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NANOPHARMACEUTICALS
EXPECTATIONS AND REALITIES OF
MULTIFUNCTIONAL DRUG
DELIVERY SYSTEMS
VOLUME 1
Edited by

RANJITA SHEGOKAR, PhD

Capnomed GmbH, Zimmern, Germany
Elsevier
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 Contributors
Hend Abd-Allah Department of Pharmaceutics and
Industrial Pharmacy, Faculty of Pharmacy, Ain
Shams University, Egypt
Mona Abdel-Mottaleb Department of Pharmaceu-
tics and Industrial Pharmacy, Faculty of Pharmacy,
Ain Shams University, Egypt; PEPITE EA4267,
Univ. Bourgogne Franche-Comté, Besançon, France
Annis Catur Adi Faculty of Health, University of
Airlangga, Surabaya, Indonesia
Mukta Agrawal Rungta College of Pharmaceutical
Sciences and Research, Bhilai, Chhattisgarh, India
Amit Alexander Rungta College of Pharmaceutical
Sciences and Research, Bhilai, Chhattisgarh, India
Mahavir Bhupal Chougule Translational Bio-
pharma Engineering Nanodelivery Research Labo-
ratory, Department of Pharmaceutics and Drug
Delivery, School of Pharmacy, University of Missis-
sippi, University, MS, United States; Pii Center for
Pharmaceutical Technology, Research Institute of
Pharmaceutical Sciences, University of Mississippi,
University, MS, United States; National Center
for Natural Products Research, Research Institute
of Pharmaceutical Sciences, University of Missis-
sippi, University, MS, United States
Juan Bueno Research Center of Bioprospecting
and Biotechnology for Biodiversity Foundation
(BIOLABB), Armenia, Quindío, Colombia
J.R. Campos Department of Pharmaceutical Tech-
nology, Faculty of Pharmacy, University of Coim-
bra (FFUC), P

olo das Ciências da Sa

ude, Coimbra,
Portugal
Anne Marie Clark CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Diana Diaz-Arévalo Molecular Biology and Immu-
nology Department, Fundaci

on Instituto de Inmu-
nología de Colombia-FIDIC, School of Medicine
vii
and Health Sciences, Universidad del Rosario,
Bogot

a, DC, Colombia
Riham I. El-Gogary Department of Pharmaceutics
and Industrial Pharmacy, Faculty of Pharmacy,
Ain Shams University, Egypt
N.B. Jadav Centre for Pharmaceutical Engineering
Sciences, Faculty of Life Sciences, University of
Bradford, Bradford, United Kingdom
AnCelka B. Kovacevi
c Department of Pharmaceu-
tical Technology, Institute of Pharmacy, Faculty of
Biological Sciences, Friedrich-Schiller University
Jena, Jena, Germany
Atsarina Larasati Research Center for Nanosciences
and Nanotechnology, Bandung Institute of Tech-
nology, Bandung, Indonesia
Peter Mattei CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Maha Nasr Department of Pharmaceutics and In-
dustrial Pharmacy, Faculty of Pharmacy, Ain
Shams University, Egypt
A. Paradkar Centre for Pharmaceutical Engineering
Sciences, Faculty of Life Sciences, University of
Bradford, Bradford, United Kingdom
Heni Rachmawati School of Pharmacy, Bandung
Institute of Technology, Bandung, Indonesia;
Research Center for Nanosciences and Nanotech-
nology, Bandung Institute of Technology, Bandung,
Indonesia
Kobra Rostamizadeh Zanjan Pharmaceutical Nano-
technology Research Center, Zanjan University of
Medical Sciences, Zanjan, Iran; Center for Pharma-
ceutical Biotechnology and Nanomedicine, North-
eastern University, Boston, MA, United States
A. Santini Department of Pharmacy, University of
Napoli “Federico II”, Napoli, Italy
viii Contributors
Shailendra Saraf University Institute of Pharmacy,
Pt. Ravishankar Shukla University, Raipur, Chhat-
tisgarh, India
Swarnlata Saraf University Institute of Pharmacy,
Pt. Ravishankar Shukla University, Raipur, Chhat-
tisgarh, India
P. Severino Universidade Tiradentes (Unit), Aracaju,
Sergipe, Brazil; Instituto de Tecnologia e Pesquisa,
Laborat
orio de Nanotecnologia e Nanomedicina
(LNMed), Aracaju, Sergipe, Brazil; Tiradentes
Institute, Dorchester, United States
Ranjita Shegokar Capnomed GmbH, Zimmern,
Germany
A.M. Silva School of Biology and Environment, Uni-
versity of Tr
as-os-Montes e Alto Douro (UTAD), Vila
Real, Portugal; Centre for Research and Technology
of Agro-Environmental and Biological Sciences
(CITAB), University of Tr
as-os-Montes e Alto Douro
(UTAD), Vila Real, Portugal
S.B. Souto Department of Endocrinology, S. Jo~ ao
Hospital, Alameda Prof. Hern^ani Monteiro, Porto,
Portugal
E.B. Souto Department of Pharmaceutical Technol-
ogy, Faculty of Pharmacy, University of Coimbra
(FFUC), P
olo das Ciências da Sa
ude, Coimbra,
Portugal; CEB - Centre of Biological Engineering,
University of Minho, Braga, Portugal
Asur Srinivasan CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Amanda Starling-Windhof CAS, a Division of the
American Chemical Society, Columbus, OH,
United States
Tina Tomeo CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Vladimir P. Torchilin Center for Pharmaceutical
Biotechnology and Nanomedicine, Northeastern
University, Boston, MA, United States
Mingtao Zeng Center of Emphasis in Infectious Dis-
eases, Department of Molecular and Translational
Medicine, Paul L. Foster School of Medicine, Texas
Tech University Health Sciences Center El Paso, El
Paso, TX, United States
 Preface

