Preparation and Characterization of Poly(Lactic Acid) Nanoparticles ...
Transcript of Preparation and Characterization of Poly(Lactic Acid) Nanoparticles ...
Division of Pharmaceutical Technology
Faculty of Pharmacy
University of Helsinki
Finland
Preparation and Characterization of
Poly(Lactic Acid) Nanoparticles
for Pharmaceutical Use
Samuli Hirsjärvi
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Pharmacy of the University of
Helsinki, for public examination in lecture room 1041, Viikki Biocenter 2,
on February 1st 2008, at 12 noon.
Helsinki 2008
Supervisors
Docent Leena Peltonen Division of Pharmaceutical Technology Faculty of Pharmacy University of Helsinki Finland Professor Jouni Hirvonen Division of Pharmaceutical Technology Faculty of Pharmacy University of Helsinki Finland
Reviewers Professor Kristiina Järvinen Department of Pharmaceutics Faculty of Pharmacy University of Kuopio Finland Docent Vesa-Pekka Lehto Laboratory of Industrial Physics Department of Physics Faculty of Mathematics and Natural Sciences University of Turku Finland
Opponent Professor A. Atilla Hıncal Department of Pharmaceutical Technology Faculty of Pharmacy Hacettepe University Ankara Turkey
© Samuli Hirsjärvi 2008
ISBN 978-952-10-4455-7 (paperback)
ISBN 978-952-10-4456-4 (PDF, http://ethesis.helsinki.fi)
ISSN 1795-7079
Helsinki University Printing House
Helsinki 2008
Finland
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Abstract
Hirsjärvi, S. (2008) Preparation and Characterization of Poly(Lactic Acid) Nanoparticles for
Pharmaceutical Use.
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki, 2/2008,
42 pp.; ISBN 978-952-10-4455-7 (paperback), ISBN 978-952-10-4456-4 (PDF), ISSN 1795-7079
Nanoparticles have versatile potential for efficient exploitation of different drug delivery
formulations and routes because of the properties provided by their small size. These
possible benefits include controlled release, protection of the active pharmaceutical
ingredient and drug targeting. Nanoparticles are expected to offer new solutions e.g. for
gene therapy and delivery of peptide drugs. Generally, nanoparticles are applied as an
injectable or oral solution, but their use as dried material in formulations such as tablets or
inhalable powders is equally conceivable. Although the research on pharmaceutical
nanoparticles has been extensive during recent years, breakthrough of products to the
market has not yet occurred. Problems like poor drug encapsulation efficiency and difficulties
in controlling and scaling up of the preparation process have inhibited progress.
In this study, nanoparticles were prepared from a biodegradable poly(lactic acid) (PLA)
polymer. Knowledge and understanding of properties of the materials used in the particle
preparation as well as behavior of the nanoparticles in different environments are essential
in order to create successful nanoparticle formulations and to predict their performance in
the body. Therefore, the effect nanoprecipitation, a nanoparticle preparation method, on
the physicochemical properties of the polymer and model drugs encapsulated in the
nanoparticles as well as the effect of the drugs on the polymer were studied by
thermoanalytical and spectroscopic methods. The suitability of capillary electrophoresis for
drug quantitation in the nanoparticle formulations was estimated. Surface pressure
measurements were applied for the assessment of stability and aggregation of the
nanoparticles. The protective ability of carbohydrates to improve the freeze-drying process of
the nanoparticles was evaluated. Finally, a layer-by-layer polyelectrolyte coating process,
which modifies the particle surface properties, was introduced. Analytical methods such as
electron microscopy, size measurements and zeta potential determinations were included in
the nanoparticle characterization.
Both the preparation process as well as the drugs affected the physicochemical
characteristics of the nanoparticles such as particle size and crystallinity of the polymer, and,
further, the stability of the PLA nanoparticles. With a combination of surface pressure
measurements and electron microscopy observations, it was possible to gain information
about conditions and progression of nanoparticle aggregation and the role of the
stabilization mechanisms. Stability of nanoparticle dispersion was a prerequisite for
completion of the layer-by-layer coating. Utilization of protective excipients, glucose and
lactose in this study, was found to be indispensable for the freeze-drying. Capillary
electrophoresis appeared to be a promising tool for the quantitation of drug encapsulation
and subsequent drug release. The results of this study provide information about the
materials used and the methods applied for further studies that aim at development of
biocompatible and biodegradable nanoparticles for human use.
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Acknowledgements
This work was carried out at the Division of Pharmaceutical Technology, Faculty of Pharmacy,
University of Helsinki.
I wish to express my gratitude to my supervisor, Docent Leena Peltonen for her support
and guidance into the world of science. I am also grateful to my other supervisor, Professor
Jouni Hirvonen, for his support and guidance as well as for providing excellent working
facilities. I would like to thank my supervisors for always being availabile and willing to help
me in the challenges of this work.
I am sincerely grateful to all my co-authors, Docent Milja Karjalainen, Docent Susanne
Wiedmer, M.Sc. Laura Kainu and Ms. Anne Helle, for their remarkable contribution to this
work. I would like to express my warmest thanks to all my colleagues in the Division of
Pharmaceutical Technology for discussions, technical assistance, friendship and for creating a
pleasant atmosphere during these years. A special word of thanks goes to my friend and
colleague Harri Nurmi for valuable advice in IT issues.
Professor Kristiina Järvinen and Docent Vesa-Pekka Lehto are acknowldeged for carefully
reviewing the manuscript and providing constructive comments and suggestion for its
improvement.
I am deeply grateful to Dr. Jan Dabek for revising the language of this thesis.
Research Foundation of the University of Helsinki, Finnish Cultural Foundation (Elli
Turunen fund), the Finnish Funding Agency for Technology and Innovation (Tekes), the
Chancellor of the University of Helsinki, the Finnish Pharmaceutical Society and the
Association of Teachers and Researchers of Pharmaceutical Sciences are acknowledged for
funding my research.
I would also like to express my gratitude to my family for endless support and love.
Finally, I want to thank the most important and loved ones, my wife Hanna and our son Oiva,
for bringing love and happiness in my life.
Helsinki, January 2008
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Contents
Abstract i Acknowledgements ii Contents iii List of original publications iv Abbreviations v 1 Introduction 1 2 Review of the literature 3
2.1 Preparation and processing of polymeric nanoparticles 3 2.1.1 Poly(lactic acid) 4 2.1.2 Nanoprecipitation 5 2.1.3 Surface modification 7 2.1.4 Freeze-drying 8
2.2 Nanoparticle characterization 9 2.2.1 Size, morphology, surface properties and stability 9 2.2.2 Drug-polymer interactions 11 2.2.3 Drug loading and release 12
2.3 Nanoparticles in drug delivery applications 13 3 Aims of the study 16 4 Experimental 17
4.1 Materials 17 4.2 Methods 18
4.2.1 Preparation, coating and freeze-drying of nanoparticles 19 4.2.2 Characterization of nanoparticles 19
5 Results and discussion 21 5.1 Characteristics of the nanoparticles 21
5.1.1 Preparation of the nanoparticles 21 5.1.2 Physical characterization of the polymer and the encapsulated drugs 22 5.1.3 Determination of drug content 24 5.1.4 Surface charge of the nanoparticles 25 5.1.5 Stability and aggregation studies by surface pressure measurements 26
5.2 Freeze-drying 27 5.3 Layer-by-layer coating 28
6 Conclusions 31 7 References 32
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List of original publications
This thesis is based on the following publications of Samuli Hirsjärvi (né Hyvönen):
I Hyvönen, S., Peltonen, L., Karjalainen, M., and Hirvonen, J. (2005) Effect of
nanoprecipitation on the physicochemical properties of low molecular weight
poly(L-lactic acid) nanoparticles loaded with salbutamol sulphate and
beclomethasone dipropionate. Int J Pharm 295(1-2): 269-281.
II Helle, A,. Hirsjärvi, S., Peltonen, L., Hirvonen, J., and Wiedmer, S. Quantitative
determination of drug encapsulation in poly(lactic acid) nanoparticles by capillary
electrophoresis. J Chromatogr A in press. III Hirsjärvi, S., Peltonen, L., and Hirvonen, J. Surface pressure measurements in
particle interaction and stability studies of poly(lactic acid) nanoparticles. Int J Pharm in press.
IV Hirsjärvi, S., Peltonen, L., Kainu, L., and Hirvonen, J. (2006) Freeze-drying of low
molecular weight poly(L-lactic acid) nanoparticles: effect of cryo- and
lyoprotectants. J Nanosci Nanotechnol 6(9/10): 3110-3117.
V Hirsjärvi, S., Peltonen, L., and Hirvonen, J. (2006) Layer-by-layer coating of low
molecular weight poly(lactic acid) nanoparticles. Colloid Surf B 49(1): 93-99.
The publications are referred to in the text by their roman numerals I-V. Reprinted with the
permission of the publishers.
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Abbreviations
AFM Atomic force microscopy
BBB Blood-brain barrier
BDP Beclomethasone dipropionate
BSA Bovine serum albumin
CE Capillary electrophoresis
CHL Chloroform
D,L-PLA
Poly(D,L-lactic acid)
DCM Dichloromethane
DLCA Diffusion-limited cluster aggregation
DSC Differential scanning calorimetry
ESCA Electron spectroscopy for chemical analysis
ESEM Environmental scanning electron microscope
FTIR Fourier transform infrared spectroscopy
GI Gastro-intestinal
GPC Gel permeation chromatography
HIC Hydrophobic interaction chromatography
HPLC High performance liquid chromatography
LbL Layer-by-layer
L-PLA Poly(L-lactic acid)
MEEKC Microemulsion electrokinetic chromatography
MPS Mononuclear phagocyte system
MW Molecular weight
NMR Nuclear magnetic resonance spectroscopy
PAH Poly(allylamine hydrochloride)
PCS Photon correlation spectroscopy
PE Polyelectrolyte
PEG Poly(ethylene glycol)
PLA Poly(lactic acid)
PLGA Poly(lactic-co-glycolic acid)
PSS Poly(styrene sulfonate)
RLCA Reaction-limited cluster aggregation
SCG Sodium cromoglycate
SEC Size exclusion cromatrography
SEM Scanning electron microscopy
SS Salbutamol sulphate
Tc Cold crystallization temperature
TEM Transmission electron microscopy
Tg Glass transition temperature
Tm Melting temperature
UV Ultraviolet
XRPD X-ray powder diffraction
Introduction
1
1 Introduction
Pharmaceutical nanoparticles are submicron-sized, colloidal vehicles that carry drugs to the
target or release drugs in a controlled way in the body. After preparation, nanoparticles are
usually dispersed in liquid. Such a system can be administered to humans for example by
injection, by the oral route, or used in ointments and ocular products. Alternatively,
nanoparticles can be dried to a powder, which allows pulmonary delivery or further
processing to tablets or capsules.
