Mid IR Imaging and Multivariate Analysis of Dynamic Processes in Pharmaceutically Relevant Microparticles

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Mid-IR Imaging and Multivariate Analysis of Dynamic

Processes in Pharmaceutically Relevant Microparticles

KELES, Hakan

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Mid-IR Imaging and Multivariate Analysis of Dynamic

Processes in Pharmaceutically Relevant Microparticles

Hakan Keles

v

A thesis submitted in partial fulfilment of the requirements of

Sheffield Hallam University

for the degree of Doctor of Philosophy

Collaborating Organisations:

EPSRC

&

Critical Pharmaceuticals Limited, Nottingham, United Kingdom

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D eclaration

The work described in this thesis was carried out by the author in the Materials and

Engineering Research Institute at Sheffield Hallam University, Sheffield, United Kingdom, between October 2010 and February 2014. The author declares that this work

has not been submitted for any other degree. The work is original except where acknowledged by reference.

Author:

(Hakan Keles)

Director o f Studies:

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Acknowledgements

I w ould like to thank people w hose support during this project was invaluable;

First o f all, Chris Sam m on for his help and scientific advice during this project which

helped me develop as a standalone researcher and for always being there w hen needed.

His great sense o f hum our and optim ism m ade my PhD experience a fruitful one since

O ctober 2010.

A ndrew N aylor (at CPL) and Francis C legg for the useful scientific discussions during

project m eetings.

CPL staff for providing the sam ples and for their help with bulk characterisation

techniques.

Chris Breen and Jack Y arw ood for their advice during PCAS G roup m eetings.

Christine Le M aitre (at The B iom edical Research Centre) for her help with cryo-

m icrotom y at the early stages o f the project.

All the M E R I staff, particularly Corrie H outon, Gillian Hill and D eeba Z ahoor, for their

professional approach and prom pt responses, w ithout their great help it probably w ould

not be possible to com plete this project on time.

Friends in the PCAS group;

V ictoria Boyes, Prakash M uthudoss, Fabio Pappini, M arianne Labet, M ichael Barw ood,

Subodh Sabnis, Trinh Pham , Kerstin M ader and T hom as Sm allw ood.

M y m other and m other in law for looking after Ela during my PhD studies.

E PSRC and CPL for the Industrial CA SE sponsorship that financially supported this 42

m onth project.

And my better half, Nilgun, for supporting m e by all m eans during this PhD , and

particularly for covering m ost o f m y responsibilities as a parent to Ela since A ugust

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List of Abbreviations

API Active pharmaceutical ingredient

ATR

B

BSA

cm' 1

CPL

CPU

d2o

DSC

FDA

FoV

FPA

FTIR

FWHM

GA

Ge

GPC

GRAS

h

h2o

HEPES

hGH

HPLC

Attenuated total reflection

Hydrated layer size

Bovine serum albumin

Wavenumber

Critical Pharmaceuticals Limited

Central processing unit

Heavy water

Differential scanning calorimetry

U.S.A Food and Drug Administration

Field of view

Focal plane array

Fourier transform infrared

Full width at half maximum

Glycolic acid

Germanium

Gel permeation chromatography

Generally recognised as safe

Hour

Water

4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid

Human growth hormone

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IR Infrared

k D egradation rate

K Kelvin

kGy Kilo Gray

LA Lactic acid

m M inute

M C R M ultivariate curve resolution

M CR -A LS M ultivariate curve resolution-alternating least squares

M CT M ercury-cadm ium -telluride

M id-IR M edium -infrared

mm M illim etre

M n A verage m olecular w eight

M w M olecular w eight

NA N um erical aperture

N L C F N on-linear curve fitting

nm N anom etre

PBS Phosphate buffer saline

PCA Principal com ponent analysis

PDI Polydispersity index

PH Peak height

PLA Poly(lactic acid) or Polylactide

PLG A Poly(lactic-co-glycolic acid)

PG SS Particles from gas saturated solutions

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RAM Random-access memory

ROI Region of interest

ScC02 Supercritical carbon dioxide

sd Spray dried

SEC Size-exclusion chromatography

SEM Scanning electron microscopy

t Time

Tc Crystallisation temperature

Tg Glass transition temperature

Tm Melting temperature

UV Ultraviolet

UV-Vis Ultraviolet-Visible

2D 2 Dimensional

3D 3 Dimensional

y Gamma

X Wavelength

° c Degree Celsius

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A bstract

Sustained release m icroparticles used for parenteral drug delivery m ust be well characterized in term s o f their size range, m orphology and function. It is w idely understood that the chem istry and m orphology o f m icroparticles have a degree o f interdependence w hich strongly affects drug release behaviour from m icroparticles. This thesis investigates, for the first tim e, the use o f m id-IR im aging along with the developm ent and optim isation o f relevant m ultivariate im age analysis m ethods for studying the real-tim e degradation o f pharm aceutically relevant biodegradable polym er m icroparticles and the real-tim e release o f protein based drugs from such m icroparticle system s.

The application o f attenuated total reflection - Fourier transform infrared spectroscopic (ATR -FTIR ) im aging and analysis to m onitor the degradation o f a single m icroparticle is optim ised and the developed m ethodology is detailed. A series o f tim e resolved im ages o f a PLG A m icroparticle undergoing hydrolysis at 70 °C are obtained using A T R -FTIR im aging for the first tim e. A novel partially supervised non-linear curve fitting (NLCF) tool is developed and the output from the N L C F is evaluated by direct quantitative com parison with a traditional peak height (PH) data analysis approach and m ultivariate curve resolution alternating least squares (M C R -A LS) analysis for the sam e im ages, in order to develop an im age analysis strategy. The N L C F m ethod is shown to facilitate the calculation o f hydrolysis rate constants for both the glycolic (kG) and lactic (kL) segm ents o f the PLG A copolym er. This results in im proved spatial resolution on tim e-resolved m icroparticle im ages, so providing better insight into the dim ensions o f hydration layers and particle dim ension changes during hydrolysis when com pared to im ages derived from both PH m easurem ents and M C R -A L S. The M C R - ALS routine is show n to be faster than N L C F and its im ages are found to provide sufficient contrast to be used for qualitative com parison.

