. Pharm. 2010, 399 (1–2), 129–

139.

(6) Wang, G. D.; Mallet, F. P.; Ricard, F.; Heng, J. Y. Pharmaceutical Nanocrystals. Curr.

Opin. Chem. Eng. 2012, 1 (2), 102–107.

(7) Buckton, G.; Beezer, A. E. The Relationship between Particle Size and Solubility. Int. J.

Pharm. 1992, 82 (3), R7–R10.

(8) Möschwitzer, J. P. Drug Nanocrystals in the Commercial Pharmaceutical Development

Process. Int. J. Pharm. 2013, 453 (1), 142–156.

(9) Van Eerdenbrugh, B.; Van den Mooter, G.; Augustijns, P. Top-down Production of Drug

Nanocrystals: Nanosuspension Stabilization, Miniaturization and Transformation into

Solid Products. Int. J. Pharm. 2008, 364 (1), 64–75.

(10) Zhang, D.; Tan, T.; Gao, L.; Zhao, W.; Wang, P. Preparation of Azithromycin

Nanosuspensions by High Pressure Homogenization and Its Physicochemical

Characteristics Studies. Drug Dev. Ind. Pharm. 2007, 33 (5), 569–575.

(11) Peltonen, L.; Hirvonen, J. Pharmaceutical Nanocrystals by Nanomilling: Critical Process

Parameters, Particle Fracturing and Stabilization Methods. J. Pharm. Pharmacol. 2010, 62

(11), 1569–1579.

(12) Malamatari, M.; Taylor, K. M. G.; Malamataris, S.; Douroumis, D.; Kachrimanis, K.

Pharmaceutical Nanocrystals: Production by Wet Milling and Applications. Drug Discov.

Today 2018, 23 (3), 534–547.

(13) Khadka, P.; Ro, J.; Kim, H.; Kim, I.; Kim, J. T.; Kim, H.; Cho, J. M.; Yun, G.; Lee, J.

Pharmaceutical Particle Technologies: An Approach to Improve Drug Solubility,

Dissolution and Bioavailability. Asian J. Pharm. Sci. 2014, 9 (6), 304–316.

(14) Qian, K. K.; Bogner, R. H. Application of Mesoporous Silicon Dioxide and Silicate in

Oral Amorphous Drug Delivery Systems. J. Pharm. Sci. 2012, 101 (2), 444–463.

(15) Wang, S. Ordered Mesoporous Materials for Drug Delivery. Microporous Mesoporous

Mater. 2009, 117 (1–2), 1–9.

(16) Hancock, B. C.; Zografi, G. Characteristics and Significance of the Amorphous State in

Pharmaceutical Systems. J. Pharm. Sci. 1997, 86 (1), 1–12.

(17) Willart, J. F.; Descamps, M. Solid State Amorphization of Pharmaceuticals. Mol. Pharm.

2008, 5 (6), 905–920.

148

(18) Yu, L. Amorphous Pharmaceutical Solids: Preparation, Characterization and Stabilization.

Adv. Drug Deliv. Rev. 2001, 48 (1), 27–42.

(19) Jackson, C. L.; McKenna, G. B. Vitrification and Crystallization of Organic Liquids

Confined to Nanoscale Pores. Chem. Mater. 1996, 8 (8), 2128–2137.

(20) Azaïs, T.; Tourné-Péteilh, C.; Aussenac, F.; Baccile, N.; Coelho, C.; Devoisselle, J.-M.;

Babonneau, F. Solid-State NMR Study of Ibuprofen Confined in MCM-41 Material.

Chem. Mater. 2006, 18 (26), 6382–6390.

(21) Azais, T.; Hartmeyer, G.; Quignard, S.; Laurent, G.; Babonneau, F. Solution State NMR

Techniques Applied to Solid State Samples: Characterization of Benzoic Acid Confined in

MCM-41. J. Phys. Chem. C 2010, 114 (19), 8884–8891.