In drug delivery, nanoparticles should readily be biocompatible (not harmful for humans)
and biodegradable (deteriorate and expulse in the body conditions). These properties, as
well as targeting and controlled release, can be affected by nanoparticle material selection
and by surface modification. Materials such as synthetic polymers, proteins or other natural
macromolecules are used in the preparation of nanoparticles. To process these materials into
nanoparticles, a variety of preparation techniques exist ranging from polymerization of
monomers to different polymer deposition methods [1, 2].
Nanoparticles in pharmaceutical applications have gained plenty of research attention
during recent decades [3]. Although the research concerning formulation of nanoparticles
into drug delivery devices has been extensive, only a few polymeric nanoparticulate products
have reached the market. One known product, Abraxane™, consist of intravenously
administered 130-nm nanoparticles prepared from the protein albumin bound with
paclitaxel, a drug used in cancer therapy [4]. Another cancer drug, Doxorubicin Transdrug®,
consisting of doxorubicin-loaded poly(isohexylcyanoacrylate) nanoparticles is currently at the
Phase II/III clinical trials [5]. Among the drugs used in nanoparticle formulations, particularly
cancer therapeutics are widely studied because the formulation might reduce toxicity of the
drug while improving efficacy of the treatment [6]. In addition to drug molecules, other
candidates to be encapsulated in or coupled with nanoparticles include macromolecules like
proteins, peptides and genes (nucleic acids) [7, 8]. These kinds of molecules tend to be
inactivated in the body by enzymatic degradation. In terms of controlled release,
nanoparticles provide protection against the body conditions resulting in sustained release
and maintenance of bioactivity before the drug reaches the target.
After intravenous administration, nanosized particles are mainly taken up by the
macrophages of the mononuclear phagocyte system (MPS) [9, 10] and, thus, can be localized
in the liver, spleen and lungs [11-13]. By modifying particle surface, e.g., by coating, defence
mechanisms of the body can be avoided to some extent leading to longer circulation times of
nanoparticles in the blood [14]. Tailored coating also enables another promising application
of nanoparticles: drug delivery across the blood-brain barrier (BBB) [15]. From the intestine,
after oral administration, intracellular uptake may occur and prior to that, the nanoparticles
can adhere to the mucosa (bioadhesion) and thus improve pharmacokinetics of the drug [16].
As a summary of the above mentioned facts, the benefits of nanoparticles include
protection of the encapsulated pharmaceutical substance, improved efficacy, fewer adverse
effects, controlled release and drug targeting. Correspondingly, potential active substances
in nanoparticle formulations could be expensive molecules applicable in small amounts.
At the present time, several successful laboratory-scale nanoparticulate drug targeting
systems are available, (reviewed e.g. in [3, 17]), and some processes have been scaled up [18].
Introduction
2
As the time approaches when an increasing number of nanoparticulate drug delivery systems
reach the market and the systems are transferred from animal tests to human use, concerns
about the safety of the products are emerging. More attention will be paid to stability and
toxicology of nanoparticles and their constituents. According to recent opinion, proper
physicochemical characterization of nanoparticles should be included in risk assessment and
toxicology considerations of nanoparticulate systems, in addition to pharmaceutical in vitro
and in vivo testing [19].
In this thesis, nanoparticles were prepared from poly(lactic acid) polymers using the
nanoprecipitation technique and model drug substances were encapsulated in the
nanoparticles. Properties of the nanoparticle surface were modified by layer-by-layer coating
whereas freeze-drying was applied to further expand the processing possibilities of
nanoparticles. The focus in this study was on physicochemical characterization and evaluation
of nanoparticle-behavior during different stages of preparation, surface modification and
stabilization in different environments. Increasing knowledge about these kinds of systems
extends the understanding and facilitates the prediction of the behavior of similar systems in
man.
Review of the literature
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2 Review of the literature
2.1 Preparation and processing of polymeric nanoparticles
Biodegradable nanoparticles for pharmaceutical use are prepared from a variety of synthetic
and natural polymers. Synthetic polymers such as polyacrylates, polycaprolactones,
polylactides and its copolymers with polyglycolides are widely used and discussed [20, 21].
Natural polymers include e.g. albumin [22, 23], alginate [24], gelatin [25, 26], and probably
the most studied natural polymer for pharmaceutical nanoparticle applications: chitosan
(reviewed in [27]).
Several methods exist for the preparation of nanoparticles. When synthetic polymers are
used, they are typically dissolved in a convenient solvent followed by precipitation in a liquid
environment leading to nanoparticle formation. The drug intended to be encapsulated in
the particles is usually incorporated in the process during the polymer solvation and
precipitation. Emulsification/solvent diffusion, emulsification/solvent evaporation,
nanoprecipitation and salting-out methods are widely applied techniques and they have
been discussed in several reviews [2, 20, 21, 28]. In emulsification/solvent evaporation, the
dissolved polymer is emulsified with aqueous phase with the help of a high-energy source
such as ultrasound or homogenization followed by solvent evaporation (vacuum, heat). In
the salting-out method, polymer precipitation is obtained via phase separation (organic
solvent - aqueous phase) by the addition of a salting-out agent, e.g. electrolytes in the case
of acetone as a solvent for the polymer. Similarly, in emulsification/solvent diffusion,
nanoparticles are formed when the saturation limit of a partially water-miscible solvent (e.g.
benzyl alcohol) is exceeded by addition of water. In both techniques, the phase separation is
accompanied by vigorous stirring. The separated solvent is removed e.g. by cross-flow
filtration. The nanoprecipitation method is presented in chapter 2.1.2. Techniques such as
supercritical fluid technologies [29, 30] are different approaches to nanosized particle
preparation. In addition to polymers, nanoparticles can be created directly from monomers
using different polymerization techniques as presented in comprehensive reviews [1, 2, 21].
The properties and fate in vivo of a nanoparticulate system depend to a great extent on
the surface properties of the particles. Thus, stability, drug release and targeting can be
tailored by surface modification. Two strategies exist. First, polymers with grafted molecules,
such as poly(ethylene glycol) (PEG), can be used at the surface (covalent attachment) [31].
Alternatively, nanoparticles can be coated by adsorbing molecules on the surface by
techniques like layer-by-layer adsorption (non-covalent attachment) [32].
Nanoparticles are usually in (aqueous) liquid dispersion after preparation, which may
cause degradation of the polymer and drug leakage into the medium. In addition, handling
(storage, transportation) of a liquid particle system is laborious. Stability and facilitated
handling as well as readiness for further processing (formulation of tablets, capsules,
powders) and conservation of the particle structure of the nanoparticulate system are
achieved by drying. Freeze-drying in appropriate conditions is a method that conserves the
structure of particles in an acceptable and efficient manner.
Review of the literature
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2.1.1 Poly(lactic acid)
Poly(lactic acid) (PLA) is a linear aliphatic polyester derived from lactic acid monomers (Fig. 1).
PLA among its copolymers with glycolic acids (PLGA) are widely used in micro- and
nanoparticulate drug delivery systems because of their biocompatibility and biodegradation
properties; they possess the Generally Regarded as Safe (GRAS) status of the Food and Drug
Administration (FDA). In addition to drug delivery devices, these polymers are used in several
medical devices such as sutures, tissue screws and tacks, tissue regeneration membranes,
bone fixation devices and systems for meniscus and cartilage repair [33-35].
Figure 1. Chemical structure of poly(lactic acid).
PLA exists in two optical forms. Polymers of L(+) lactic acid and D(-) lactic acid are partially
crystalline, while the racemic poly(D,L-lactic acid) is amorphous. Of the two enantiomers, the
polymeric L-lactic acid is commonly employed because it is the one degrading into the
endogenous lactic acid in the body. Low molecular weight (MW) PLAs (< 3 000 g/mol) are
produced by direct condensation of lactic acid, whereas higher MW PLA polymers are
obtained usually from ring opening polymerization of lactide [36]. PLAs are soluble in
organic solvents such as chloroform and dichloromethane, but insoluble in common alcohols
like ethanol [36]. They are also insoluble in water, although low MW PLAs are more
hydrophilic and contain oligomers with some aqueous solubility [37, 38]. Unlike racemic D,L-
PLA, enantiomeric PLAs are insoluble in some water miscible organic solvents like acetone
and acetonitrile, which makes the use of co-solvents indispensable e.g. in the
nanoprecipitation process.
The presence of ester linkages in the polymer backbone (Fig. 1) allows hydrolytic
degradation of the polymer in an aqueous environment [39, 40]. The degradation products
are biocompatible and metabolizable; they are removed from the body by the citric acid
cycle. The degradation rate is dependent on several parameters such as crystallinity
(crystalline PLA degrades slower), MW (low MW PLAs degrade faster) environment (pH, ionic
strength, temperature) and particle morphology (porous particles degrade faster) [38, 41,
42]. At neutral pH (in vitro liquid environment without enzymes), PLA nanoparticles [41] and
microparticles [38] remain relatively stable over tens of days. On the contrary, polyester
microparticles are reported to degrade substantially faster when injected subcutaneously or
intravenously to rats compared to in vitro tests in buffer solutions [43, 44]. Also in gastric and
intestinal fluids, degradation of D,L-PLA nanoparticles in vitro [45] and in vivo [46], and PLGA
nanoparticles in vivo [47] was fast due to enzymatically catalyzed hydrolysis. Drugs
encapsulated in the particles are known to affect the degradation. Both acidic and basic
drugs may catalyze the degradation. For example, basic drugs can catalyze ester linkage
Review of the literature
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scission [48, 49] but, on the other hand, they may also neutralize the carboxylic acid chain
end groups of the polymer and slow down the hydrolysis [50].
2.1.2 Nanoprecipitation
In nanoprecipitation, introduced by Fessi and co-workers [51], the particle formation is based
on precipitation and subsequent solidification of the polymer at the interface of a solvent
and a non-solvent. Thus, the process is often called solvent displacement or interfacial
deposition. The polymer is dissolved in a water miscible organic solvent (or solvent mixture)
and added to an aqueous solution, in which the organic solvent diffuses (Fig. 2). Particle
formation is spontaneous, because the polymer precipitates in the aqueous environment.
According to the current opinion, the Marangoni effect is considered to explain the process
[28]: solvent flow, diffusion and surface tensions at the interface of the organic solvent and
the aqueous phase cause turbulences, which form small droplets containing the polymer.
Subsequently, as the solvent diffuses out from the droplets, the polymer precipitates. Finally,
the organic solvent is typically evaporated with the help of a vacuum. No emulsification step
(which is usually part of a nanoparticle preparation process), laborious processing conditions
or special laboratory ware is needed. The size of the nanoparticles prepared by
nanoprecipitation varies typically from 100 to 500 nm.
Figure 2. Schematic illustration of the nanoprecipitation process.