The optim ised m id-IR -A T R procedures are then applied to investigate several factors influencing the hydrolytic degradation o f a fam ily o f PLG A m icroparticles. D egradation rate constants for glycolic and lactic units are show n to increase (w hilist m aintaining a -1 .3 ratio betw een each other) with increasing initial glycolic content o f the copolym er, tem perature or y-radiation exposure. D ifferential scanning calorim etry (DSC) and gel perm eation chrom atography (GPC) results indicate a chain scission based degradation in PLGA upon y exposure. The distribution o f lactic acid is probed with IR during the hydrolysis o f a PLA m icroparticle for the first tim e, show ing a diffusional pathw ay from the degrading m icroparticle outw ards into surrounding water.

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1

Introduction

This thesis investigates the use o f M id-IR spectroscopic im aging and relevant

m ultivariate data analysis techniques to characterise the degradation kinetics of

biodegradable polym er m icroparticles and the release of protein drugs from such

m atrices.

The w ork presented w ithin this thesis was conducted at Sheffield H allam U niversity in

collaboration w ith Critical Pharm aceuticals Ltd. This chapter provides a b rief

introduction into the drug delivery system s investigated within this thesis. It includes

inform ation about polym ers, proteins and controlled release drug delivery system s along

with production m ethods for such system s with particular attention to supercritical C O 2

based processes am ong which a m odified particles from gas saturated solutions (PG SS)

m ethod (C riticalM ix™ ) was used to produce the sam ples studied here by the

collaborating com pany.

In this chapter a very brief initial insight is given for M id-IR spectroscopic im aging,

including its relevance for studying polym er degradation and drug release before the

aim s o f this w ork are discussed in detail.

In C hapter 2, the theory o f the m ain m ethod o f study, FTIR spectroscopy and im aging,

is detailed and relevant data analysis techniques are discussed. O ther characterisation

m ethods including scanning electron m icroscopy, chrom atography, differential scanning

calorim etry, U V -V is dissolution testing and m icroscopic sam ple preparation m ethod

(ultra-m icrotom y) are also detailed including inform ation regarding the param eters and

m ethods used.

C hapter 3 dem ostrates the optim isation o f A T R im aging m ethod coupled w ith relevant

im age analysis techniques for studying degradation in single polym eric m icroparticles.

A direct num erical com parison o f three data analysis m ethods: peak height

m easurem ents, soft and hard m odelling, is studied using the sam e IR im age set o f a

PLGA 75/25 m icroparticle undergoing hydrolysis at 70 °C.

In C hapter 4, a range o f single PLG A m icroparticles are studied using the A T R im aging

m ethodology developed in Chapter 3. Effect o f C riticalM ix™ process, copolym er ratio

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PLG A based m icroparticle system s) on hydrolytic degradation behaviour o f individual

PLG A m icroparticles is investigated and results are discussed.

In C hapter 5, a lead controlled release form ulation that consists o f PLG A /PLA polym ers,

an excipient (Poloxam er 407) and hum an grow th horm one (hGH) is initially

investigated in term s o f its com ponents’ spatial distributions in dry form in order to

validate the form ulation m odel. The release o f hG H from scuh com plex form ulation is

m onitored using A T R -FT IR im aging. Finally the effect o f y- irradiation on the stability

o f PLG A m icroparticles and on the release o f the hGH protein from the m icroparticles

is investigated using FTIR im aging, SEM analyses and using a com bination o f

conventional bulk m ethods including; differential scanning calorim etry (DSC) to

understand therm al properties o f irradiated and un-irradiated pure polym ers, gel

perm eation chrom atography (GPC) to m onitor effect o f irradiation on the m olecular

w eight o f the polym ers, U V -V is spectrophotom etry to m onitor in vitro API release from

the m icroparticles and size exclusion chrom atography (SEC) to determ ine protein

aggregation and stability.

The overall conclusions and possible further w ork are sum m arised in C hapter 6 .

1.1 Drug Delivery

The concept o f drug delivery is the adm inistration o f active pharm aceutical ingredients

(APIs) to patients, in sufficient am ounts that they reach and treat the targeted region o f

body w ithout causing any adverse effect(s). A lthough this can be achieved by several

routes including parenteral (which is achieved thorugh injection; into a vein, under the

skin or into a m uscle, or through inhalation or through absorption by intact skin), oral,

pulm onary or nasal application, the adm inistration o f proteins are lim ited to the

parenteral (often subcutaneous) type as their high m olecular w eight or poor stability

blocks their transderm al, nasal or oral delivery.

A nother problem in the delivery o f proteins and peptides is their short half-life in the

hum an body. Therefore their unfavourable pharm acokinetics m eans that they are

usually adm inistered by frequent injections, for exam ple for hum an grow th horm one

deficiency every 3 days, which is inconvenient for patients and can affect patient

com pliance. How ever, the developm ent o f sustained release form ulations can overcom e

these issues as controlled release drug delivery is used to delay the release o f drugs

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1.2 Controlled Release Drug Delivery

T he use o f biodegradable polym ers is one o f the fundam ental w ays o f controlling

release system s. There are three different types o f m icro-scale controlled release

system s; ( 1) reservoir devices, w here the drug is released upon degradation o f a

biopolym er that is used to encapsulate it avoiding im m ediate release, (2 ) diffusion

devices, w here the drug is released through pores o f the biopolym er m atrix and (3)

erosion, w here the drug release is based up on degradation o f the biopolym er m atrix.

H ow ever for porous biopolym er m atrices it is often the case that com bination o f last

two hypotheses occurs as initially the surface attached drug m olecules will be released

upon contact with aqueous m edia by diffusion through pores and finally the degradation

o f biopolym er will result in release o f the drug m olecules entrapped deeper in the

biopolym er m atrix (Figure 1.1).

A)

Reservoir device

B) Diffusion device (porous)

C) Erosion device (degradation)

W ater Water + drug

Polymer Drug

Polymer Drug Wate

Water

o

°

o o

Figure 1.1 Controlled release scenarios for polymer microparticles; (a) reservoir

devices, (b) diffusion of drug from porous polymer matrix and (c) erosion

mechanism where the drug molecules are released as they become free from

degrading polymer.