The book series titled Expectations and Real-
ities of Multifunctional Drug Delivery Systems
covers several important topics on drug-delivery
systems, regulatory requirements, clinical studies,
intellectual properties trends, new advances,
manufacturing challenges, etc. written by leading
industry and academic experts. Overall, the
chapters published in this series reflect the broad-
ness of nanopharmaceuticals, microparticles,
other drug carriers and the importance of the
respective quality, regulatory, clinical, GMP scale
up, and regulatory registration aspects.
This series is destined to fill the knowledge
gap through information sharing and with orga-
nized research compilation between diverse
areas of pharma, medicine, clinical, regulatory
practices, and academics.
Expectations and Realities of Multifunctional Drug
Delivery Systems is divided into four volumes:
Volume 1: Nanopharmaceuticals
Volume 2: Delivery of Drugs
Volume 3: Drug Delivery Trends
Volume 4: Drug Delivery Aspects
The specific objectives of this book series are
to:
(1) provide a platform to discuss opportunities
and challenges in development of
nanomedicine and other drug-delivery
systems;
(2) discuss current and future market trends;
(3) facilitate insight sharing within various
areas of expertise; and
(4) establish collaborations between academic
scientists, and industrial and clinical
researchers.
Innovative cutting-edge developments in
micro-nanotechnology offer new ways of pre-
venting and treating diseases like cancer, ma-
laria, HIV/AIDS, tuberculosis, and many more.
The application of micro-nanoparticles in drug
delivery, diagnostics, and imaging is vast.
Hence, Volume 1: Nanopharmaceuticals, in
the book series mainly reviews advances in
drug delivery area via targeted therapy with
improved drug efficiency at a lower dose, trans-
portation of the drug across physiological bar-
riers as well as reduced drug-related toxicity.
One of the contributions by Campos et al.
(Chapter 1) discusses the influence of physico-
chemical factors affecting long-term stability,
release and toxicological profiles of solid lipid
nanoparticles. This chapter also reviews the
importance of composition and administration
routes studied for lipid nanocarrier systems.
In another manuscript by Rachmawati et al.
(Chapter 2), the authors highlight the current
status of drug delivery development for herbal
bioactives. Along with various mucosal bio-
carriers, the authors describe biokinetic and
clinical translation challenges with herbal deliv-
ery and limitations with regulatory procedures.
In this chapter herbal nanocarriers like lipid
nanoparticles, nanosuspensions etc. are
discussed in detail.