Usually, surfactants or stabilizers are included in the process to modify the size and the
surface properties, or to ensure the stability of the nanoparticle dispersion (especially during
the early stages of the precipitation). However, presence of surfactants/stabilizers is not
indispensable for the formation of the particles. The drug substance to be encapsulated is,
depending on its solubility, dispersed as an aqueous solution or dissolved in the organic
Solventevaporation
Inner phase
Polymer and drugin solution
Outer phase
Precipitation of the polymer
Nanoparticles
Inner phase
Solventevaporation
Inner phase
Polymer and drugin solution
Outer phase
Precipitation of the polymer
Nanoparticles
Inner phase
Review of the literature
6
solvent before the fusion of the phases. The nanoprecipitation technique suffers from poor
encapsulation efficacy of hydrophilic drugs, because the drug can diffuse to the aqueous
outer phase during polymer precipitation [52]. By modifying the solubility of the drug by
changes in pH, the encapsulation has been increased [53-55]. For example, decrease of
solubility of procaine hydrochloride (pKa 8.8) by increasing the aqueous phase pH from 5.8 to
9.3. increased the drug entrapment from 11% to 58% [54]. Correspondingly, increased
encapsulation was achieved by decreasing the aqueous solubility of sodium cromoglycate
(pKa 2.0) by lowering the pH [55]. An accelerated precipitation rate of the polymer, modified
solvent composition and increase in the MW of the polymer are among the other means to
improve the encapsulation efficiency. Examples of different nanoprecipitated formulations
are listed in Table 1.
Table 1. Examples of polymeric nanoparticles prepared by the nanoprecipitation method.
Polymer Drug/study and reference
Eudragit L100-55 A study on the preparation variables [56]
Hydroxypropyl methylcellulose phthalate (HP55)
A study on the preparation variables [57]
Poly(D,L-lactic acid) Isradipine [58], tyrphostin [59], cloricromene [53], indomethacin [51, 60], diclofenac [61], atovaquone [62], acyclovir [63], clofibride [64], primaquine [65], docetaxel [66], a model poorly water-soluble crystalline drug [67], a study on the preparation variables [68, 69], a stability study [70]
Poly(L-lactic acid) Sodium cromoglycate [55, 71, 72]
Poly(D,L-lactic acid) -poly(ethylene glycol)
Procaine hydrochloride [73], a study on the preparation variables [74],
Poly(D,L-lactic-co-glycolic acid)
Isradipine [58], xanthone/3-methoxyxanthone [75], atovaquone [62], benzathine penicillin G [76], nifedipine [77], procaine hydrochloride [54], docetaxel [66], flurbiprofen [78], rose bengal [79], DNA plasmids [80], a study on the preparation variables [68], a stability study [70]
Poly(D,L-lactic-co-glycolic acid) - poly(ethylene glycol)
Docetaxel [81], paclitaxel [82]
Poly(D,L-lactic-co-glycolic acid) - poly(vinyl alcohol)
DNA [83]
Poly(sebaic acid) terminated with lithocholic acid and poly(sebaic acid-co-lithocholic acid)
A model poorly water-soluble crystalline drug [67]
Poly(ε-caprolactone) Cyclosporin A [12, 84], isradipine [58], atovaquone [62], primidone [85], Parsol MCX (a sunscreen agent) [86], griseofulvin [87], rhodamine (a fluorescent dye) [88], a stability study [70]
Poly(ε-caprolactone) -poly(ethylene glycol)
Rhodamine (a fluorescent dye) [88]
Review of the literature
7
Overall, the challenge in nanoprecipitation is to find a drug/polymer/solvent/non-solvent
combination, which allows successful nanoparticle production and drug encapsulation.
However, scale-up of nanoprecipitation from laboratory-scale to pilot-scale has been recently
reported [18].
2.1.3 Surface modification
Surface modification or coating by biocompatible (hydrophilic) polymers protects
nanoparticles against uptake of the MPS and enhances stability. Polymers for this purpose
include e.g. PEG, and the ethylene oxide / propylene oxide block copolymers poloxamers and
poloxamines [31, 79, 88-93]. Mucoadhesive coatings by polymers like chitosan, poly(acrylic
acid), sodium alginate and poloxamers improve bioavailability of the encapsulated drug by
prolonging the residence time of nanoparticles at the absorption site (mucus layer) [88, 94-
96]. Polysorbates and PEG on the particle surface are known to enable the transport across
the blood-brain barrier [15]. In addition, the drug release profile may be modulated by
surface modification.
Surface modifiers can be attached on the nanoparticles by adsorption (e.g. hydrophobic
insertion) [90-93, 97, 98], grafting via covalent bonds [31, 99, 100], or by electrostatic
interactions [32, 101, 102].
In layer-by-layer (LbL) coating, based on electrostatic interactions, polyelectrolytes (PEs)
are adsorbed sequentially on charged substrates (Fig. 3) [32, 103]. For example, PLA
nanoparticles are good substrates for LbL coating because of the carboxylic acid groups on
the particle surface (generally negatively charged in dispersion). In LbL coating, the
physicochemical properties of the resulting, coated nanoparticles can be controlled by the
conditions of assembly such as pH, ionic strength, number of layers and type of PEs. Examples
of polycations (positively charged PEs) applied to LbL coating are poly(allylamine
hydrochloride) (PAH), poly(ethylenimine) and chitosan, while poly(styrene sulfonate) (PSS),
dextran sulphate, poly(acrylic acid), chitosan sulphate, alginic acid and DNA molecules
represent polyanions (negatively charged PEs) [32, 101, 102, 104-109].
Figure 3. Sequential adsorption of polyelectrolytes on a charged particle.
+ +
+
+
+
+
__
_
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+
+
+
++
+
+
A positivelycharged particle
Addition of polyanion, incubation
Addition of polycation, incubation
+ +
+
+
+
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+
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A positivelycharged particle
Addition of polyanion, incubation
Addition of polycation, incubation
Review of the literature
8
The most central challenge in an LbL process is to remove the unadsorbed PEs among the
particles prior to the next layer. To overcome that, two methods have been suggested: (1)
utilization of exact amounts of PEs with regards to the particle surface charge [32]; (2)
intermediate removal of excess PEs by ultracentrifugation [32, 105, 110], or ultrafiltration
[101] before each new layer. Determination of exact amounts might be difficult, and in some
cases nanoparticle structure does not tolerate the forces of ultracentrifugation. However,
once the processing conditions are optimized, the coating process can possibly be continued
to tens of layers. Several LbL coating studies have been carried out using non-biodegradable
polymers such as PAH and PSS [32, 101, 107, 109]. In the future, biocompatible and functional
PEs are needed to develop surface-modified nanoparticles with MPS-avoidance and
controlled release properties.
2.1.4 Freeze-drying
Briefly, in freeze-drying (lyophilization), solvent (e.g. water) in the system is frozen followed
by its sublimation in vacuum. At present, freeze-drying is the method of choice to remove
water from the system in nanoparticle formulations [12, 111-120]. However, freeze-drying of
nanoparticles is a complicated process because the stability of the colloidal nanoparticle
dispersion is, in most cases, disturbed, leading to aggregation before the particulate
population is dry. Several variables affect the final result: freezing rate and temperature,
drying temperatures, pressure, use of excipients like bulking agents or cryo- and
lyoprotectants, and changes in pH or ionic strength. After successful drying, one should be
able to re-disperse particles in their initial dispersing medium without causing significant
changes in their size and drug content.
Freezing (temperature and rate) is the most crucial part of the process for particle
survival. Freezing temperature affects the rate, e.g. if the product is frozen with the help of
liquid nitrogen (fast freezing), but usually the freezing rate can be controlled. In general
terms, slow freezing rates lead to a smaller number of large ice crystals and high freezing
rates lead to a larger number of small ice crystals. It is possible that large ice crystals destroy
the particles mechanically, which promotes the use of faster freezing [112]. On the contrary,
in another study, slow freezing enabled survival and efficient drying of poly(caprolactone)
nanoparticles [114], which could be explained on the basis of the more porous and
permeable structure (for the water vapor) of the slowly frozen material [121]. In addition to
the freezing protocol, changes in the dispersant phase may cause particle instability. During
freezing, the nanoparticle concentration of the non-frozen phase is increased (more
interactions between the particles) accompanied by possible changes in ionic strength and
pH, which may induce aggregation.
Appropriate protective excipients are indispensable for a successful freeze-drying process,
and the choice of the excipients has to be evaluated for each different nanoparticulate
composition [12, 111, 115, 116, 119, 120]. In the case of poly(caprolactone) nanoparticles,
when optimal amounts of protectants were applied, even the freezing rate did not
significantly affect the result [122]. Cryoprotectants protect the product during freezing,
whereas lyoprotectants provide stability during the drying step. Glycine, sucrose, glucose and
poly(vinyl pyrrolidone) are cryoprotectants, while disaccharides such as trehalose, sucrose and
Review of the literature
9
lactose act usually as lyoprotectants [123-125]. The protective mechanism of disaccharides is
understood to originate from an amorphous matrix, which is formed when the sugar is in
contact with water [126]. Protection arises, when the amorphous matrix forms hydrogen
bonds with the nanoparticulate product and act as a water substitute inhibiting the
destructive effect of ice (water) crystals. Therefore, it is essential to maintain the amorphous
material in the glassy state (below the glass transition temperature, Tg), to avoid
crystallization of the sugar. For example, the Tg of water-plasticized trehalose is
approximately -30°C [126], which is higher than Tgs of most of the other sugars (disaccharides
and monosaccharides). Thus, the use of trehalose enables a wider temperature scale for
drying.
Examples of successfully freeze-dried nanoparticles are presented in a comprehensive
review article [127].
2.2 Nanoparticle characterization
Colloidal systems like nanoparticles differ from macroscopic objects because of sub-micron
properties such as high surface area and energy, and movement of the particles by diffusion
(Brownian motion). The different behavior of nanoparticles leads, to some extent, to the use
of a different pattern of characterization methods. Extensive characterization of a
nanoparticle system is essential for understanding and prediction of the performance of the
system in the body. As the field of pharmaceutical nanoparticles is evolving constantly, the
need for more thorough characterization and comprehensive understanding of the systems
increases.
Size, morphology and physical state of the encapsulated drug as well as MW and
crystallinity of the polymer influence drug release and degradation of the nanoparticles.
Meanwhile, size, surface charge and hydrophobicity/hydrophilicity are parameters that affect
the body distribution and interactions with the biological environment. Stability of
nanoparticles is also a general issue governing the above mentioned properties. In the
following chapters, some common characterization methods of pharmaceutical nanoparticles
are presented.