In recent years research in m olecular and cell biology to m eet clinical needs has

intensified the interest in biologies, a variety o f therapeutics such as vaccines,

recom binant proteins and peptides, genes, viruses and synthetic tissues, ensuing a strong

m arket grow th [1]. As a result, currently hundreds o f recom binant proteins and peptides

[image:17.612.102.511.276.486.2]

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There are num erous candidate biodegradable polym ers for carrying and releasing

biologies as shown in Figure 1.1 how ever concerning biocom patibility to hum an body,

the FD A approved ones are few [2,3].

Poly (lactide-co-glycolide) (PLGA ) is a FD A approved biodegradable synthetic

polyester that is physically strong and highly processable [4]. H ence PLG A has been the

m ost studied carrier m atrix for m acrom olecules such as proteins, DN A, RN A , vaccines

and peptides for treatm ent of im portant diseases such as cancer, hum an growth

deficiency and m ultiple sclerosis [3].

A nother advantage o f PLG A is that its degradation in vivo is controllable by choice o f

polym er m olecular w eight and copolym er ratio (lactide to glycolide (L /G)) and yields

lactic and glycolic acids, which are biocom patible and rapidly cleared from the body via

the renal system [2 ].

The num ber o f PLGA based form ulations that are readily adm inistered through a

syringe, has been researched increasingly resulting in m any form ulations that are

m arketed today including ones given in Table 1.1.

Table 1.1 PLGA based microparticle formulations available in the market [1,3].

1

Lupron Depot® Leuprolide TAP Prostate cancer, endometriosis

Enantone Depot® Leuprolide Takeda Prostate cancer, endometriosis

Trenantone Leuprolide Takeda Prostate cancer, endometriosis

Nutropin Depot® Growth hormone Genetech Pediatric growth hormone deficiency

Suprecur® MP Buserelin acetate Aventis Prostate cancer

Decapeptyl® Triptorelin pamoate Ferring Prostate cancer

Sandostatin LAR® Depot Octreotide acetate Novartis Acromegaly

Somatuline® LA Lanreotide Ipsen Acromegaly

Decapeptyl SR. Triptorelin Ipsen Prostate cancer

Trelstarn : Depot Triptorelin pamoate Pfizer Prostate cancer

Arestin® Minocycline Orapharma Periodontal disease

Risperidal® Constan: Risperidone Johnson & Johnson Antipsychotic

Parlodel® LAR Bromocriptine Novartis Parkinsonism

From the pharm aceutical m anufacturing technology perspective, the key goal;

controlling the release o f the drug for longer periods w ithout causing any degradation o f

the drug at production stage, can be achieved by obtaining a hom ogenous distribution o f

the drug within the m atrix. The follow ing sections will give relevant details o f the

[image:18.612.96.510.385.598.2]

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protein loaded polym er m icroparticle system s, with a focus on relation to the novel

m icroparticle system s characterised in this thesis.

1.3 Polymers

The self-definition o f ‘polym ers’ can be derived from the G reek w ords ‘p o ly ’ m eaning

‘m any’ and ‘m ono’ m eaning ‘o n e ’, as polym ers are m acrom olecules built up from

m onom ers covalently bonded together form ing a chain type structure. Repeating

m onom er units connected together m ay form linear, branched or 3D structured (cross-

linked polym ers in w hich one chain is bonded with m onom ers o f another chain) types

as illustrated in Figure 1.2.

c)

Figure 1.2 Linear (a), branched (b) and 3-dimensional (c) structures of polymers.

Since the initial understanding o f their structure by Herm ann Staudinger [5], polym ers

becam e the m ost versatile m aterial type in our daily lives ranging from the sim plest

form as a recyclable carrier bag, to arguably m ore com plicated and im portant form s

such as long life batteries used in m obile electronic devices or biodegradable m atrices

for carrying drugs.

Polym ers can be grouped m ost generally in to two; biopolym ers (polym ers w ith natural

origin) such as chitosan and proteins, or synthetic polym ers including biodegradables

[image:19.612.200.405.227.490.2]

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poly(|3-malic acid)) and non-biodegradables such as silicones and some acrylic

polym ers [6]. Step grow th polym erisation and chain growth polym erisation are the two

principal m ethods for synthesising m an-m ade polym ers [7]. Step grow th polym erisation

occurs as m ultifunctional m onom ers react to form dim ers, trim ers longer oligom ers and

long chain polym ers at the end o f the reactions. Chain grow th polym erisation occurs as

the unsaturated m onom ers continuously add onto the grow ing polym eric chains.

Polym ers exhibit two types o f m orphology; crystalline in w hich m olecules are packed

in regular arrays and am orphous in which m olecules are oriented random ly and are

entangled and m ost polym ers contain both crystalline and am orphous regions

(sem icrystalline). At low tem peratures the m olecules o f an am orphous polym er vibrate

at low frequency, exhibiting solid state properties, this is term ed as the ‘glassy sta te ’.

U pon heating an am orphous polym er, due to the increase in m olecular energy, a

transition occurs from the glassy state to a rubbery state and the inflection tem perature

at which this effect occurs is called the glass transition tem perature (Tg). C rystalline

polym ers show a clear phase transition upon heating; m elting. The tem perature at w hich

this therm odynam ic effect occurs is called m elting tem perature (Tm).

Differential scanning calorim etry (DSC) which is discussed in C hapter 2 can be used to

determ ine the m elting tem perature and the glass transition tem perature o f polym ers.

Therm al properties o f polym ers are often dependent on their m olecular w eight w hich

can be given as;

M w = Equation 1.1

or their m olecular num ber w hich can be given as;

M n = ~ ~‘Mt Equation 1.2

where A, is the num ber o f m olecules with length i and M, is the m ass o f polym er chain

with length i, both define the size o f a m olecule and can both be m easured using gel

perm eation chrom atography (GPC) [8] as discussed in C hapter 2.

M ost polym ers vary in their chain lengths. Their distribution in the length o f the chains

is defined as polydispersity index (PD I) which is m olecular num ber per m olecular

weight. The decrease in m olecular w eight due to degradation, m easured w ith GPC, can

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C opolym ers are substances that are usually produced by step polym erisation from

chains in which two or m ore different m onom ers are connected through covalent bonds.