(22) Ha, J.-M.; Hamilton, B. D.; Hillmyer, M. A.; Ward, M. D. Phase Behavior and

Polymorphism of Organic Crystals Confined within Nanoscale Chambers. Cryst. Growth

Des. 2009, 9 (11), 4766–4777.

(23) Ha, J.-M.; Wolf, J. H.; Hillmyer, M. A.; Ward, M. D. Polymorph Selectivity under

Nanoscopic Confinement. J. Am. Chem. Soc. 2004, 126 (11), 3382–3383.

(24) Ha, J.-M.; Hillmyer, M. A.; Ward, M. D. Thermotropic Properties of Organic

Nanocrystals Embedded in Ultrasmall Crystallization Chambers. J. Phys. Chem. B 2005,

109 (4), 1392–1399.

(25) Tran, T. H.; Ramasamy, T.; Truong, D. H.; Choi, H.-G.; Yong, C. S.; Kim, J. O.

Preparation and Characterization of Fenofibrate-Loaded Nanostructured Lipid Carriers for

Oral Bioavailability Enhancement. AAPS PharmSciTech 2014, 15 (6), 1509–1515.

(26) Yang, X.; Ong, T. C.; Michaelis, V. K.; Heng, S.; Huang, J.; Griffin, R. G.; Myerson, A.

S. Formation of Organic Molecular Nanocrystals under Rigid Confinement with Analysis

by Solid State NMR. CrystEngComm 2014, 16 (39), 9345–9352.

(27) Brettmann, B.; Bell, E.; Myerson, A.; Trout, B. Solid-State NMR Characterization of

High-Loading Solid Solutions of API and Excipients Formed by Electrospinning. J.

Pharm. Sci. 2012, 101 (4), 1538–1545.

(28) Ahern, R. J.; Hanrahan, J. P.; Tobin, J. M.; Ryan, K. B.; Crean, A. M. Comparison of

Fenofibrate–mesoporous Silica Drug-Loading Processes for Enhanced Drug Delivery.

Eur. J. Pharm. Sci. 2013, 50 (3–4), 400–409.

(29) Rengarajan, G. T.; Enke, D.; Steinhart, M.; Beiner, M. Size-Dependent Growth of

Polymorphs in Nanopores and Ostwald’s Step Rule of Stages. Phys. Chem. Chem. Phys.

2011, 13 (48), 21367–21374.

(30) Ulman, A. Formation and Structure of Self-Assembled Monolayers. Chem. Rev. 1996, 96

(4), 1533–1554.

(31) Singh, A.; Sung Lee, I.; Kim, K.; S. Myerson, A. Crystal Growth on Self-Assembled

Monolayers. CrystEngComm 2011, 13 (1), 24–32.

(32) Yang, X.; Sarma, B.; Myerson, A. S. Polymorph Control of Micro/Nano-Sized Mefenamic

Acid Crystals on Patterned Self-Assembled Monolayer Islands. Cryst. Growth Des. 2012,

12 (11), 5521–5528.

(33) Kulkarni, S. A.; Weber, C. C.; Myerson, A. S.; ter Horst, J. H. Self-Association during

Heterogeneous Nucleation onto Well-Defined Templates. Langmuir ACS J. Surf. Colloids

2014, 30 (41), 12368–12375.

149

(34) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Self-Assembled

Monolayers of Thiolates on Metals as a Form of Nanotechnology. Chem. Rev. 2005, 105

(4), 1103–1169.

(35) Hiremath, R.; Basile, J. A.; Varney, S. W.; Swift, J. A. Controlling Molecular Crystal

Polymorphism with Self-Assembled Monolayer Templates. J. Am. Chem. Soc. 2005, 127

(51), 18321–18327.

(36) Chen, J.; Myerson, A. S. Pasteur Revisited: Chiral Separation by Crystallization on Self-

Assembled Monolayers. CrystEngComm 2012, 14 (24), 8326.

(37) Zhou, G. X.; Fujiwara, M.; Woo, X. Y.; Rusli, E.; Tung, H.-H.; Starbuck, C.; Davidson,

O.; Ge, Z.; Braatz, R. D. Direct Design of Pharmaceutical Antisolvent Crystallization

through Concentration Control. Cryst. Growth Des. 2006, 6 (4), 892–898.