2.2.1 Size, morphology, surface properties and stability
Conventional light microscopy is not suitable for nanoparticle characterization because its
resolution is limited to about 1 µm. Instead, techniques for the characterization of
nanoparticle size and morphology are scanning electron microscopy (SEM) [53, 56, 58, 68, 80]
(Fig. 4) and transmission electron microscopy (TEM) [51, 54, 59, 64, 69, 74, 75, 99]. Nowadays,
nanoparticles are usually studied with 5 000 – 30 000-fold magnifications. In a SEM setup, the
nanoparticulate sample, coated to be conductive (e.g. platinum), is scanned in a high vacuum
chamber with a focused electron beam [128]. Secondary electrons, emitted from the sample,
are detected and the image formed. The environmental scanning microscope (ESEM) allows
working even with moist samples, without conductive coatings and high vacuum. In TEM,
Review of the literature
10
electrons scattered by the sample are detected; the sample is between the electron gun and
the detector [129]. Another technique in pharmaceutical nanoparticle characterizations is
atomic force microscopy (AFM) (scanning probe microscopy) [101, 130-133].
Photon correlation spectroscopy (PCS), a technique based on dynamic (laser) light
scattering, is widely used in size determinations of nanoparticles [54, 56, 58, 59, 64, 67, 70,
73, 77, 79, 80]. PCS measures the intensity variation (because of the Brownian motion of
nanoparticles) of scattered light, and relates it to the particle size with the help of an
autocorrelation function [134]. As a result, a hydrodynamic diameter is obtained (PCS
presumes all the particles being spherical). If the nanoparticle population is not
monodisperse, the data can be further processed with the help of algorithms to give size
distributions. PCS is a fast technique, sensitive to nanoscale particles, and provides
information about the whole particulate population. On the other hand, the dispersion has
to be diluted, filtered and the results are based on mathematical calculations (dispersant
viscosity, temperature and refractive index should be known). Therefore, one should be
vigilant when interpreting size information from PCS experiments. Instead, SEM/TEM
provides visual and descriptive information, a real overview about the nanoparticle
population. However, this information is not usually quantitative (size distributions). As a
conclusion, micrographic images (electron microscopy) and a computational technique (light
scattering) should be used together in the initial size determinations.
Figure 4. An SEM image of poly(styrene) nanoparticles with the size of 460 nm, 5 000-fold magnification.
Surface charge of nanoparticles, for its part, determines the performance of the
nanoparticle system in the body, e.g. interactions with cell membranes. Zeta (ζ) potential
measurements provide information about the particle surface charge [135]. ζ-Potential is the
charge at the electrical double layer, created by ions of the liquid, which exists around each
particle. The mobility of charged particles is determined with the help of electric potential
(electrophoretic mobility) and transferred to ζ-potential, e.g. with the help of
Smoluchowski’s equation. ζ-Potential is a function of conditions of the dispersing medium
such as pH and electrolyte concentration [136]. E.g. polystyrene nanoparticles are found to
exhibit the most extreme ζ-potential values at very low electrolyte concentrations [137].
Correspondingly, carboxylic acid groups at the PLA nanoparticle surface are well ionized at
basic pH which decreases the ζ-potential value. ζ-Potential can also be altered by surface
Review of the literature
11
modification [32] or stabilizer concentration [138]. ζ-Potential measurements are useful e.g.
in the LbL coating to verify the success of the process [32, 101].
Nanoparticles dispersed in aqueous solutions can be stabilized either by electrostatic
stabilization (surface charge) or by steric stabilization (surfactants or other molecules at the
particle surface), or by a combination of both [139, 140]. ζ-Potential represents a measure of
an electrostatically stabilized colloidal dispersion: adequately high ζ-potential absolute values
provide stability for nanoparticle dispersion. Values beyond +/-30 mV are considered
characteristics for a stable colloidal dispersion [141]. According to the DLVO theory,
aggregation occurs when attractive van der Waals forces between the particles become
dominant. This loss of stability leads in an increase in polydispersity (detected e.g. by PCS)
and, finally, in visual cloudiness. Aggregation is usually determined by turbidity
measurements (ultraviolet (UV) spectroscopy); increasing turbidity indicates decreased
stability [73, 105, 142].
The amount of the functional, charged groups on the surface of nanoparticles can be
determined by e.g. acid number titration [143] or by conductometric titration [144].
Hydrophobicity of nanoparticles is characterized by hydrophobic interaction chromatography
(HIC): hydrophilic particles pass the column faster while elution of hydrophobic particles is
retarded [92, 98, 99, 145]. As an example of another technique, coating of polystyrene and
PLA nanoparticles by a Vitamin E derivative [131, 146], and PLA nanoparticles by PEG [147]
were verified by x-ray photoelectron spectroscopy (XPS). This technique, also known as ESCA
(electron spectroscopy for chemical analysis), reveals the elemental and chemical composition
at the surface. Other surface chemistry analysis methods include Fourier-transform infrared
spectroscopy (FTIR) [148] and nuclear magnetic resonance spectroscopy (NMR) [149].
2.2.2 Drug-polymer interactions
Drug loading can be performed during the preparation of nanoparticles or by
adsorbing/absorbing in preformed particles. Within the particle-forming polymer, drug can
be present as a solid solution (individual drug molecules) or as a solid dispersion
(amorphous/crystalline drug). It can be adsorbed on the particle surface [150] or bound
chemically within the nanoparticles [151]. The preparation process can also modify the crystal
structure of the drug. The polymer is usually amorphous or semi-crystalline. Differential
scanning calorimetry (DSC), (powder) x-ray diffractometry (XRPD) and FTIR are commonly
used techniques to reveal the physicochemical state and possible interactions of the drug and
the polymer in pharmaceutical micro- and nanoparticles. Polymer MW is determined e.g. by
size exclusion chromatography (SEC) [58, 70] (the term gel permeation chromatography
(GPC) is interchangeably used [61, 152]).
DSC detects phase transitions such as glass transition, (exothermic) crystallization and
(endothermic) melting: the nanoparticle sample is heated and changes in heat flow,
compared to reference, are registered [153]. Crystallinity/amorphicity properties are obtained
from XRPD analysis when diffraction pattern of the x-ray from the sample is determined as a
function of scattering angle [154]. In FTIR, a vibrational spectrum, characteristics for a given
crystal structure, is obtained [155].
Review of the literature
12
Absence of the drug melting peak and diffraction peaks of the crystal structure of the
drug in DSC thermogram and XRPD pattern, respectively, are usually signs of amorphous or
molecularly dispersed drug within the polymer [156-164]. It can also indicate that the amount
of drug is lower than the detection limit of the instrument [50, 160, 161, 165, 166]. Drug-
polymer interactions (e.g. plasticizing effect of drug on polymer) or polymorph change of the
drug can be detected as peak shifts in DSC thermogram, band shifts in FTIR spectra or as new
reflections in XRPD pattern [162, 166-169]. Correspondingly, smoothened XRPD pattern,
increased cold crystallization exotherms (DSC) or some band shifts to higher wavenumbers
(FTIR) indicate increased amorphicity of the polymer [167].
2.2.3 Drug loading and release
Drug entrapment or encapsulation efficiency is a percentage value that describes the
quantity of the drug material in the nanoparticles out of the total amount used in the
process. The drug content (or drug loading) percentage is the drug amount compared to the
nanoparticle mass. Because of the colloidal nature of nanoparticles, determination of drug
encapsulation, drug loading and in vitro release may be problematic. However, the
encapsulation is determined e.g. by separating nanoparticles from the dispersion medium by
ultracentrifugation or ultrafiltration. Drug content is then quantified from the supernatant
or after solvation of the nanoparticle pellet [12, 64, 80, 84, 88]. In the case of drug
quantitation after freeze-drying, the polymer and drug are first dissolved [54, 63, 73, 94,
170]. For example, PLGA nanoparticles containing procaine hydrochloride were dissolved in
acetonitrile and the drug was quantitied from the solution by UV spectroscopy, because the
polymer was found not to exhibit absorbance interference at the wavelengths used [54]. In
another case, D,L-PLA nanoparticles containing acyclovir were dissolved in dichloromethane
followed by extraction of the drug by an aqueous buffer [63]. The drug quantitation was
performed by high performance liquid chromatography (HPLC).
Drug release from nanoparticles may occur by diffusion through the particle, by
desorption from the surface or after degradation. Although the drug release environment in vivo is complex and may be difficult to simulate, important information about the release in vitro can be collected by several techniques. Ultracentrifugation is often exploited [63, 79, 85,
91, 94, 99, 170]. E.g. D,L-PLA nanoparticles containing savoxepine were stirred in a buffer
solution [91]. At predetermined intervals, a sample was withdrawn and ultracentrifugated.
Subsequently the drug content was analyzed from the supernatant by UV spectroscopy.
Similarly, the withdrawn sample can be treated by ultrafiltration [54, 171]. In a dialysis setup,
nanoparticles are placed either in small dialysis bags in a stirred receptor (aqueous) medium
[53, 58, 86] or in the medium containing drug-free dialysis bags (reverse dialysis) [64, 75, 88].
Because the nanoparticles do not permeate the dialysis bags, the released drug is
quantitated from the compartment not containing the particles. In addition to dialysis bags,
diffusion cells separated with a membrane provides a possibility to monitor the drug
diffusion (excluding particle diffusion) from one compartment to another [172, 173].
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13
2.3 Nanoparticles in drug delivery applications
While the research in the field on pharmaceutical nanoparticles is extensive (reviewed e.g. in
[20, 21, 152, 174-177]), introduction of commercialized products has not yet occurred. At the
same time, several microparticulate drug delivery systems prepared from the same
biodegradable materials are already on the market. Using as an example the polymer used in
this study, PLA, and its copolymers with glycolic acid (PLGA), marketed microparticulate
systems are listed in Table 2. These products offer mostly extended duration of therapeutic
effect when injected into tissue (intramuscularly, subcutaneously). Instead, research in the
field of biodegradable nanoparticles has concentrated on the formulation of systems that
take advantage of their smaller size. The following paragraphs present examples of recent, in vivo tested nanoparticulate drug delivery applications based on PLA and PLGA polymers.
Table 2. Examples of marketed PLA and PLGA microparticulate drug delivery systems.
Product Drug / polymer Indication Company
Arestin® minocycline / PLGA periodontitis, powder administered into periodontal pocket
OraPharma
Decapeptyl® Depot triptorelin / PLGA prostate cancer, edometriosis, i.m.
Ferring
Decapeptyl® SR triptorelin / PLA, PLGA prostate cancer, endometriosis, i.m.
Ipsen
Lupron Depot® leuprolide / PLA, PLGA prostate cancer, endometriosis, i.m.
TAP
Nutropin® human growth hormone / PLGA
treatment of growth failure, s.c.