C opolym ers are useful in that therm odynam ic and physicochem ical properties o f

polym ers therein can be im proved and m odified. C onsidering their form , copolym ers

can be grouped as; ‘ran d o m ’ in which two or m ore different repeating units are

distributed random ly, in which sequences o f the different m onom ers alternate w ithout

any regular pattern and block in w hich long sequences o f m onom ers are follow ed by

another o f the same or graft in w hich one type o f m onom er chain is connected to

another as branches at different points. The form ation o f copolym ers results in a

com bination o f the properties o f the original hom opolym ers and this can im prove the

properties o f the individual polym ers such as tensile strength, reactivity and

therm odynam ic properties [9].

Tw o or m ore polym ers or co-polym ers can be m ixed, typically; by heating and/or

stirring or by dissolving each in a co-solvent adding together and letting solvent

evaporate or using one m onom er as a solvent for another, resulting in a ‘b len d ’ in w hich

polym ers are connected by interm olecular forces, but not covalent bonds. D epending on

their hom ogeneity at a m olecular level, polym er blends are classed as m iscible (very

hom ogenous) or im m iscible (phase separated) show ing single or m ultiple T g in DSC

analysis, respectively.

1.3.1 Polymers in Sustained Release Drug Delivery

The use o f biodegradable and biocom patible (i.e. that can degrade in to m olecules that

are not toxic to hum an body and therefore are ejected w ithout causing any dam age)

polym ers enables the extended release o f drugs.

D uring this project, two polym ers have been used as the drug carrier m atrix, poly(lactic

acid) (PLA) and poly (lactide-co-glycolide) (PLG A ). Poloxam er407, accepted as a

GRAS (generally regarded as safe) has also been used as a processing aid in the

production o f these m icroparticles as it enhances the drug release [ 10].

1.3.1.1 Poly (lactic acid) {PLA)

PLA has 3 stereoisom ers that are the crystalline 1-PLA and d-PLA form s and the

am orphous D L -PL A [11]. The m ost com m on synthetic route to produce PLA is via the

ring opening polym erisation o f lactide w hich is catalysed by stannous octanoate (Figure

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o

JL*

q

Stannous octanoate

►

* V V ],

Figure 1.3 Synthesis of PLA where chiral centres are indicated with X.

PLA are m ore suited as im plants than controlled release delivery devices as their

crystalline nature retards their w ater uptake causing long degradation tim es (typically

m ore than a year depending on m olecular w eight). Upon contact with aqueous m edia,

PLA undergoes hydrolysis and breaks down to lactic acid fragm ents. PLA has a T g o f

about 60-65 °C and a m elting tem perature o f about 173-178 °C [11].

1.3.1.2 Poly(lactide-co-glycolide) (PLGA)

PLG A is synthesised by the ring opening polym erisation o f the cyclic diesters o f lactic

acid and glycolic acid, catalysed by stannous octanoate (Figure 1.4) [12].

0

Y ^ °

o „

1

0

+ f

"0

\

0.

1

r

]

(1

0

Stannous

octanoate O

. J '

ir

o

I n f

O

°+«-m

Figure 1.4 Synthesis of PLGA.

A lthough PG A is not useful as a drug delivery device due to its high crystallinity and

high m elting tem perature, as m entioned earlier in this chapter, PLG A , a copolym er o f

glycolic acid (GA) and lactic acid (LA), is the m ost used biodegradable polym er in

delivery o f biologies [13]. D epending on the ratio o f lactide to glycolide used for the

polym erization, different form s o f PLG A can be obtained. These are usually identified

by the m onom er ratio used. For exam ple PLG A 75:25 identifies a copolym er

com position o f 75% lactic acid and 25% glycolic acid. All PLG A s are am orphous rather

than crystalline and show a glass transition tem perature in the range o f 40-60 °C [12].

U nlike the hom opolym ers o f lactic acid (polylactide) and glycolic acid (polyglycolide),

which show poor solubilities, PLG A has the property o f hydrophobicity and therefore

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acetate. The production o f a 50:50 m ixture o f these two polym ers also reduces the

degradation tim e o f PLA from several m onths to few weeks.

o

PLGA

Figure 1.5 Structures of PLA, PGA and PLGA.

A s show n in Figure 1.5, PLA contains an additional m ethyl group over PG A which

creates the am orphous nature by m aking chain packing m ore difficult w hereas the

absence o f this group allow s PG A to be crystalline. As PLGA is a copolym er o f these

two m onom ers it is less crystalline com pared to PGA, due to the addition o f the methyl

group. And this provides the observed differences in the degradation rates. D egradation

rates o f bulk PLG A s can be calculated using R am an and IR spectroscopies [14]. PLGA

can degrade via hydrolysis o f the ester linkage into small, w ater soluble fragm ents.

1.3.1.2.1 PLG A H ydrolysis

W hen PLG A is exposed to an aqueous solution or vapour, the ester carbonyl is subject

to nucleophilic attack by water, as show n in Figure 1.6 [15]. The ester group in these

polym ers will be hydrolysed via a tetrahedral interm ediate to give prim ary O H and

carboxyl end groups, resulting in chain cleavage. Esters are vulnerable to hydrolysis

because the electron w ithdraw ing effects o f the attached oxygen atom s result in a

partially positive ester carbon. The accum ulation o f carboxylic acid hydrolysis products

in PLG A m atrices results in autocatalysis o f the hydrolysis reaction w hich is follow ed

by addition o f a proton to carbonyl oxygen, w hich facilitates the reaction by stabilizing

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HO OH

OH

0 HO

OH

n -m

HO, _■OH

0

HO OH

Figure 1.6 PLGA hydrolysis mechanism via ester links. R groups represent CH3

for lactide and H for glycolide. The resulting alcohol can abstract a proton from

the carboxylic acid during the chain cleavage step as shown, or a proton addition

to alcohol and removal from carboxylic acid can be provided by water (from [15]).