(38) Kulkarni, S. A.; Myerson, A. S. Reversible Control of Solubility Using Functionalized

Nanoparticles. Chem. Commun. 2017, 53 (8), 1429–1432.

(39) Kim, K.; Lee, I. sung; Centrone, A.; Hatton, T. A.; Myerson, A. S. Formation of

Nanosized Organic Molecular Crystals on Engineered Surfaces. J. Am. Chem. Soc. 2009,

131 (51), 18212–18213.

(40) Zuo, B.; Sun, Y.; Li, H.; Liu, X.; Zhai, Y.; Sun, J.; He, Z. Preparation and in Vitro/in Vivo

Evaluation of Fenofibrate Nanocrystals. Int. J. Pharm. 2013, 455 (1–2), 267–275.

(41) Junghanns, J.-U. A. H.; Müller, R. H. Nanocrystal Technology, Drug Delivery and

Clinical Applications. Int. J. Nanomedicine 2008, 3 (3), 295–310.

(42) de Waard, H.; Hinrichs, W. L. J.; Frijlink, H. W. A Novel Bottom–up Process to Produce

Drug Nanocrystals: Controlled Crystallization during Freeze-Drying. J. Controlled

Release 2008, 128 (2), 179–183.

(43) Bleich, J.; Müller, B. W. Production of Drug Loaded Microparticles by the Use of

Supercritical Gases with the Aerosol Solvent Extraction System (ASES) Process. J.

Microencapsul. 1996, 13 (2), 131–139.

(44) Lee, S.; Nam, K.; Kim, M. S.; Jun, S. W.; Park, J.-S.; Woo, J. S.; Hwang, S.-J. Preparation

and Characterization of Solid Dispersions of Itraconazole by Using Aerosol Solvent

Extraction System for Improvement in Drug Solubility and Bioavailability. Arch. Pharm.

Res. 2005, 28 (7), 866–874.

(45) Rogers, T. L.; Johnston, K. P.; III, R. O. W. Solution-Based Particle Formation of

Pharmaceutical Powders by Supercritical or Compressed Fluid Co2 and Cryogenic Spray-

Freezing Technologies. Drug Dev. Ind. Pharm. 2001, 27 (10), 1003–1015.

(46) Sarkari, M.; Brown, J.; Chen, X.; Swinnea, S.; Williams III, R. O.; Johnston, K. P.

Enhanced Drug Dissolution Using Evaporative Precipitation into Aqueous Solution. Int. J.

Pharm. 2002, 243 (1–2), 17–31.

(47) Chen, X.; Young, T. J.; Sarkari, M.; Williams III, R. O.; Johnston, K. P. Preparation of

Cyclosporine A Nanoparticles by Evaporative Precipitation into Aqueous Solution. Int. J.

Pharm. 2002, 242 (1–2), 3–14.

(48) de Waard, H.; Grasmeijer, N.; Hinrichs, W. L. J.; Eissens, A. C.; Pfaffenbach, P. P. F.;

Frijlink, H. W. Preparation of Drug Nanocrystals by Controlled Crystallization:

Application of a 3-Way Nozzle to Prevent Premature Crystallization for Large Scale

Production. Eur. J. Pharm. Sci. 2009, 38 (3), 224–229.

(49) Jiang, Q.; Ward, M. D. Crystallization under Nanoscale Confinement. Chem. Soc. Rev.

2014, 43 (7), 2066–2079.

150

(50) Hilden, J. L.; Reyes, C. E.; Kelm, M. J.; Tan, J. S.; Stowell, J. G.; Morris, K. R. Capillary

Precipitation of a Highly Polymorphic Organic Compound. Cryst. Growth Des. 2003, 3

(6), 921–926.

(51) Beiner, M.; Rengarajan; Pankaj, S.; Enke, D.; Steinhart, M. Manipulating the Crystalline

State of Pharmaceuticals by Nanoconfinement. Nano Lett. 2007, 7 (5), 1381–1385.