Genetech
Risperdal® Consta™ risperidone / PLGA schizophrenia, i.m. Janssen-Cilag
Sandostatin® LAR octreotide / PLGA-glucose acromegaly, i.m. Novartis
Somatuline® PR lanreotide / PLGA acromegaly, i.m. Ipsen
Suprecur® MP buserelin / PLGA endometriosis, i.m. Sanofi-Aventis
Trelstal™ LA / Depot triptorelin / PLA prostate cancer, i.m. Pfizer
Vivitrol® naltrexone / PLGA alcohol depence treatment, i.m.
Alkernes
i.m. = intramuscularly injected; s.c. = subcutaneously injected
After oral administration, nanoparticles (usually in suspension) are uptaken and
transported across the mucosal epithelium by enterocytes or the M cells of Peyer’s patches
because of their small size [16]. Before reaching these locations, nanoparticles protect the
active ingredient in the gastro-intestinal (GI) tract and/or prolong the residence time of its
contents on the mucous membrane. For example, oral absorption of peptides such as salmon
calcitonin (osteoporosis treatment) using PLGA nanoparticles [178] and elcatonin using
chitosan-coated PLGA nanoparticles [94] have been reported. Similarly, improved oral
bioavailability (sustained release) of e.g. mifepristone [179] and estradiol [47] loaded in PLGA
Review of the literature
14
nanoparticles has been shown due to the enhanced mucoadhesion. The animals used in these
studies were rats. Delivery of antigens, such as tetanus toxoid with PLA-PEG nanoparticles
[106], ovalbumin with PLGA-PEG nanoparticles [180] and bovine serum albumin (BSA) with
PLGA nanoparticles [181] has led to better immunization responses because of the targeted
uptake in the M cells of the test animals (mice). These kinds of systems are promising for oral
vaccination.
When nanoparticles have reached the blood circulation, e.g. after intravenous injection
or absorption from the GI tract, they are rapidly recognized and uptaken as foreign objects
by macrophages. Therefore, strategies to escape the clearance mechanism and thus to
improve the in vivo biodistribution are developed. Most of these approaches are based on
nanoparticle surface engineering. Several examples can be found in the field of tumor/cancer
treatment. Due to the prolonged time in blood circulation, e.g. camptothecin-loaded
PEG/poly(propylene glycol)-coated PLA nanoparticles prolonged the mean residence time of
the drug in mice to 25 hours (compared to 0.6 h of the free drug) [182]. Paclitaxel-loaded
methoxy-PEG-PLA nanoparticles provided approximately 3-fold longer half life and effective
concentration of the drug in rats, without concentration peaks above toxic level compared to
a conventional formulation [82]. On the other hand, sustained drug release by nanoparticles
may not always be a key factor determining the drug efficacy: PEG-PLA nanoparticle-
delivered doxorubicin has performed similar anti-cancer effects in mice compared to the free
doxorubicin [183]. However, reduced toxicity of the drug was emphasized also in this case.
Targeting of drugs by the natural body distribution of nanoparticles is thought to be an
alternative to treat tumors in fenestrated and phagocytically active tissues such as liver, lung
and spleen. For example, oridonin-loaded PLA nanoparticles have maintained the drug
concentration high in these tissues of mice (36 h) compared to the free drug (0.5 h) [13].
Recently, tumor targeting, independent of body distribution, has been reported: paclitaxel-
loaded poly(vinyl alcohol)-PLGA nanoparticles have been successfully targeted to prostate
tumors of mice with the help of an RNA aptamer (binds to the tumors) on the particle
surface [81].
Another studied application of nanoparticles is drug delivery across the blood-brain
barrier (BBB). For that purpose, the particles should be coated by suitable surfactants (e.g.
Polysorbate 80, PEG) [15]. Thus far, most of these studies have been carried out with
poly(alkylcyanoacrylate) nanoparticles [184]. Brain localization has also been achieved using
the probably safer poly(lactic acids): intranasally administered (in rats) PLA-PEG nanoparticles
functionalized by lectin [185], intravenously injected (in mice) PLA-PEG nanoparticles bearing
cationic BSA coating [186], PLA-polysorbate 80 nanoparticles (in mice) [187], and PLGA-
peptide nanoparticles (in rats) [188]. Recently, loperamide has been delivered to the central
nervous system of rats with sub-200 nm PLGA-peptide nanoparticles and sustained effect of
the drug was observed [189]. However, the mechanisms of particle transport across the BBB
are not entirely clarified; several different processes are probably involved depending on the
nanoparticle characteristics [15].
In gene delivery studies, DNA is coupled to nanoparticles to create non-viral vectors
protecting the DNA in body conditions. A size of around 100 nm and the positive charge of
the particles facilitate the delivery and interactions with the cell membrane, respectively. For
example, orally administered PLA-PEG nanoparticles have protected the encapsulated DNA
and resulted in high efficiency transfection in mice [190]. With chitosan coating on the
Review of the literature
15
particles, the gene expression was even more pronounced. Intramuscularly injected PLGA
nanoparticles in mice have been reported to release DNA in a prolonged manner which
promoted the antigen presentation [191]. Increasing the amount of DNA encapsulated in the
particles, while maintaining the small nanoparticle size, is one of the key challenges in
nanoparticulate gene delivery applications.
Nanoparticles with mucoadhesive properties can improve ocular drug delivery by
prolonging the residence time of the drug in the tear film, controlling release and reducing
irritation after topical administration. Because of these benefits, e.g. flurbiprofen (an anti-
inflammatory drug) encapsulated in PLGA nanoparticles showed higher anti-inflammatory
effect than the corresponding commercial eye-drops [78]. Similarly, acyclovir-loaded PEG-PLA
nanoparticles provided enhanced ocular bioavailability compared to the free drug: a 12.6-
fold increase in the total drug concentration was detected and the concentration remained
at an effective level three times longer [63]. These experiments were performed using
rabbits.
Despite the successful in vivo tests with animals, the relevance of these findings to human
body conditions as well as long-term effects of the formulations is uncertain. However,
biodegradable colloidal drug delivery systems seem to provide promising treatment for
various diseases using different administration routes.
Aims of the study
16
3 Aims of the study
The purpose of this study was to prepare nanoparticles from poly(lactic acid) polymer and to
characterize physicochemical properties of the starting materials and the nanoparticles by
different methods. The aim was to evaluate and understand the effect of different process
conditions and environments on the nanoparticle characteristics.
Specifically, the following themes were examined:
• Effect of the nanoparticle preparation method, nanoprecipitation, on the
physicochemical properties of the polymer and the encapsulated drugs
• Application of capillary electrophoresis, a novel method in the field of
biodegradable pharmaceutical nanoparticles, in the quantitation of the
encapsulated drugs
• Evaluation of stability and aggregation behavior of the prepared nanoparticles
with the help of surface pressure measurements – an alternative method to
characterize pharmaceutical nanoparticles
• Improvement of the freeze-drying process of the nanoparticles by the use of cryo-
and lyoprotectants
• Introduction and development of a layer-by-layer coating process for poly(lactic
acid) nanoparticles
Materials and methods
17
4 Experimental
4.1 Materials
Materials used in this work and references to the corresponding publications are summarized
in Table 3. All other used materials were of analytical grade.
Table 3. Materials used in this work.
Material Publication Source
Acetone II, III Riedel-de-Haën, Seelze, Germany
Beclomethasone dipropionate (BDP) I, II Sigma Chemical Co., St. Louis, USA
Chloroform (CHL) I, IV, V Riedel-de-Haën, Seelze, Germany
D-glucose IV Fischer Scientific UK Ltd., Leicestershire, UK
Dichloromethane (DCM) I, V Riedel-de-Haën, Seelze, Germany
Ethanol (96% v/v) I, IV, V Primalco, Rajamäki, Finland
Hydrochloric acid (HCl) III, V Shannon Co., Clare, Ireland
Lactose IV DMV International, Veghel, The Netherlands
Paper filters (11 µm) II-V Whatman, Brentford, UK
Poloxamer 188 (Lutrol® F 68) III BASF, Ludwigshafen, Germany
Poly(allylamine hydrochloride), MW 70 000 g/mol (PAH)
V Sigma-Aldrich, Steinheim, Germany
Poly(L-lactic acid) (L-PLA), MW 2 000 g/mol I, IV, V ICN Biomedicals Inc., Aurora, USA
Poly(D,L-lactic acid), IV 0.20 dl/g (D,L-PLA) II, III PURAC Biomaterials, Gorinchem, The Netherlands
Poly(sodium 4-styrenesulfonate), MW 70 000 g/mol (PSS)
V Sigma-Aldrich, Steinheim, Germany
Polycarbonate membrane filters (0.2 µm) III, V Millipore, Molsheim, France
Polysorbate (Tween) 80 IV Fluka Chemie, Buchs, Switzerland
Propylene glycol (PG) I, II, IV, V YA Kemia, Helsinki, Finland
Salbutamol sulphate (SS) I, II A donation from Orion Pharma, Espoo, Finland
Sodium chloride (NaCl) III, V Riedel-de-Haën, Seelze, Germany
Sodium cromoglycate (SCG) II, IV ICN Biomedicals Inc., Aurora, USA
Sodium hydroxide (NaOH) III, V Shannon Co., Clare, Ireland
Water (ultrapurified) I-V Millipore, Molsheim, France
Materials and methods
18
4.2 Methods
Experimental equipment and methodology used in this work together with references to the
corresponding publications are summarized in Table 4. Detailed descriptions of the methods
are given in the original publications or references therein.
Table 4. Methods and equipment used in this work.
Method / equipment Publication Equipment
Capillary electrophoresis (CE) II 3DHP CE equipped with a photodiode array detector (Hewlett-Packard, Waldbronn, Germany)
Differential scanning calorimetry (DSC) I, V DSC822e with STARe software (Mettler Toledo, Columbus, USA)
Fourier transform infrared spectroscopy (FTIR)
I Spectrum One with Universal ATR Sampling Accessory (Perkin-Elmer, Boston, USA)
Freeze-drying IV HETO LyoPro 3000 (Heto-Holten A/S, Allerød, Denmark)
Layer-by-layer coating (LbL) V Vacuum filtration equipment (Millipore, Molsheim, France)
Photon correlation spectroscopy (PCS) II-V Zetasizer 3000HS (Malvern Instruments, Worcestershire, UK)
Scanning electron microscope (SEM) I, III-V Agar Sputter Coater (Agar Scientific Ltd., Essex, UK) and DSM 962 (Zeiss, Jena, Germany)
Surface pressure measurements III KSV Minitrough (KSV Instruments, Helsinki, Finland)
X-ray powder diffractometry (XRPD) I Bruker axs D8 (Bruker, Karlsruhe, Germany)
ζ-Potential measurements III-V Zetasizer 3000HS equipped with MPT-1 titrator (Malvern Instruments, Worcestershire, UK)
Materials and methods
19
4.2.1 Preparation, coating and freeze-drying of nanoparticles
Nanoparticles were prepared either by the nanoprecipitation method (II, III) [51] or by a
modified nanoprecipitation method (I, IV, V) [72]. In the nanoprecipitation method, 25 mg
of D,L-PLA was dissolved in acetone (2 ml). If beclomethasone dipropionate (BDP) was
included in the process, it was dissolved with the polymer whereas salbutamol sulphate (SS)
and sodium cromoglycate (SCG) were first dissolved in water (0.15 ml) before mixing the
solutions together. This inner phase was then added into the outer phase, 4 ml of water,
with a syringe and a gauge. If Poloxamer 188 was included in the formulation, it was
dissolved (20 mg) in the outer phase. In the modified method, 25 mg of L-PLA together with
175 mg of propylene glycol (stabilizer) were dissolved in 2 ml of chloroform (CHL) or
dichloromethane (DCM). In the formulations containing BDP, the drug was dissolved with the
polymer and the stabilizer. In the case of SS and SCG, the drug was dissolved in 0.15 ml of
water and 0.7 ml of 96% ethanol was added (a co-solvent). In all the formulations prepared
by the modified method (drug or no drug), the polymeric solution was mixed to the water-
ethanol solution. The formed dispersion was added dropwise to 5 ml of 70% ethanol
solution under mild stirring. In the both methods (I-V), the organic solvents were evaporated
in a fume cupboard at room temperature. Prior to filtration through a paper filter, the
dispersions were diluted with water or NaCl solution (Zetasizer analysis and LbL coating), or
with isopropanol (surface pressure experiments).