A lteration o f either the PLA :PG A ratio, m olecular w eight or PD I can change the

degradation rate o f PLG A. G lycolic acid is slightly m ore hydrophilic than lactic acid,

therefore increased glycolic acid content m ay lead to an increase in the hydrolysis rate

for the same m orphology and size o f particles [13]. Typical ratios o f lactide to glycolide

range from 50:50 to 100:0 with m olecular weights typically ranging from 10 to 100 kD a

(or 10' unified atom ic m ass units). The hydrolysis rate o f PLG A also depends on its end

groups w hich m ay be carboxylic acid, m aking the polym er m ore hydrophilic or ester-

capped, decreasing the rate.

1.3.1.3 Poloxamer 407

Poloxam er 407 (also know n as Lutrol or Pluronic F I 27), poly(ethylene

oxide)/poly(propylene oxide)/poly (ethylene oxide) (P E O -P P O -P E O ) triblock

copolym ers, is a hydrophilic surfactant and best know n for its use in m outhw ash and

contact lens solutions w here its function is to help rem ove organic m olecules such as

lipids by increasing their solubility in aqueous m edia.

y L

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Surfactants are often used to increase the w ater solubility o f poorly soluble drugs, in

pharm aceutical form ulations.

In controlled release drug delivery, poloxam er 407 is shown to enhance solubilisation o f

biologies and also prolong their release [16].

1.4 Proteins

Like other bio-m acrom olecules such as nucleic acids, proteins are essential parts o f

organism s and participate in m any processes within cells. W ith varying sizes and

m olecular w eights, proteins have a range o f functions as they are able to bind to other

biom olecules, nucleic acids and metal ions [17]

Linkage betw een am ino acids result in long polym er chains called proteins. Proteins

consist o f carbon, hydrogen, nitrogen, oxygen and sulphur atom s, for exam ple,

C 990H 1532N 262O 300S7, nam ely hum an grow th horm one.

R

OH

Figure 1.8 Structure of an amino acid showing the amine in the left hanside and

carboxyl group in the right hand side circles, respectively.

There are tw enty com m on types o f am inoacids that vary in their R groups (Figure 1.8).

A m inoacids can be grouped as non-polar, uncharged polar and charged p olar [17]. Non

polar am ino acids tend to be unreactive. U ncharged polar am ino acids contain either

hydroxyl or am ine groups, w hich are capable o f hydrogen bonding to other residues and

the third group can bind to metal ions and w hich is im portant in biology [17].

The prim ary structure o f a protein is the sequence o f am inoacids o f this backbone. The

secondary protein structure is the specific geom etric shape caused by intram olecular and

interm olecular hydrogen bonding o f am ide groups. Proteins have three com m on

secondary structures, a-helix, p-sheet and turn [17]. The secondary structure o f proteins

can readily be characterised by FTIR spectroscopy [18]. The tertiary structure o f

proteins is the bending and tw isting o f the polypeptide backbone into a com pact

structure. Several polypeptide chains m ay exhibit w eak interactions form ing large

(26)

detect proteins when dissolved in a solution as proteins absorb UV light at 200nm

(peptide bonds) and - 2 8 0 nm (am inoacids with arom atic rings, Tyrosine and

T ryptophan) [19].

1.4.1 Bovine Serum Albumin (BSA)

W ith its 582 amino acids and 66267 D a total m olecular w eight, BSA is a large and

w idely used m odel protein as it is not only cheap but also stable at 60 °C up to 10 hours

[20], BSA has a total charge o f -18 (at pH 7) which is high due to its charged am ino

acids such as arginine and aspartic acid. BSA has been used as a m odel protein in this

project for the prelim inary investigations of a single polym er form ulation.

140

A

H---N et Charge -10 -8 0

a \ i

^ r

Figure 1.9 BSA structure showing the three charge domains.

1.4.2 Human growth hormone (hGH)

H um an grow th horm one (hGH) has been used to treat various diseases including

children with grow th horm one deficiency (paediatric hypopituitary dw arfism ), m uscle-

w asting disease associated with HIV, Turner's syndrom e for girls and chronic kidney

failure [21]. For children’s grow th deficiency treatm ent it is delivered via a painful

injection - 3 tim es a week, consum ing valuable hospital resources and resulting in poor

patient com pliance.

hG H contains 191 am ino acid residues and is a single chain peptide with a m olecular

w eight o f -2 2 .1 3 kD a and m elting point o f 76 °C [22]. Its secondary structure, show n in

Figure 1.10, contains four helices connected with random loops and chains and

internally cross linked by two disulfide bonds that provide stability and enable

(27)

Random loops

Random chains

Figure 1.10 hGH secondary structure (from [22]).

1.5 Methods of Producing Controlled Release Drug Delivery Systems

In order to produce m icroparticles that follow one or both o f the hypothesised scenarios

show n in Figure 1.1b and Figure 1.1c, the drug needs to be m ixed with the polym er(s)

hom ogenously and this is m ainly dependent on liquefication o f the polym er(s) during

the form ation o f the particles. C om m on m ethods for producing sustained release drug

delivery system s differ m ainly in how they are liquefied, as outlined in this section.

1.5.1 Spray Drying

Spray drying is an established m ethod in the food industry for producing a range o f dry

pow der form products including coffee and m ilk pow der [23]. The use o f spray drying

is also com m on in m icronisation (the process o f reducing particles to a size o f less than

typically 10 pm ) o f drug-polym er form ulations in the pharm aceutical industry. D uring a

typical m icronisation process a solvent such as dichlorom ethane (D CM ), in w hich the

polym er o f interest is dissolved initially, is m ixed w ith the drug, form ing a solvated

m ixture. This m ixture is then induced to atom ise and form s droplets as it is forced

through a nozzle as show n in Figure 1.11. The spray is dried by applying a hot (-6 5 °C)

[image:27.612.199.408.35.281.2]

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A TO M IZER

CVCLONE / SEPAR A TO R

HOT rtIR

M O S T A IF

PRCDUCT C O W E 'rIN G

=r c d u:t d i s c h a r g e

Figure 1.11 Schematic diagram of spray dryer (from [24]).

The spray drying m ethod has been studied w idely for producing PLG A based sustained

release devices as carriers for various biologies and has been show n to be successful in

producing <100 pm m icrospheres with encapsulation efficiencies up to -8 0 % [3,25,26].