(52) Pines, A.; Gibby, M. G.; Waugh, J. S. Proton-Enhanced Nuclear Induction Spectroscopy.

A Method for High-Resolution NMR of Dilute Spins in Solids. J. Chem. Phys. 1972, 56

(4), 1776–1777.

(53) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin, R. G. Heteronuclear

Decoupling in Rotating Solids. J. Chem. Phys. 1995, 103 (16), 6951–6958.

(54) K.S. Rao; Keshar, N. K.; Jena, N.; Rao, M. E.; Patnaik, A. K. Stability Indicating Liquid

Chromatographic Method for the Determination of Fenofibrate and Its Application to

Kinetic Studies. Int. J. Pharm. Sci. Nanotechnol. 2013, 5 (4), 1866–1874.

(55) Bhise, S. D. Effect of Hydroxypropyl β- Cyclodextrin Inclusion Complexation on Solubility

of Fenofibrate; 2011.

(56) Heinz, A.; Gordon, K. C.; McGoverin, C. M.; Rades, T.; Strachan, C. J. Understanding the

Solid-State Forms of Fenofibrate--a Spectroscopic and Computational Study. Eur. J.

Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Pharm. Verfahrenstechnik EV 2009, 71 (1),

100–108.

(57) Henry, R. F.; Zhang, G. Z.; Gao, Y.; Buckner, I. S. Fenofibrate. Acta Crystallogr. Sect. E

Struct. Rep. Online 2003, 59 (5), o699–o700.

(58) Sliwinska-Bartkowiak, M.; Dudziak, G.; Gras, R.; Sikorski, R.; Radhakrishnan, R.;

Gubbins, K. E. Freezing Behavior in Porous Glasses and MCM-41. Colloids Surf.

Physicochem. Eng. Asp. 2001, 187–188, 523–529.

(59) O’Mahony, M.; Leung, A. K.; Ferguson, S.; Trout, B. L.; Myerson, A. S. A Process for

the Formation of Nanocrystals of Active Pharmaceutical Ingredients with Poor Aqueous

Solubility in a Nanoporous Substrate. Org. Process Res. Dev. 2015, 19 (9), 1109–1118.

(60) Andersson, J.; Rosenholm, J.; Areva, S.; Lindén, M. Influences of Material Characteristics

on Ibuprofen Drug Loading and Release Profiles from Ordered Micro- and Mesoporous

Silica Matrices. Chem. Mater. 2004, 16 (21), 4160–4167.

(61) Badruddoza, A. Z. M.; Godfrin, P. D.; Myerson, A. S.; Trout, B. L.; Doyle, P. S. Core–

Shell Composite Hydrogels for Controlled Nanocrystal Formation and Release of

Hydrophobic Active Pharmaceutical Ingredients. Adv. Healthc. Mater. 2016, 5 (15),

1960–1968.

(62) Deen, W. M. Hindered Transport of Large Molecules in Liquid-Filled Pores. AIChE J.

1987, 33 (9), 1409–1425.

(63) Jamzad, S.; Fassihi, R. Role of Surfactant and PH on Dissolution Properties of Fenofibrate

and Glipizide—A Technical Note. AAPS PharmSciTech 2006, 7 (2), E17–E22.

(64) Beck, C.; Dalvi, S. V.; Dave, R. N. Controlled Liquid Antisolvent Precipitation Using a

Rapid Mixing Device. Chem. Eng. Sci. 2010, 65 (21), 5669–5675.

(65) Stegemann, S.; Leveiller, F.; Franchi, D.; de Jong, H.; Lindén, H. When Poor Solubility

Becomes an Issue: From Early Stage to Proof of Concept. Eur. J. Pharm. Sci. Off. J. Eur.

Fed. Pharm. Sci. 2007, 31 (5), 249–261.

(66) Lipinski, C. A. Drug-like Properties and the Causes of Poor Solubility and Poor

Permeability. J. Pharmacol. Toxicol. Methods 2000, 44 (1), 235–249.