LbL coating (V) was performed on a membrane using a vacuum filtration setup (Fig. 1 in
V). 10 ml of nanoparticle dispersion in 0.02 M NaCl, stirred gently throughout the process,
was used in the coating experiments. PEs were dissolved in 0.02 M NaCl (1 mg/ml). Particles
were exposed to each PE solution, 200 µl, for 5 min followed by two washing cycles: vacuum
filtration until the volume of the dispersion was about 2 ml followed by expansion of the
volume to 10 ml by 0.02 M NaCl. A weak overpressure under the membrane together with
gentle brushing was applied to detach the particles from the membrane after filtration. The
membrane was replaced by a new one after each coating layer.
In freeze-drying (IV), the protective excipients glucose (50-400 mg) and lactose (100-300
mg), as aqueous solutions, were added to the nanoparticle dispersions after evaporation of
the organic solvents. The nanoparticle dispersions were frozen at -32 °C for a minimum of 12
h and freeze-dried at -55 °C and 0.5 kPa for 24 h. Additional freeze-thawing (freezing and
subsequent melting at room temperature) experiments were conducted to evaluate the
performance of the protectants during the freezing step.
4.2.2 Characterization of nanoparticles
ζ-Potentials and size distributions were determined with Zetasizer (II-V). Electrophoretic
mobilities were converted to ζ-potentials using Smoluchowski’s equation. In PCS, the results
were analyzed by the CONTIN algorithm and the sizes were presented as intensity
distributions.
Materials and methods
20
Surface pressure versus surface area (π vs. A) isotherms on different aqueous solutions
(subphases) were recorded by the Wilhelmy plate technique using a Langmuir trough (III). 40-75 µl of the particle dispersion was spread dropwise on the subphase surface with a
Hamilton microsyringe. The surface was compressed at a speed of 10 mm/min. For the SEM
analysis, particle populations were deposited on trichloro octadecyl silane -funtionalized
silicon plates by touching the surface with a horizontal plate (Langmuir-Schaefer deposition).
Other SEM samples of nanoparticles were prepared by depositing a drop of particle
dispersion (I, IV) or by attaching (with two-sided tape) a piece of a filter membrane
containing the particles (V) on a metal plate. The metal and silicon plates were sputtered for
20 s with platinum prior to the SEM analysis.
Drug encapsulation in the nanoparticles was determined by capillary electrophoresis (CE)
(II). The samples were injected and analyzed in fused-silica capillaries. In the case of SS and
SCG, the nanoparticles (in water), filtrates or water solutions obtained after particle
decomposition with chloroform were detected at a wavelength of 200 nm (UV detection).
For BDP, the nanoparticles as such (in water), and the filtrate (particles separated by filtration
through 100 nm membrane filter) or dispersing medium of the particles (methanol,
microemulsion or aqueous sodium dodecyl sulphate solution) were detected at a wavelength
of 254 nm (UV detection).
Before DSC experiments (I, III), dispersions were allowed to dry and weighed (2-4 mg) in
aluminium pans. Powder samples were used as such. The pans were sealed with aluminium
caps. The samples were heated at the rate of 10 °C/min. Prior to 2nd scan (I), the samples were
cooled down after the 1st scan to -10 °C at a rate of 20 °C/min. The locations of the thermal
transitions and surface areas of the peaks (V) were obtained from the thermograms.
Similarly, dry powder samples were also used in FTIR experiments (I). Samples for XRPD analysis (I) were prepared by depositing and drying the nanoparticle
dispersions on silicon plates. Powder samples were analyzed as such. The experiments were
performed in symmetrical reflection mode. The scattering was recorded from 5° to 30° with
steps of 0.1° (20 s per step). Crystallinity of the samples was estimated by comparing the
intensities of crystalline and amorphous samples. The single crystal data of the Cambridge
Structural Database (CSD) was used in the identification of crystal structures.
The success of the freeze-drying (IV) was also evaluated by visual inspection of the dried
formulations (volume and porosity of the cakes), as well as by redispersibility of the
nanoparticles. The cake was redispersed in 10 ml of water or 2.5 ml of aqueous Tween 80
solution (1.2 mg/ml). Aggregate formation and the Tyndall effect in the redispersions were
observed.
Results and discussion
21
5 Results and discussion
5.1 Characteristics of the nanoparticles
5.1.1 Preparation of the nanoparticles
Generally, the PLA nanoparticles prepared were smooth and spherical (Fig. 5). The
polydispersity indices obtained from the PCS measurements were around 0.1 or lower
indicating narrow deviations in sizes.
Figure 5. Examples of PLA nanoparticles. SEM images of empty nanoparticles prepared with DCM (left) or CHL (middle) as solvents for L-PLA, and SS containing L-PLA nanoparticles prepared with CHL as a solvent (right).
Low MW polymers were selected for this study because of their faster biodegradation
compared to higher MW polymers (MWs well above 10 000 g/mol) [38, 41]. Fast degradation is
desirable if the retention of the polymer in the body is unfavourable, e.g. after pulmonary
delivery. However, production of particles from low MW PLA sets up certain challenges
compared to higher MW polymers. Low MW PLA has higher solubility in some commonly used
organic solvents and, therefore, precipitation of the polymer occurs more slowly and more
solvent diffusion is needed to precipitate the polymer during preparation [143, 165]. The
surface of the low MW PLA nanoparticles is reported to be more adhesive than that of the
particles from polymers with higher MW, which causes an aggregation tendency [164]. The
particle yield has also been reported to decrease as the water soluble fraction was increased
by the addition of low MW PLA in the composition [143].
The L-form of PLA was selected for this study (I, IV, V) in order to evaluate the properties
of this semi-crystalline polymer with drugs and in different processes. Moreover, the L-PLA
polymer, compared to the D,L-PLA polymer, is seldom used in nanoparticulate applications.
Preparation of the nanoparticles from L-PLA was performed by a method optimized for this
particular polymer [71, 72] because of the solubility limitations of the semi-crystalline L-form:
it is soluble only e.g. in CHL or DCM. A co-solvent, ethanol in this case, was needed to
increase the mutual miscibility between the solvent of the polymer and the outer aqueous
phase. The nanoparticles were prepared using either CHL (IV, V) or DCM (I, V) as solvents for
Results and discussion
22
L-PLA. The (empty) particles using DCM were slightly smaller, approximately 350 nm
compared to 400 nm in the case of CHL (V). Solubility of PLA polymers is higher in DCM than
in CHL [192] and DCM has slightly higher miscibility in water [193]. This obviously led to
faster diffusion of DCM into the outer phase (70% ethanol), which created smaller polymer-
containing droplets ready to precipitate as smaller nanoparticles compared to the
formulations with CHL as a solvent. Correspondingly, the lower the interfacial tension is [69]
or, similarly, the interaction parameter [56, 81] between the organic solvent and the outer
phase, the smaller are the nanoparticles formed.
The nanoparticles from D,L-PLA (II, III) were prepared by the original nanoprecipitation
method [51] with or without a surfactant, Poloxamer 188. Nanoparticles with the surfactant
were larger than the surfactant-free nanoparticles (approximately 290 nm compared to 240
nm), probably due to surfactant adsorption on the particle surfaces (II). The D,L-PLA
nanoparticles were smaller than those prepared from the L-PLA polymer. Acetone, the
solvent used in the preparation in this case, is miscible in water in all proportions, which
affected the droplet size in the outer phase, polymer precipitation and, thus, led to smaller
particle size. The D,L-PLA nanoparticles were prepared by injecting the inner phase into the
outer phase using a fine gauge needle. In contrast to the dropwise addition, this approach
was expected to spread the organic solvent better into the outer phase as the density of
acetone is smaller than that of water [193].
When SS or SCG were encapsulated in the nanoparticles, the particle size was bigger (~50
nm difference) compared to the BDP-loaded or empty nanoparticles (Fig. 5). When the
phases were fused during the nanoprecipitation, water soluble drugs (such as SS and SCG) in
the inner phase created a solvent flow of the outer aqueous phase towards the inner phase
because of a concentration gradient (no drug initially in the outer phase) [194]. The osmotic
pressure thus formed increased the polymeric droplet size before polymer precipitation
leading to bigger particles.
Although the nanoprecipitation process is relatively simple to perform, the spontaneous
particle formation process involves complex interfacial phenomena that can be described as
outer phase/solvent, outer phase/polymer and solvent/polymer interactions [28, 51, 56]. In
more detail, the organic (polymer) phase viscosity, stabilizer, drug, solvent properties and
polymer properties (e.g. MW and ability to precipitate), among others, give their contribution
to the final particle properties. This variety enables speculative discussion concerning the role
of each parameter e.g. on the particle size, as was the case of the PLA nanoparticles in this
study.