H ow ever, considering the efficacy o f proteins that are very sensitive to tem perature and

the biocom patibility o f the produced form ulation, the high process tem peratures

(beyond 60 °C) and the possible existence of toxic solvent residues within

m icroparticles caused by insufficient drying, exist as critical draw backs.

1.5.2 Emulsion

E m ulsification processes can be grouped into two; single and double [3]. T he form er

involves tw o m ain steps; firstly, dissolving the polym er in an organic solvent such as

chloroform and adding the drug m olecules to this solution and secondly, adding this

m ixture into a w ater- em ulsifier m ixture and stirring. U pon evaporation o f the solvent,

the drug loaded polym er solidifies in the aqueous phase. As one can expect by the

description o f the m ethod, single em ulsification is not suitable for w ater soluble drugs

[4].

T he double em ulsion m ethod is sim ilar to the single em ulsion one, the only difference

being that the drug m olecules are dissolved in w ater before being added to the polym er

solution, such that a prim ary em ulsion is form ed on m ixing. The rest o f the procedure

follow s as in single em ulsification. A stabilizer such as polyvinyl alcohol (PV A ) m ay

(29)

Since the solvents used can cause degradation, em ulsification is not suitable for the

encapsulation o f sensitive drugs such as proteins into biodegradable polym ers such as

PLGA. R equiring the use o f large am ounts o f solvents, this m ethod is also expensive

and has potential environm ental hazards.

1.5.3 Supercritical Fluid Technologies

Substances becom e supercritical fluids (SCF) when placed above their critical point (i.e.

above their critical tem perature and critical pressure) exhibiting the flow properties o f a

gas (i.e. show ing low viscosity) and the dissolving pow er o f a liquid, thus becom ing

ideal solvents [27].

SCF, first observed by C agniard de la Tour in 1822 [28], can easily penetrate through

m aterials because they do not exhibit significant surface tension, and their solvent

pow er can be controlled by altering tem perature and/or pressure as it is related to their

density [29].

A m ong m any other m olecules including propane, ethane, am m onia and water, carbon

dioxide has becom e the fluid o f choice in SC processes because it is cheap, no n

flam m able, has GRAS (generally regarded as safe) status, is environm entally acceptable

and m ay be recycled. ScCC>2 is best know n for its ‘fam ous’ application o f caffeine

extraction from coffee beans, but perhaps m ore im portantly, its easily achievable critical

point (3 1 .1°C and 73.8 bar) also m akes it ideal for processing biologies w ithout causing

denaturation [30].

D uring the last decade, drug encapsulation o f polym er m icroparticles using supercritical

fluid technology has been reported using a num ber o f m ethods. These include expansion

o f supercritical solutions (RESS), gas anti-solvent techniques (G A S), supercritical an ti

solvent technique (SAS) and particles from gas saturated solutions (PG SS) [31]. One

disadvantage o f SCCO2 considering applicability for w ide use is that only non-polar and

som e low polar m olecular substances can dissolve in SCCO2. H ow ever for the latter

m ethod, PG SS, this is not a concern, particularly for biodegradable polym ers and m ost

biologies as they do not need to be dissolved in C O 2 as detailed in section 1.5.3.4.

1.5.3.1 Rapid Expansion o f Supercritical Solutions (RESS)

This process requires both the polym er and drug to be dissolved in SCCO2, before being

m ixed and then forced through a nozzle (typically <150 pm ) into a low er pressure zone.

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process how ever since only a few polym ers are soluable in SCCO2 this m ethod has not

been w idely applied. In the case o f having to use a poor SCCO2 soluble polym er, organic

co-solvents can be added to im prove solubility; how ever this m ay cause protein

degradation. Also, the processes m ay have to be perform ed at tem peratures over 100 °C

and 200 bar, to im prove polym er solubility, which m akes it im possible to process

biologies w ithout dam aging them [31].

1.5.3.2 Gas Antisolvent Technique (GAS)

In this process, SCCO2 acts as an anti-solvent (a solvent in w hich the product is insoluble)

therefore the polym er and drug are not required to dissolve in SCCO2. A solution o f the

drug and polym er in an organic solvent is produced beforehand and SCCO2 is then

gradually added. As the polym er and drug are insoluble in the SCCO2 they com e out as

m icroparticles.

This process has been used to prepare insulin and insulin/PE G loaded PLLA

nanoparticles with -9 4 % encapsulation efficiency and very low residual content -3 0 0

ppm ) [33]. How ever, solvent residue issues cannot be overcom e w ith this technique.

1.5.3.3 Supercritical Antisolvent Technique (SAS)

A lthough this is a sim ilar process to G A S, based on SCCO2 being used as an antisolvent,

it is different in that the polym er-drug solution is forced through a nozzle or capillary

into the SCCO2 phase. The solvent then evaporates form ing encapsulated polym er

m icroparticles. U sing this process D uarte et al. produced naproxen-loaded m icrospheres

from ethylcellulose/m ethylcellulose blends for oral delivery that show ed a sustained

release o f the drug [34],

1.5.3.4 Particles from Gas Saturated Solution (PGSS)

This process, first developed for the coatings industry [35], is sim ply analogous to

painting w ith a spray coater using small particles. A fter its reported suitability for

m icronisation o f nifedipine [36], a small m olecule drug, m ore recent developm ents have

focused on production o f drug particles entrapped within polym ers in m icroparticle [37],

im plant [38] or scaffold [39] form . U nlike the aforem entioned m ethods, PG SS has the

advantage in that it w orks in the absence o f organic solvents and the polym er and drugs

do not need to be soluble in SCCO2. H ow ever the polym er needs to be sufficiently

plasticised by SCCO2. This is achievable at m oderate tem peratures (typically <40 °C)

and pressures (<150 bar) w hich have been show n to have no adverse effect on the

(31)

PGSS procedure for producing a protein encapsulated polym er form ulation is illustrated

in Figure 1.12. The polym er and drug are initially added to a high pressure vessel

(Figure 1.12-step 1). C O 2 is added and the tem perature and pressure are set to the critical

conditions o f C O 2 (Figure 1.12-step2). Then as a result, as show n in Figure 1.12-step3,

the polym er liquefies. The plasticised m ixture is then stirred with a helical im peller

(Figure 1.12-step4) to m ix the drug m olecules with liquefied polym er until a

hom ogenous m ixture is form ed as depicted in Figure 1.12-step5. This polym er/drug

m ixture is then let through a nozzle generating particles as shown in Figure 1.12-step6.