ix
x Preface
Chapter 3 by Rostamizadeh et al. describes
the development of polymeric micelles for multi-
ple drug delivery in oncology. The co-loading of
two or more drugs is possible using polymeric
micelles. The authors describe the types of
polymers employed, preparation methods, and
characterization techniques for such carrier
systems. Furthermore, wider applications like
chemotherapeutic delivery, stimuli-responsive
drug delivery, and targeted-drug delivery via
such carriers is discussed in detail. On the other
hand, hyaluronic acid nanoparticles are being
widely explored in nanomedicines. The promising
nanocarriers to deliver drugs in conjugated, self-
assembled, or in nanocomplex form are discussed
in the book chapter by Nasr et al. in Chapter 4. The
authors confirm that the current research
shows impressive research findings in areas like
osteoarthritis, tissue engineering, cancer target-
ing, theranostic applications, and so on, which
are under further exploration by industry.
The work by Jadav and Paradkar (Chapter 5)
is aimed at discussing widely studied drug
delivery systems in academics and in industry,
i.e. solid dispersions. Various aspects like classi-
fication of solid dispersions, their formulation
optimization, processing and physicochemical
characterization are reviewed in this chapter.
Chapter 6 by Bueno highlights the impor-
tance of understanding nanotoxicity at early
stages of development. Although nanocarriers
have shown promising results in delivering
drugs at target sites or locations, the accumula-
tion of nanoparticles at cellular and tissue
levels in excess causes toxicity. Current literature
contains very limited information on this topic.
This chapter reviews various aspects of nanotox-
icity and provides information on key concepts
for evaluation of the toxicity.
The topic presented by Diaz-Arévalo et al.
(Chapter 7) describes the systemic review of
nanoparticles-based vaccine development. Initial
results of nanocarriers like liposomes, virus like
particles, metallic and nonmetallic particles, and
polymeric nanoparticles in vaccine therapy are
promising although some limitations like stability
and cytotoxicity needs to be overcome.
Chapter 8 by Kovacevic discusses delivery of
poorly soluble and low-permeable drugs via
lipid nanocarriers. An overview of available
mechanistic studies in model and in real cell
membranes for better understanding of cell
internalization processes is provided.
The chapter by Alexander et al. (Chapter 9)
reviews approaches for effective brain drug deliv-
ery using nasal mucosa. This route can deliver
drugs effectively at target sites with improved
therapeutic performance of drugs. In addition,
the authors explain limitations of this drug deliv-
ery route and regulatory market approval
challenges.
Starling-Windhof et al. (Chapter 10) address
industry and technology trends in the intellec-
tual property (IP) landscape of pharmaceutical
drug delivery over 3 decades. It is fascinating
to see the global picture; it makes scientists
aware of the trends and special interests of
specific geographical regions or markets.
This chapter provides information on IP trends
in oral, topical, and parenteral drug delivery
area. Guidance on emerging trends and
IP-monitoring strategies are also presented.
In summary, I am sure this book volume and
the complete book series will provide you great
insights in areas of micro-nanomedicines, drug
delivery sciences, new trends, and regulatory
aspects.
O. Farokhzad, R. Langer, and the National
Cancer Institute are gratefully acknowledged for
the book cover image, which represents the po-
tential of nanopharmaceuticals in targeting drug
molecules. This photograph presented on cover
page captures interactions of surface-modified
polymeric nanoparticles with prostate cancer
cellsdit is an ideal example for drug targeting.
All the efforts of experts, scientists, and
authors are highly acknowledged for sharing their
knowledge, ideas, and insights about the topic.
Ranjita Shegokar, PhD
Editor
 C H A P T E R

1
Solid lipid nanoparticles (SLN):
prediction of toxicity, metabolism, fate
and physicochemical properties
J.R. Campos1, P. Severino2,3,4, A. Santini5, A.M. Silva6,7,
Ranjita Shegokar8, S.B. Souto9, E.B. Souto1,10
1
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), P
olo
ude, Coimbra, Portugal; 2Universidade Tiradentes (Unit), Aracaju, Sergipe, Brazil;
das Ci^encias da Sa