5.1.2 Physical characterization of the polymer and the encapsulated drugs
The physical state of the PLA polymer and the encapsulated drugs was studied by DSC, XRPD
and FTIR (I, V). These analytical methods provide extensive information about the
interactions between the polymer and the drugs, as well as about the influence of the
nanoparticle preparation process on the materials. A selection of DSC thermograms
describing the behavior of the PLA polymers is presented in Figure 6 (also Fig. 4 in I and Fig. 6
in V). In the thermograms of L-PLA, an endothermic event at 152 °C is indicative for the
melting of the crystalline polymer. Correspondingly, the only detectable thermal event of the
Results and discussion
23
amorphous D,L-PLA is glass transition, which is located at 45 °C. Generally, the
nanoprecipitation process decreased the crystallinity of L-PLA because the polymer network
did not have enough time to be organized prior to the precipitation during the solvent
diffusion into the outer phase [167]. This could be observed as reappearance of the glass
transition and cold-crystallization (at 100 °C, crystallization of amorphous material) thermal
events (Fig. 6). The same observation could also be noticed from the XRPD data: the
calculated crystallinity proportion of L-PLA decreased from 59% to 39% as a consequence of
nanoprecipitation (Table 2 in I). The L-PLA nanoparticles prepared with CHL were more
crystalline than the particles prepared with DCM (V). Again, the solvent diffusion probably
explains the difference: slower diffusion of CHL to the outer phase during nanoprecipitation
left more time for the polymer to obtain favourable conformation for the crystallization.
Figure 6. Examples of the PLA thermograms.
Salbutamol sulphate and beclomethasone dipropionate were used as model drugs in the
evaluation of the nanoprecipitation process on the properties of L-PLA and the drugs (I). No
interactions between the drugs and PLA were detected. However, the physical state of the
components was affected. Effect of the nanoprecipitation was studied by increasing the
amount of the drug used in the process.
In the case of SS, although the amount of the drug (the drug being more crystalline than
L-PLA according to the XRPD studies) was increased with respect to the polymer, the relative
amount of the crystalline PLA remained high (Table 2 in I). As this was opposite to the
finding that the nanoprecipitation decreased the crystallinity of L-PLA, it seemed obvious
that the presence of SS enhanced the crystallinity of L-PLA. During the DSC experiments,
when these samples were cooled down after the thermogram registration, the polymer
exhibited a tendency to crystallize even though the cooling rate was 20 °C/min. In addition,
cold-crystallization exotherm of L-PLA was decreased during re-heating as the amount of SS
was increased, which indicated increased crystallinity. SS acted as plasticizer for L-PLA by
enhancing the polymer chain mobility and thus promoting crystallization tendency [195,
196]. Instead, although amine drugs such as SS are known to interact electrostatically with
carboxylic acids (chain ends of PLA) [197], no such interactions were found in this study.
0 20 40 60 80 100 120 140 160 180 200
Temperature (°C)
< E
nd
oth
erm
Exo
ther
m >
L-PLA
L-PLA nanoparticles
D,L-PLA nanoparticles
Results and discussion
24
When BDP was used as a model drug, it did not seem to modify the crystallinity of L-PLA
compared to the empty nanoparticles. BDP is reported to form solvates with a variety of
solvents such as alcohols [198] and halogenated hydrocarbons [199]. In this study, the
anhydrous BDP used was changed to monohydrate as a result of nanoprecipitation (Figs. 3 &
9 and Table 2 in I). BDP-containing samples were also treated by cooling and re-heating.
Contrary to the SS-PLA samples, neither the polymer nor the drug crystallized during the
cooling. The Tg and Tc of an amorphous material (a portion of L-PLA in this study) can be
increased by the addition of another amorphous compound having a higher Tg (BDP) [200].
Indeed, during the re-heating, the amorphous BDP increased the Tg and Tc of the polymer.
It should be noted that the crystallininty values calculated from the XRPD results were
estimations based on a mathematical model and the error marginal in the values was
approximately 10%. However, comparison of the values e.g. as a result of increased drug
amount provided useful information about the changes in the crystalline/amorphous nature.
FTIR results supported the findings of the DSC and the XRPD studies: no detected
interactions between SS and PLA and transformation of the anhydrous BDP to the
monohydrate form.
Overall, these findings indicated that the nanoprecipitation process influences the
physical properties of the polymer and the drugs. Even though the drug was molecularly
dispersed within PLA in the nanoparticles, the drug may modify the polymer and
nanoparticle properties. Also phenomena such as drug crystallization, affecting the drug
properties, may occur during storage or further processing.
5.1.3 Determination of drug content
In paper II, applicability of CE in quantitation of drugs encapsulated in the D,L-PLA
nanoparticles was estimated. CE is a versatile and sensitive technique for the separation and
determination of various substances [201].
A method for the determination of water soluble SS and SCG could be developed. The
results demonstrated that, with the loading protocol used, no drugs (SS or SCG) could be
found inside the nanoparticles at the detection moment; all the drugs used for the
preparation could be found outside the nanoparticles in the water phase. These results are
consistent with the observation that the entrapment of hydrophilic drugs inside
nanoparticles by the nanoprecipitation method is problematic [52]. No increase in the
encapsulation could be achieved when the solubility of SS (pKa 9.3) in aqueous outer phase
was decreased by increasing the pH of the phase from neutral to 8 (Fig. 4 in II). To
successfully entrap hydrophilic drugs inside the PLA nanoparticles, the preparation method
should be further optimized by changing the compositions of the outer and inner phases (of
nanoprecipitation). However, significant changes e.g. in pH and ionic strength expose the
particles to aggregation as reported in the chapter 5.1.5 and in paper III. A suitable method for the determination of hydrophobic BDP was developed using
microemulsion electrokinetic chromatography (MEEKC) [202]. In MEEKC, hydrophobic
substances are solubilized and detected from the microemulsion phase. In more detail, the
optimized microemulsion of this study consisted of octane, 1-butanol, sodium dodecyl
sulphate, and sodium tetraborate buffer. Approximately 30% of the drug used in the
Results and discussion
25
nanoparticle preparation was found inside the particles. The results with the BDP-loaded
nanoparticles suggest that the quantitation technique could even detect the drug inside the
nanoparticles: BDP was detected from the nanoparticulate dispersion (in water) but not from
the filtrate (Fig. 5 in II). Instead, when the nanoparticles were mixed with the microemulsion,
almost 90% of the successfully encapsulated BDP was found in the filtrate. This finding may
provide a novel approach to drug encapsulation determination as the existing conventional
techniques require separation of the particles from the dispersing medium.
Overall, electromigration techniques based on CE can be successfully used in the
determination of different drugs used in nanoparticle preparation processes.
5.1.4 Surface charge of the nanoparticles
Stability of the PLA nanoparticles was evaluated by ζ-potential measurements (III, V). ζ-Potential, determined by the state of the functional groups (carboxylic acid in the case of
PLA) at the surface of nanoparticles, is a function of environment, i.e. ionic strength and pH.
ζ-Potentials of the PLA nanoparticles as a function of the added electrolyte concentration
and pH are presented in Figure 7. Generally, the nanoparticles were most charged at low
ionic strengths (0.02 mol/l), when NaCl concentration of the dispersing medium was
increased gradually from zero (Fig. 3 in V). The lowest ζ-potential values of the L-PLA and
D,L-PLA nanoparticles were approximately -56 mV (V) and -76 mV (III), respectively. The
difference between the ζ-potential values of the two PLA nanoparticles probably originated
from the difference of the polymer chain: the D,L-PLA in question was tailored to carry
uncapped chain ends (free carboxylic acids).
Figure 7. ζ-Potential of the L-PLA (●) and the D,L-PLA (○) nanoparticles as a function of NaCl addition (left) and pH (right).
At some point during the electrolyte addition, PLA nanoparticles are known to start to
aggregate [203]. When the electrolyte concentration was increased towards 0.1 mol/l and
beyond, ζ-potential approached zero and the particles started to aggregate. At low
electrolyte concentrations, the range of the electrical double layer around the nanoparticles
was high (ζ-potential maximum) and according to the DLVO theory, repulsive forces
extended to a large distance [140]. At high electrolyte concentrations, free ions in the
-80
-70
-60
-50
-40
-30
-20
0,00 0,04 0,08 0,12 0,16
NaCl (mol/l)
ζ-p
ote
nti
al (m
V)
-50
-40
-30
-20
-10
0
2 4 6 8 10
pH
ζ-p
ote
nti
al (
mV
)
-80
-70
-60
-50
-40
-30
-20
0,00 0,04 0,08 0,12 0,16
NaCl (mol/l)
ζ-p
ote
nti
al (m
V)
-50
-40
-30
-20
-10
0
2 4 6 8 10
pH
ζ-p
ote
nti
al (
mV
)
Results and discussion
26
medium decreased the double layer magnitude and the particles could approach each other
giving rise to attractive van der Waals forces leading to aggregation. Decreasing pH to acidic,
ζ-potential approached zero as the carboxylic acid groups on the surface were deionized.
When pH is titrated to acidic values e.g. by HCl addition, the dissociated H+ and Cl- ions may
act first as electrolytes, which surpass the surface charge-decreasing effect of the pH
decrease. This was observed in the case of D,L-PLA nanoparticles (Fig. 1 in III). Variation in the ζ-potential values was also observed if the processing conditions were
modified although the polymer type remained the same. Nanoparticles prepared from L-PLA
using CHL as the solvent were more charged (ζ-potential ~ -50 mV) than the particles
prepared with DCM (ζ-potential ~ -30 mV) (V). An explanation could be based on the slower
diffusion rate of CHL to the outer phase (compared to DCM) and subsequent slower
precipitation of PLA, as the hydrophilic chain ends of the polymer (ionized carboxylic acid
groups) had more time to be organized at the solvent - outer phase interface towards the
aqueous outer phase making the formed surface more charged. In the case of D,L-PLA
nanoparticles prepared with the surfactant, Poloxamer 188 (III), adsorption of the surfactant
molecules on the nanoparticle surfaces was detected as minor ζ-potential (absolute) values
compared to the surfactant-free nanoparticles.
5.1.5 Stability and aggregation studies by surface pressure measurements
Behavior of colloidal particles (stability, aggregation, film forming ability etc.) at interfaces
such as water-air or water-oil has been a topic of several studies during recent years. Surface
pressure can be studied by the Wilhelmy plate technique when the particle population is
compressed as a monolayer at an interface (π vs. A isotherms). Surface pressure
determinations are a widely used tool in the characterization of inorganic particles, but its
application in the studying of organic nanoparticles has been less frequent. Thus far, mainly
polystyrene nanoparticles have gained such research attention [131, 204-206]. In paper III, stability and aggregation behavior of D,L-PLA nanoparticles in different environments were
characterized by surface pressure measurements and SEM visualizations.
The π vs. A isotherms and corresponding SEM images of the surfactant free PLA
nanoparticles clearly revealed that the particle aggregation had started at pH 4.0 during the
pH decrease (Fig. 8 and also Figs. 2 & 6 in III). The isotherms registered on subphases at pH
4.0 or below exhibited increase in the surface pressure at larger surface area and higher
surface pressure in the end of the compression. The formed particle aggregates obviously
resisted (mechanically) the compression more than the nanoparticle population consisting of
the more charged individual particles (pH above 4.0), which could be rearranged in a more
flexible way.