Upon spraying, the C O 2 evaporates setting the shape o f the particles as the polym er

solidifies [31]. U sing different nozzles, the particle shape and size can be controlled.

An im portant com plication o f this process may be that the escaping C O 2 can influence

polym er m orphology, for exam ple generating m acroporosity [40]. Further this process

requires high precision equipm ent which is expensive both to m odify for drug

encapsulation and to m aintain.

Figure 1.12 Schematic diagram of the processing steps of PGSS method (from

[41]).

1.5.3.4.1 A Custom Built PG SS A pparatus-C riticalM ix™

A high pressure particle rig was developed at U niversity o f N ottingham im plem enting

the PGSS process to encapsulate drugs within polym er m icroparticles [42].

The C riticalM ix™ m ethod is a novel PG SS approach developed using this rig, and was

[image:31.612.121.478.324.526.2]

(32)

process with 100% encapsulation efficiency [37]. The w orkings o f the m ethod are

briefly outlined below.

W hen suitable polym ers, such as PLGA, PLA and protein particles, such as BSA, hGH,

are exposed to SCCO2 in a pressure vessel, the polym er is liquefied, thereby allow ing the

protein to be m ixed efficiently with the polym er. Follow ing m ixing, the m ixture is

depressurised through a nozzle w hereby the C O 2 returns to a gaseous state and

evaporates w hilst the polym er solidifies around the protein, resulting in the production

o f m icroparticles with the protein distributed throughout.

For the form ulations investigated in this thesis, a typical batch was prepared by adding

2.1 g o f a pre-w eighed com bination o f polym er, protein and excipients such as

Poloxam er407 to the PG SS apparatus. The apparatus was sealed, pressurised w ith C O 2

to 700 psi (48 bar) and heated to 40 °C. Once at tem perature, the pressure was increased

to 2030 psi (140 bar). The liquefied protein/polym er m ixture was then stirred at 150

rpm for 1 hour, after w hich tim e stirring was stopped and the m ixture was depressurised

through a nozzle generating m icroparticles. These w ere collected in a cyclone and

recovered as a free flow ing w hite pow der.

1.6 Mid-IR Spectroscopic Imaging and Analysis Concept

The subject o f this thesis is the developm ent o f m id-IR spectroscopic im aging and

m ultivariate analysis applied to controlled release polym er m icroparticles to investigate

kinetic processes, including degradation, drug release and particle m orphology,

‘visually’ and ‘quantitatively’.

A brief introduction is given here particularly to introduce the subject area for readers

unfam iliar with chem ical im aging and analysis concepts and to help better define the

aim s o f the project.

Natural contrast arises from m olecular vibrations in vibrational spectroscopy, m agnetic

effects in nuclear m agnetic resonance im aging, electronic transitions in electron and x-

ray m icroscopy, from chem ical labels in near field optical m icroscopy, auto or enhanced

fluorescence in fluorescence m icroscopy or surface topography (m echanically) in

scanning probe m icroscopy (Figure 1.13). For polym ers and proteins, chem ical

inform ation is richest at m id-IR wavelengths, w ithout the need o f any dyes, com plicated

specim en preparation or radiation dam age concerns. A lso obtaining spatiotem poral

(33)

been ‘th e ’ other non-invasive and non-destructive im aging m odality that facilitates

internal and surface im ages (2D and 3D) o f pharm aceuticals and living organism s on

both m icro and m acro scale [43]. Figure 1.13 dem onstrates that SEM is also well suited

to the study o f m orphology and it requires relatively sim ple sam ple preparation.

High

Chemical Information

Low

10 pm 1 pm 50 nm 1 nm 1 A

Spatial Resolution

Figure 1.13 A comparison plot of well-established microscopy methods that

particle systems can be studied with micron scale or better spatial resolution

versus chemical information. Magnetic resonance imaging (MRI), vibrational

chemical imaging (Raman and Infrared microscopic imaging, RM and IR,

respectively), x-ray photoelectron spectroscopy (XPS), energy dispersive x-ray

spectroscopy (EDS), scanning transmission x-ray microscopy (STXM), electron

energy loss spectroscopy (EELS), transmission electron microscopy (TEM),

scanning electron microscopy (SEM), fluorescence microscopy (FM), optical

microscopy (OM), nearfield scanning optical microscopy (NSOM) and atomic

force microscopy (AFM) (adapted from [44]).

M id-IR im aging facilitates the generation o f chem ical im ages using an FPA detector

w here each o f the pixels o f the focal plane array (FPA) detector acts as an individual

detector, allow ing the collection o f thousands o f IR spectra sim ultaneously. A stack o f

2D im ages can be collected within a few m inutes, proving good tem poral resolution in

slow processes with a spatial resolution dow n to 5 pm depending on the sam pling

m ethod used (Figure 1.14). H ow ever regardless o f the sam pling m ethod (transm ission,

reflection etc.) or w avelength range used (IR, X -rays etc.), particularly for kinetic

‘perfect’ chemical ^ 7

microscopy

MRI

RM

STXM

XPS

EDS EELS

FM

TEM

NSOM SEM

OM

[image:33.612.174.420.111.348.2]

(34)

experim ents that last longer than a few hours, hyperspectral im ages often require

detailed analysis once collected.

Formulation in D20

0.6 0.5 c 0.4

n

0.3 .a 0.2

< 0.1

3950 2950 1950 W av en u m b ers [c m 1]

950

B) 0.6 0.2

0.5

0.6 0.5

Polymer Protein

g 0.15 w

c 0.4

ra

| 0.3

Ja0.2

ei

c 0.4

0.1 _|_0-3

a 0.2 <

0.1

0

< 0.05

< 0.1

0

3950 2950 1950 950

3950 2950 1950 950 3950 2950 1950 950 W av en u m b ers [c m 1] W av en u m b ers [ c m 1] W av en u m b ers [ c m 1]

Figure 1.14 Mid-infrared spectroscopic image stack of a protein loaded polymer

microparticle in water, showing a pixel spectrum on the right (A) and pure

component spectra (B) therein to be extracted from formulation spectra for each

pixel.