3
Instituto de Tecnologia e Pesquisa, Laborat
orio de Nanotecnologia e Nanomedicina (LNMed), Aracaju,
Sergipe, Brazil; 4Tiradentes Institute, Dorchester, United States; 5Department of Pharmacy, University of
Napoli “Federico II”, Napoli, Italy; 6School of Biology and Environment, University of Tr
as-os-Montes e
Alto Douro (UTAD), Vila Real, Portugal; 7Centre for Research and Technology of Agro-Environmental
and Biological Sciences (CITAB), University of Tr
as-os-Montes e Alto Douro (UTAD), Vila Real,
Portugal; 8Capnomed GmbH, Zimmern, Germany; 9Department of Endocrinology, S. Jo~ao Hospital,
Alameda Prof. Hern^ani Monteiro, Porto, Portugal; 10CEB - Centre of Biological Engineering,
University of Minho, Braga, Portugal

1. Introduction formulations [2]. Various controlled drug deliv-

Solid lipid nanoparticles (SLNs) gained
greater attention as a drug delivery system
when in 1991 M€ uller developed them [1]. This
promising drug carrier system is at the interface
in the preexisting lipid systems (emulsions and
liposomes) and polymeric nanoparticle systems.
Lipid nanoparticles, known as SLNs or nano-
structured lipid carriers (NLCs), have specific
features of structure and composition, showing
benefits in comparison to conventional
ery systems like polymer-based controlled-
release systems, hydrogels, as well as nano-
and microparticles have been introduced in
recent years in order to improve solubility, sta-
bility and bioavailability of poorly water-soluble
drugs. In this context, lipid nanoparticles offer
attractive and ideal properties for drug or gene
delivery. These particles (either composed of
solid lipids only in SLNs, or of a blend of solid
and liquid lipids in NLCs) stabilized with surfac-
tants have the advantages of other colloidal