151

(67) Kalepu, S.; Nekkanti, V. Insoluble Drug Delivery Strategies: Review of Recent Advances

and Business Prospects. Acta Pharm. Sin. B 2015, 5 (5), 442–453.

(68) Otto, D. P.; Villiers, M. M. de. Physicochemical Principles of Nanosized Drug Delivery

Systems. In Nanotechnology in Drug Delivery; Villiers, M. M. de, Aramwit, P., Kwon, G.

S., Eds.; Biotechnology: Pharmaceutical Aspects; Springer New York, 2009; pp 3–33.

(69) Müller, R. H.; Peters, K. Nanosuspensions for the Formulation of Poorly Soluble Drugs: I.

Preparation by a Size-Reduction Technique. Int. J. Pharm. 1998, 160 (2), 229–237.

(70) Mullin, J. W. 3 - Solutions and Solubility. In Crystallization (Fourth Edition);

Butterworth-Heinemann: Oxford, 2001; pp 86–134.

(71) Keck, C. M.; Müller, R. H. Drug Nanocrystals of Poorly Soluble Drugs Produced by High

Pressure Homogenisation. Eur. J. Pharm. Biopharm. 2006, 62 (1), 3–16.

(72) List, M.; Sucker, H. Hydrosols of Pharmacologically Active Agents and Their

Pharmaceutical Compositions Comprising Them. US5389382 A, February 14, 1995.

(73) de Waard, H.; Grasmeijer, N.; Hinrichs, W. L. J.; De Beer, T.; Frijlink, H. W. A Process

Suitable for Large-Scale Production of Drug Nanocrystals. Pharm. Technol. 2011, 35 (8),

58–62.

(74) Panagiotou, T.; Mesite, S. V.; Fisher, R. J. Production of Norfloxacin Nanosuspensions

Using Microfluidics Reaction Technology through Solvent/Antisolvent Crystallization.

Ind. Eng. Chem. Res. 2009, 48 (4), 1761–1771.

(75) Chan, H.-K.; Kwok, P. C. L. Production Methods for Nanodrug Particles Using the

Bottom-up Approach. Adv. Drug Deliv. Rev. 2011, 63 (6), 406–416.

(76) Khan, S.; Matas, M. de; Zhang, J.; Anwar, J. Nanocrystal Preparation: Low-Energy

Precipitation Method Revisited. Cryst. Growth Des. 2013, 13 (7), 2766–2777.

(77) Thomas, M. J. K.; Slipper, I.; Walunj, A.; Jain, A.; Favretto, M. E.; Kallinteri, P.;

Douroumis, D. Inclusion of Poorly Soluble Drugs in Highly Ordered Mesoporous Silica

Nanoparticles. Int. J. Pharm. 2010, 387 (1–2), 272–277.

(78) Azad, M.; Moreno, J.; Bilgili, E.; Davé, R. Fast Dissolution of Poorly Water Soluble

Drugs from Fluidized Bed Coated Nanocomposites: Impact of Carrier Size. Int. J. Pharm.

2016, 513 (1–2), 319–331.

(79) Geszke-Moritz, M.; Moritz, M. APTES-Modified Mesoporous Silicas as the Carriers for

Poorly Water-Soluble Drug. Modeling of Diflunisal Adsorption and Release. Appl. Surf.

Sci. 2016, 368, 348–359.

(80) Martín, A.; García, R. A.; Karaman, D. S.; Rosenholm, J. M. Polyethyleneimine-

Functionalized Large Pore Ordered Silica Materials for Poorly Water-Soluble Drug

Delivery. J. Mater. Sci. 2014, 49 (3), 1437–1447.

(81) Dwyer, L.; Michaelis, V.; O’Mahony, M.; Griffin, R.; Myerson, A. Confined

Crystallization of Fenofibrate in Nanoporous Silica. CrystEngComm 2015, 17 (41), 7922–

7929.