When the nanoparticles were prepared with Poloxamer 188, the surfactant molecules
located on the nanoparticle surface created a steric barrier that resisted the compression at
longer distances compared to the surfactant-free nanoparticles. This could be seen as an
earlier increase in the surface pressure during the compression (Fig. 8 and also Fig. 3 in III). Repulsion (pressure) was similar in proximity of the most compressed state in the cases of the
both particles. Therefore, contribution of the surface charge was probably dominant at these
identical sections of the isotherms. The surfactant, i.e. steric stabilization, protected the
Results and discussion
27
nanoparticles against pH-induced aggregation, which could be seen as a non-aggregated
particle population even at pH 4.0 (Fig. 7 in III). These findings promote the role of both the
surface charge and the surfactant in the nanoparticle stabilization [203].
Figure 8. π vs. A isotherms (left) and SEM images (right, 1 000-fold magnification) of the surfactant-free nanoparticles (except * indicating the isotherm of the nanoparticle prepared with Poloxamer 188) on different subphases.
Increased surface charge (ζ-potential) and, thus, increased repulsion between the
surfactant-free nanoparticles, induced by electrolyte addition (0.02 mol/l NaCl), caused a rise
in the surface pressure values (Fig. 4 in III). Cluster formation as a sign of aggregation was
observed in SEM images, when the electrolyte concentration was futher increased to 0.15
mol/l. Also this time, Poloxamer 188 prevented particle aggregation at the higher NaCl
concentration.
Aggregate formation of the nanoparticles was treated by the classification system of
Robinson and Earnshaw [207]. If the aggregation occurs spontaneously by particle diffusion
and collisions, the process is referred to diffusion-limited cluster aggregation (DLCA).
Correspondingly, if the probability for the aggregation is lower, e.g. due to repulsion or
steric barriers between the particles, the process occurs by reaction-limited cluster
aggregation (RLCA). The aggregation due to the pH decrease occurred by DLCA and resulted
in percolated particle networks (Fig. 8 and also Fig. 6 in III), if the surfactant was not present.
With surfactant, or if the surface charge was high enough as a result of electrolyte addition,
the aggregation followed the RLCA regime. Under such conditions, the formation of
networks was prevented and the aggregates remained as clusters (Figs. 7 & 8 in III).
5.2 Freeze-drying
Protective excipients, such as carbohydrates, are widely used in freeze-drying to ensure
redispersibility and to avoid aggregation or size changes of nanoparticles [12, 111, 113, 115,
116]. In paper IV, glucose and lactose were evaluated as cryo- and lyoprotectants for the L-
0
2
4
6
8
10
12
14
16
18
0 50 100 150 200 250
Trough area (cm2)
Surf
ace
pre
ssu
re (
mN
/m) pH 4.0
pH 6.5*pH 6.5
pH 6.5
pH 2.6
0.15 mol/l NaCl0
2
4
6
8
10
12
14
16
18
0 50 100 150 200 250
Trough area (cm2)
Surf
ace
pre
ssu
re (
mN
/m) pH 4.0
pH 6.5*pH 6.5
pH 6.5
pH 2.6
0.15 mol/l NaCl
pH 6.5
pH 2.6
0.15 mol/l NaCl
Results and discussion
28
PLA nanoparticles because these nanoparticles could not survive during the drying process
without protectants.
Even the smallest tested amount of glucose (weight ratio glucose:nanoparticles 1:4) was
found to protect the nanoparticles (Fig. 3 and Table 2 in IV), although the appearance of the
dried material was translucent and sticky, and its redispersibility was poor. When lactose was
used as a protectant, it enhanced the appearance of the cake (the dried material) as a white
powder, eligible for a freeze-dried formulation. Redispersion of the nanoparticle was
possible, but as a form of visible aggregates. Further freeze-thawing experiments revealed
that already the freezing step (with lactose) destroyed the particles (Fig. 4 in IV). Next, the
two carbohydrates were used together to combine the cryoprotective functionality of
glucose and the lyoprotective functionality of lactose. The best result, prolonged Tyndall
effect (opalescence in the dispersion) after redispersion of the dried formulation and good-
quality nanoparticles (Fig. 6 in IV) were obtained, when the amount of lactose was double
the amount of glucose. The weight ratios of glucose and lactose to the nanoparticles were
1:2 and 1:1, respectively. Additionally, when an extra stabilizer, Tween 80, was used during
the nanoparticle preparation or during the redispersion, the freeze-dried cake could be
redispersed more easily with increased stability (prolonged Tyndall effect).
The good cryoprotective results with glucose probably arise from its ability to bind water
molecules to the amorphous phase which it forms during the freezing step [12]. Part of the
water in the frozen glucose remained non-frozen (even 32% w/w). That water acted as a
plasticizer and as a spacing matrix reducing the pressure of ice crystals against the
nanoparticles and preventing harmful aggregation caused by freeze concentration,
respectively. At the same time, insufficient cryoprotective function of lactose derived most
likely from its lower water binding activity. However, as a combination with glucose, lactose
reduced the amount of water to a level where the interaction of glucose with water was
reduced [208] and, thus, the formation of ice crystal was slightly promoted. This enabled
sufficient evaporation of water during the drying and formation of a proper cake. Tween 80
improved the freeze-drying result as it acted as a steric stabilizer and increased the
hydrophilicity of the nanoparticles. An hydrophilic surface enhances the redispersion
properties of the freeze-dried nanoparticles [115, 122].
5.3 Layer-by-layer coating
To successfully perform the LbL coating process, the core particles should be stable enough
and carry sufficient surface charge. These properties are needed to enable adsorption of PEs
and to avoid particle aggregation and destruction during different stages of the coating
process. Therefore, in paper V, influence of process parameters including polymer solvent,
coating medium, PE concentration and filtration membrane type, were evaluated to obtain
suitable conditions in relation to nanoparticle surface charge and PE adsorption.
PAH (+) and PSS (-) were selected as model coating PEs because of their known usability
in LbL processes [32, 101, 209]. Knowing their nature as non-biodegradable plastics, they
should be replaced by biocompatible PEs in further studies. Suitable macromolecules for that
could be e.g. chitosan and alginic acid.
Results and discussion
29
Although the nanoparticle preparation process with either of the solvents (CHL or DCM)
was successful producing spherical particles (Fig. 2 in V), coating of the particles prepared
with DCM failed (instant aggregate formation). The reason for the failure was the less
charged (surface) nature of the nanoparticles in question, which led to unwanted electrical
interactions between the particles and the PEs.
0.02 mol/l NaCl solution at pH ~7 was selected as the medium for coating because the
nanoparticles possessed the highest surface charge in these circumstances (Fig. 7 and also
Figs. 3 & 5 in V). Low ionic strength of the medium was also favourable in relation to the
flexibility of the PEs [210, 211]. 1 mg/ml concentration of PEs in the coating solutions applied
as a volume of 200 µl provided excess amounts of PEs [103]. It was possible to perform the
LbL process using several types of membranes. However, 0.2 µm polycarbonate membranes
were preferred due to their durability and finely tailored structure (uniform, black dots in
the background of the nanoparticles in Fig. 9 (particles after coating, below) are membrane
pores). The particles blocked the membrane during subsequent filtrations, if they were not
properly detached with the help of pressure and gentle brushing with a hair pencil.
Otherwise, the vacuum below the membrane was increased because of the poor flow of the
coating/washing medium. This led to stress on the particles, which promoted aggregation.
Figure 9. Left: ζ-Potential (●) and size (○) as a function of LbL coating layers. Right: Nanoparticles before coating (above) and after coating (below).
The success of the coating process was studied by monitoring the alternation of the ζ-potential and increase in size (PCS, SEM) as a function of coating layers (Fig. 9 and also Figs. 7
& 8 in V). After six coating layers, the size of the nanoparticles was increased to above 500
nm. The size distribution was slightly broadened during the coating; the polydispersity index
increased from ~0.1 to ~0.3. In addition, adsorption of PEs (PLA-CHL nanoparticles) and the
failure of the coating (PLA-DCM nanoparticles) was verified by heats of thermal transitions
detected by DSC. In the case of the CHL particles, heats of Tc and Tm of PLA decreased from
3.07 J/g and 15.6 J/g to 1.19 J/g and 5.64 J/g, respectively, when two layers were adsorbed
(PAH+PSS) (Table 1 in V). Such a decreasing tendency was not observed with the PLA-DCM
particles. The decrease of heats indicated adsorption of PEs, because the relative amount of
PLA (of the sample mass) was decreased due to the presence of the PEs. In the case of PLA-
DCM particles, the PEs were not adsorbed and were most probably filtered away during the
-80
-60
-40
-20
0
20
40
60
0 1 2 3 4 5 6
Number of coating layers
ζ-p
ote
nti
al (
mV
)
0
100
200
300
400
500
600
Size
(n
m)
-80
-60
-40
-20
0
20
40
60
0 1 2 3 4 5 6
Number of coating layers
ζ-p
ote
nti
al (
mV
)
0
100
200
300
400
500
600
Size
(n
m)
Results and discussion
30
washing step. No new thermal events were found in the thermograms indicating that the
structure of PLA was not affected by any interactions between the polymer and the PEs.
Conclusions
31
6 Conclusions
Nanoparticles could be successfully prepared by the nanoprecipitation method using either
semi-crystalline poly(L-lactic acid) or amorphous poly(D,L-lactic acid). Applying different
characterization methods, notably scanning electron microscopy, X-ray powder diffraction,
differential scanning calorimetry and infrared spectroscopy, changes could be detected in the
physicochemical characteristics of the nanoparticles induced by the preparartion process and
by the model drugs used in the preparation process. These characteristics included size,
crystallinity of the polymer and state of the drugs. Capillary electrophoresis could be used in
the quantitative concentration determination of the model drugs.
Stability of the nanoparticle dispersion was affected by the dispersion medium conditions
such as pH and ionic strength. Surface pressure measurements together with scanning
electron microscopy imaging proved to be a suitable tool in the evaluation of the stability
and the aggregation properties of these biodegradable nanoparticles. During the freeze-
drying process, stability of the nanoparticles could be enhanced by the addition of
carbohydrates, which acted as cryo- and lyoprotectants inhibiting aggregation of the
particles and facilitating the redispersion properties.
Surface properties of the nanoparticles could be modified by the layer-by-layer
polyelectrolyte coating technique. A filtration approach in the coating together with the
optimized process conditions enabled successful attachment of the polyelectrolytes on the
nanoparticle surface.
Overall, the main focus in this work was in the physicochemical characterization of
nanoparticles during preparation, in different processes and in changing environments. Some
of the applied techniques – capillary electrophoresis, surface pressure measurements and
layer-by-layer coating – bring novel aspects to the characterization of poly(lactic acid)
nanoparticles and other biodegradable pharmaceutical nanoparticles. All the results of this
thesis provide useful information for the future studies aiming at development of drug
delivery formulations consisting of nanoparticles.
References
32
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