Nanopharmaceuticals
https://doi.org/10.1016/B978-0-12-817778-5.00001-4 1 © 2020 Elsevier Inc. All rights reserved.
2 1. Solid lipid nanoparticles (SLN)
particles (polymeric nanoparticles, fat emul-
sions, and liposomes) by overcoming their limi-
tations [3,4]. Taking account their polymeric
and lipid raw materials, several modifications
of drug delivery systems have been proposed
to increase the bioavailability of loaded drugs.
SLNs are made of a solid lipid matrix and a sur-
factant layer and they can load poorly water-
soluble drugs, delivering them at defined rates
and with improved bioavailability [5]. These
colloidal drug delivery systems protect the
drug against chemical degradation and modify
its release profile since the drug is entrapped in
the solid lipid matrix [6,7]. These nanoparticles
of spherical shape have a mean size of
40e1000 nm [8,9]. The lipid matrix is composed
of a solid lipid (or a mixture of solid and liquid
lipids) in a 0.1%e30% (w/w) concentration
dispersed in aqueous medium, and their stability
is ensured by the presence of a surfactant in a
0.5%e5% (w/w) concentration [8] and can be
used for lipophilic or hydrophilic drugs [9]. Tri-
glycerides (tristearin), sterols (cholesterol), par-
tial glycids (glyceryl monostearate), fatty acids
(stearic acid), as well as waxes (cetylpalmitate)
are especially used as lipids in the SLNs. In these
systems emulsifiers and polymers are used as
stabilizers in order to avoid aggregation of the
particles. Examples of stabilizers are bile salts
(e.g., taurodeoxycholate), lecithins, and copoly-
mers of polyoxyethylene and polyoxypropylene
(Poloxamer) [10].
It is clear that lipid nanocarriers are ideal for
sensitive bioactive compounds. SLNs exhibit
biocompatibility, matrix with lipophilic nature
protecting active compounds of chemical degra-
dation, drug targeting, controlled release profile,
and high drug payload [9,12]. Moreover, they
are suitable for industrial production mainly
because they are easy to scale up, are stable under
sterilization conditions, and they have the advan-
tage of being non-toxic or of very low toxicity,
because of their composition in Generally
Recognized as Safe (GRAS) excipients [4,6].
SLNs are biodegradable (fulfilling the require-
ments of preclinical safety) and are also stable in
blood, with prolonged lifetime in the bloodstream
[13e15]. In addition, compared to liposomes,
SLNs exhibit high encapsulation efficiency, stabil-
ity against light and oxygen, do not need organic
solvents for their preparation, and have high
drug-loading capacity (mainly for lipophilic com-
pounds) [12,15,16]. On the other hand, SLNs have
also limitations, mainly attributed to the risk of
polymorphic transitions (from a to b0 , and from
b0 to b) which causes stability challenges during
administration or storage, resulting in drug
expulsion from the particles and eventual particle
size increase [6,10,17]. These disadvantages are
related to the crystallization behavior and lipid
matrix’s polymorphic transitions, which depend
on the type of lipids used for the production of
SLNs [6]. There are various methods described
in the literature to produce SLNs based on
solidified emulsion technologies: high shear ho-
mogenization and ultrasound, high pressure
(hot and cold) homogenization, oil/water (O/
W) and water/oil/water (W/O/W) microemul-
sions, as well as solvent evaporation [10].
These techniques interfere with various character-
istics of the particles, mainly in morphology. Ac-
cording to the literature, the most commonly
applied methods are those that use high pressure
homogenizers (HPH). M€ uller and Luck devel-
oped the HPH technique (European Patent No.
0605497) for obtaining nanoemulsions for large-
scale parenteral nutrition [10]. There are diverse
kinds of equipment with various sizes, prices as
well as different capacities. The different equip-
ments work by pulling the liquid in high pressure
(100e2000 bar) through a narrow piston (nano-
meter scale), which is accelerated over a small
distance at a high speed (over 1000 km/h). This
fluid is subjected to high stress, disrupting the
macroscopic oil droplets by cavitation forces
and thus generating the nanodroplets [10]. In
 2. Toxicity profiling
the hot process, the hot preemulsion is passed
through the hot homogenizer to obtain nanoe-
mulsions, which are then cooled down in order
to solidify and crystallize the hot inner liquid
phase to obtain SLNs. In the cold process, the
drug is firstly ground milled in a mortar mill
with the solid lipid at room temperature, and
then the obtained powder is dispersed in an
aqueous surfactant solution, which is then sub-
jected to HPH. The influence of the type of
homogenizer, pressure, and number of cycles
employed, and the temperature used to obtain
the ideal particle size, have been intensively stud-
ied. Depending on the type of lipid, it is possible
to use lipid concentrations above 40% and obtain
the particle size distribution in a low range
(polydispersity index <0.2) [18]. To obtain SLNs
from microemulsions, Gasco and collaborators
developed a technique that has been modified
by numerous researchers. In this technique,
SLNs are produced by diluting a hot oil-in-water
(O/W) microemulsion in high volume of cold-
water (0e4o C). The internal phase of this microe-
mulsion is composed of low-melting lipids and,
when in contact with the cold water, they suffer
crystallization and form SLNs [18e20]. The type
of lipids used to make the microemulsion, the
preparation parameters (stirring and tempera-
ture), rate of microemulsion’s addition in the
cold water, volume of water, and the technique
used to remove the excess water, all affect the
characteristics of obtained nanoparticles [18].
This technique is therefore difficult to scale up.
Marengo has developed a device that processes
100 mL of microemulsion and can produce
SLNs of mean size below 100 nm [20].
The biomedical applications of lipid nanopar-
ticles are manyfold. Indeed, they can be used as
drug and gene carriers, and as contrast agents
for imaging analysis [21]. In recent years,
different administration routes (e.g., oral, paren-
teral, dermal, pulmonary, rectal, ocular) have
3
been explored for drug delivery using lipid
nanoparticles. Components of lipid nanopar-
ticles (lipids and surfactants) determine the
product quality, its physicochemical properties,
as well as the administration route [4] (Table
1.1). In this chapter, the concept behind the
SLNs and their physicochemical properties,
pharmacokinetics, and biopharmaceutics and
their toxicological testing are discussed.
2. Toxicity profiling
SLNs are known to be stable in aqueous
dispersion, allows the encapsulation of hydro-
philic and lipophilic drugs, are adaptable to
several administration routes, can modify the
release profile and avoid their adverse effects
(protecting the drug from undesirable interac-
tions or directing it to its target) [30]. However,
their toxicological profile has to be very well
characterized in vitro before any pre-clinical
and clinical studies [31]. There is a relationship
between the size of the particles and their
toxicity, as the lower the size, the higher the sur-
face area and the higher the reactivity. A pre-
requisite to be marketable is the GRAS status
of the excipients of SLNs [32], but additional
nanotoxicological studies are needed to allow
the understanding of the effect of nanoparticles
in the body [14,33,34]. Nanotoxicology helps in
identifying the SLN formulations to be selected
for preclinical studies by evaluating their safety,
tolerance, and cytotoxicity.
Preclinical toxicological studies allow to
determine the concentration of substances that
cause toxic effects, and allow identification of
target organs predisposed to these effects. Pre-
clinical safety tests require the appropriate selec-
tion of the animal species, age, physiological
status, the administration route, dosage form as
well as the treatment regime. Preclinical
TABLE 1.1 Commonly used lipids in the composition of SLNs/NLCs.