(82) Sonnenberger, N.; Anders, N.; Golitsyn, Y.; Steinhart, M.; Enke, D.; Saalwächter, K.;

Beiner, M. Pharmaceutical Nanocrystals Confined in Porous Host Systems – Interfacial

Effects and Amorphous Interphases. Chem. Commun. 2016, 52 (24), 4466–4469.

(83) Graubner, G.; Rengarajan, G. T.; Anders, N.; Sonnenberger, N.; Enke, D.; Beiner, M.;

Steinhart, M. Morphology of Porous Hosts Directs Preferred Polymorph Formation and

Influences Kinetics of Solid/Solid Transitions of Confined Pharmaceuticals. Cryst.

Growth Des. 2014, 14 (1), 78–86.

152

(84) Mehanna, M. M.; Motawaa, A. M.; Samaha, M. W. Tadalafil Inclusion in Microporous

Silica as Effective Dissolution Enhancer: Optimization of Loading Procedure and

Molecular State Characterization. J. Pharm. Sci. 2011, 100 (5), 1805–1818.

(85) Van Speybroeck, M.; Barillaro, V.; Thi, T. D.; Mellaerts, R.; Martens, J.; Van Humbeeck,

J.; Vermant, J.; Annaert, P.; Van den Mooter, G.; Augustijns, P. Ordered Mesoporous

Silica Material SBA-15: A Broad-Spectrum Formulation Platform for Poorly Soluble

Drugs. J. Pharm. Sci. 2009, 98 (8), 2648–2658.

(86) Hillerström, A.; Stam, J. van; Andersson, M. Ibuprofen Loading into Mesostructured

Silica Using Liquid Carbon Dioxide as a Solvent. Green Chem. 2009, 11 (5), 662–667.

(87) Zhang, Y.; Zhi, Z.; Jiang, T.; Zhang, J.; Wang, Z.; Wang, S. Spherical Mesoporous Silica

Nanoparticles for Loading and Release of the Poorly Water-Soluble Drug Telmisartan. J.

Controlled Release 2010, 145 (3), 257–263.

(88) Junyaprasert, V. B.; Morakul, B. Nanocrystals for Enhancement of Oral Bioavailability of

Poorly Water-Soluble Drugs. Asian J. Pharm. Sci. 2015, 10 (1), 13–23.

(89) Alvarez, A. J.; Singh, A.; Myerson, A. S. Crystallization of Cyclosporine in a Multistage

Continuous MSMPR Crystallizer. Cryst. Growth Des. 2011, 11 (10), 4392–4400.

(90) Peña, R.; Nagy, Z. K. Process Intensification through Continuous Spherical Crystallization

Using a Two-Stage Mixed Suspension Mixed Product Removal (MSMPR) System. Cryst.

Growth Des. 2015, 15 (9), 4225–4236.

(91) Li, J.; Trout, B. L.; Myerson, A. S. Multistage Continuous Mixed-Suspension, Mixed-

Product Removal (MSMPR) Crystallization with Solids Recycle. Org. Process Res. Dev.

2016, 20 (2), 510–516.

(92) Lai, T.-T. C.; Cornevin, J.; Ferguson, S.; Li, N.; Trout, B. L.; Myerson, A. S. Control of

Polymorphism in Continuous Crystallization via Mixed Suspension Mixed Product

Removal Systems Cascade Design. Cryst. Growth Des. 2015, 15 (7), 3374–3382.

(93) Kawabata, Y.; Wada, K.; Nakatani, M.; Yamada, S.; Onoue, S. Formulation Design for

Poorly Water-Soluble Drugs Based on Biopharmaceutics Classification System: Basic

Approaches and Practical Applications. Int. J. Pharm. 2011, 420 (1), 1–10.

(94) Hong, S.; Shen, S.; Tan, D. C. T.; Ng, W. K.; Liu, X.; Chia, L. S. O.; Irwan, A. W.; Tan,

R.; Nowak, S. A.; Marsh, K.; et al. High Drug Load, Stable, Manufacturable and

Bioavailable Fenofibrate Formulations in Mesoporous Silica: A Comparison of Spray

Drying versus Solvent Impregnation Methods. Drug Deliv. 2016, 23 (1), 316–327.