1.7 Project Aims

The prelim inary aim o f this project has been to investigate and fine-tune the application

o f m id-IR im aging to characterise scCC>2 processed ‘d ry ’ m icroparticle form ulations as

received. This is based on determ ining their changing com ponents’ distributions,

particularly the drug distribution throughout individual m icroparticles using available

sam pling m ethods (described in C hapter 2) and relevant m ultivariate data analysis

m ethods (described in C hapter 3).

Follow ing dry state characterisation o f m odel drug loaded PLG A m icroparticles, the

next objective was to optim ise the experim ental conditions such that the first kinetic

im aging study on real-tim e hydrolysis o f a single PLG A m icroparticle w as conducted.

Further objectives related to this novel experim ent were to im prove this tem poral

hyperspectral data set w hich had decreasing SN R over tim e, in term s o f spatial

resolution and SNR, using soft m ultivariate m ethods and developing a hard m odelling

m ethod to obtain and com pare the m ost im portant inform ation o f such experim ent,

(35)

physicochem ical phenom ena such as sw elling, shrinking and eroding was also

investigated using soft and hard m odelling approaches.

D eveloping this pro o f o f concept, a further objective, em erging im m ediately, was to

study PLG A copolym er m icroparticles with L/G m olar com positions o f 100/0, 75/25

and 50/50 at 50 °C and 70 °C to investigate and com pare effects on their degradation

rates, again for the first tim e utilising m id-IR im aging and m ultivariate analysis.

A T R -FTIR im aging has been a fast progressing m ethod used to study drug release in

sem isolid form ulations, drug penetration, and the influence o f penetration m odifiers in

situ com m only at sam ple-solution interfaces. H ow ever there has been lack o f literature

in studying evolution o f single m icroparticles during dissolution. H ence it was o f

interest to see and m onitor drug redistribution and release in such system s and to

correlate such data to conventional drug dissolution m ethods.

Consequently an aim was set to dem onstrate the feasibility o f the A T R geom etry for

m onitoring drug release in situ from PLG A m icroparticles and to utilise data analysis

m ethods to extract associated drug release profiles.

Pharm aceuticals often need to be sterilised before use. Finally m id-IR im aging and

analysis w ere used for visualising and rationalising the effect o f gam m a irradiation (a

com m on sterilisation m ethod for biodegradable polym er based drugs) on a pipeline

product o f the collaborating com pany that has show n successful in vivo and in vitro

sustained release for the delivery o f hGH.

The overall aim o f this project can be sum m arised as the developm ent o f m id-IR

im aging and analysis as a standalone toolbox for studying kinetic processes in

biopolym eric m icroparticles. This was assisted m ainly by verification o f m orphology

using SEM , therm al properties using D SC, dissolution testing using U V -

(36)

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[2] S.S. Davis, L. Ilium, S. Stolnik, Polym ers in drug delivery, C urrent O pinion in

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[3] R.C. M undargi, V.R. Babu, V. R angasw am y, P. Patel, T.M . A m inabhavi,

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[4] C. W ischke, S.P. Schw endem an, Principles o f encapsulating hydrophobic drugs in

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[21] J. M. Tanner, R. H. W hitehouse, P. C. R. H ughes and F. P. Vince, E ffect o f H um an

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loaded m icrospheres prepared by spray-drying poly(D ,L -lactide) and poly(D ,L -lactide-

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Mid IR Imaging and Multivariate Analysis of Dynamic Processes in Pharmaceutically Relevant Microparticles

Mid-IR Imaging and Multivariate Analysis of Dynamic

Published version

Mid-IR Imaging and Multivariate Analysis of Dynamic

D eclaration

Acknowledgements

Contents

PA PER I

List of Abbreviations

API Active pharmaceutical ingredient

HEPES

Attenuated total reflection

Glycolic acid

RAM Random-access memory

y Gamma

A bstract

Introduction

1.1 Drug Delivery

1.2 Controlled Release Drug Delivery

Figure 1.1 Controlled release scenarios for polymer microparticles; (a) reservoir

Table 1.1 PLGA based microparticle formulations available in the market [1,3].

1.3 Polymers

Figure 1.2 Linear (a), branched (b) and 3-dimensional (c) structures of polymers.

M w = Equation 1.1

1.3.1 Polymers in Sustained Release Drug Delivery

1.3.1.1 Poly (lactic acid) {PLA)

Figure 1.3 Synthesis of PLA where chiral centres are indicated with X.

1.3.1.2 Poly(lactide-co-glycolide) (PLGA)

0

0

Figure 1.5 Structures of PLA, PGA and PLGA.

Figure 1.6 PLGA hydrolysis mechanism via ester links. R groups represent CH3

1.3.1.3 Poloxamer 407

1.4 Proteins

Figure 1.8 Structure of an amino acid showing the amine in the left hanside and

1.4.1 Bovine Serum Albumin (BSA)

H---N et Charge -10 -8 0

Random loops

1.5.1 Spray Drying

A TO M IZER

CVCLONE / SEPAR A TO R

HOT rtIR

1.5.2 Emulsion

1.5.3 Supercritical Fluid Technologies

1.5.3.1 Rapid Expansion o f Supercritical Solutions (RESS)

1.5.3.2 Gas Antisolvent Technique (GAS)

1.5.3.3 Supercritical Antisolvent Technique (SAS)

1.5.3.4 Particles from Gas Saturated Solution (PGSS)

Figure 1.12 Schematic diagram of the processing steps of PGSS method (from

1.6 Mid-IR Spectroscopic Imaging and Analysis Concept

Figure 1.13 A comparison plot of well-established microscopy methods that

energy loss spectroscopy (EELS), transmission electron microscopy (TEM),

‘perfect’ chemical ^ 7

microparticle in water, showing a pixel spectrum on the right (A) and pure

1.8 References