(95) O’Neil, M. The Merck Index-An Encyclopedia of Chemicals, Drugs, and Biologicals.;

Royal Society of Chemistry: Cambridge, UK, 2013.

(96) Hu, G.; Li, H.; Wang, X.; Zhang, Y. Measurement and Correlation of Griseofulvin

Solubility in Different Solvents at Temperatures from (281.95 to 357.60) K. J. Chem. Eng.

Data 2010, 55 (9), 3969–3971.

(97) Elworthy, P. H.; Lipscomb, F. J. The Effect of Some Non-Ionic Surfactants and a

Polyoxyethylene Glycol on the Dissolution Rate of Griseofulvin. J. Pharm. Pharmacol.

1968, 20 (12), 923–933.

(98) Limnell, T.; Santos, H. A.; Mäkilä, E.; Heikkilä, T.; Salonen, J.; Murzin, D. Y.; Kumar,

N.; Laaksonen, T.; Peltonen, L.; Hirvonen, J. Drug Delivery Formulations of Ordered and

Nonordered Mesoporous Silica: Comparison of Three Drug Loading Methods. J. Pharm.

Sci. 2011, 100 (8), 3294–3306.

(99) Vekilov, P. G. Nucleation. Cryst. Growth Des. 2010, 10 (12), 5007–5019.

153

(100) Lee, A. Y.; Lee, I. S.; Dette, S. S.; Boerner, J.; Myerson, A. S. Crystallization on Confined

Engineered Surfaces: A Method to Control Crystal Size and Generate Different

Polymorphs. J. Am. Chem. Soc. 2005, 127 (43), 14982–14983.

(101) Lee, A. Y.; Ulman, A.; Myerson, A. S. Crystallization of Amino Acids on Self-Assembled

Monolayers of Rigid Thiols on Gold. Langmuir 2002, 18 (15), 5886–5898.

(102) Ko, S.; Kim, H. Y.; Choi, I.; Choe, J. Gold Nanoparticles as Nucleation-Inducing

Reagents for Protein Crystallization. Cryst. Growth Des. 2017, 17 (2), 497–503.

(103) Nicoud, L. H.; Dwyer, L. M.; Myerson, A. S. Heterogeneous Nucleation of Small

Molecule Crystals in the Presence of Nanoparticles. AIChE Proc. 2017.

(104) Rohani, S.; Horne, S.; Murthy, K. Control of Product Quality in Batch Crystallization of

Pharmaceuticals and Fine Chemicals. Part 1: Design of the Crystallization Process and the

Effect of Solvent. Org. Process Res. Dev. 2005, 9 (6), 858–872.

(105) Ganapathy, R.; Sood, A. K. Crystallization: Brought to the Surface. Nat. Phys. 2017, 13

(5), 421–422.

(106) Tierney, T. B.; Rasmuson, Å. C.; Hudson, S. P. Size and Shape Control of Micron-Sized

Salicylic Acid Crystals during Antisolvent Crystallization. Org. Process Res. Dev. 2017,

21 (11), 1732–1740.

(107) Padrela, L.; Zeglinski, J.; Ryan, K. M. Insight into the Role of Additives in Controlling

Polymorphic Outcome: A CO2-Antisolvent Crystallization Process of Carbamazepine.

Cryst. Growth Des. 2017, 17 (9), 4544–4553.

(108) Matsumoto, M.; Ohno, M.; Wada, Y.; Sato, T.; Okada, M.; Hiaki, T. Enhanced Production

of α-Form Indomethacin Using the Antisolvent Crystallization Method Assisted by N2

Fine Bubbles. J. Cryst. Growth 2017, 469, 91–96.

(109) USP Monograph Diphenhydramine Hydrochloride; Official Monographs; United States

Pharmacopeial Convention, 2018; pp 3802–3803.

Nanocrystallization confined to porous matrices with and ...

Documents

Transcript of Nanocrystallization confined to porous matrices with and ...