Therapeutic
Name Chemical structure Surfactant System Drug application References

Tristearin Dihexadecyl SLNs e Pulmonary [22]

phosphate delivery
PEGs SLNs e Oral delivery [23]

Glyceryl Mixture of Tween SLNs e e [24]

monostearate 80 and Span 80

Stearic acid Omega-3 PUFA SLNs Resveratrol Oral [25]

delivery
Cystamine SLNs e e [26]
Tween 80 NLCs e e [27]
Cholesterol Pluronic F-68 NLCs Simvastatin Oral [28]
delivery

Vitamin E O D-a-tocopheryl NLCs Rapamycin Ocular [29]

O n polyethylene glycol delivery
O
R O succinate (TPGS)
O O
 3. Physicochemical properties
toxicological evaluation of a new compound can
provide information about acute, subacute, sub-
chronic, and chronic toxicity. Before any preclin-
ical study, cytotoxicity assessment is carried out
in cell culture, also allowing the study of the
interaction between nanoparticles and cells.
The use of primary cells (isolated directly from
the animals) provides more realistic toxicological
results. On the other hand, in vivo studies eval-
uate the organism as a whole. Therefore, in vivo
studies are relevant to determine the location
and concentration of the drug in the tissues, and
systemic toxicity [35]. Systems with large quan-
tities of surfactants (e.g., nanoemulsions) have
higher risk of exhibiting cytotoxicity, since these
agents interact with cell membranes composed
of phospholipids. Likewise, their production re-
quires the use of organic solvents, which may
also increase the risk of toxicological events if
poorly removed. Although precise determina-
tion of the toxicity can only be quantified
in vivo, there are several in vitro toxicological
tests that provide preliminary information
[10,14,36]. In vitro studies have shown that
SLNs are acceptable at concentrations <1 mg/
mL (total lipids), and with particle diameter
>500 nm can be less tolerated, which can be
explained by their aggregation. It was also
shown that the stabilized formulations
composed of several surfactants are less biocom-
patible in comparison to those based on one sur-
factant only. For polysorbate 80 and poloxamer
188, two surfactants mostly used in SLNs formu-
lations, enough evidence has been found to
determine their safety [14]. It is clear that the
knowledge of the toxicological profile of any ma-
terial and the biocompatibility of the drug deliv-
ery systems are crucial for the implementation of
drug therapies, but the information of
Amount ðmgÞ of loaded drug determined experimentaly
EEð%Þ ¼
Theoretical amount of drug ðmgÞ in formulation
5
nanoparticle-based drug therapies available is
still very limited [8].
3. Physicochemical properties
The characterization of SLNs usually re-
quires the determination of the particle size
and zeta potential, morphological evaluation,
determination of the loading capacity and
encapsulation efficiency, kinetics of drug
release, as well as the nanoparticles over
time and storage temperature.
3.1 Encapsulation parameters
Determination of the amount of drug associ-
ated with the nanoparticles is a crucial parameter
for the characterization of SLNs. This task is
however difficult because of the small size of
the particles, which compromises the separation
of the free drug fraction from the associated frac-
tion. Ultracentrifugation is the most commonly
used separation procedure, after which the
non-loaded drug is quantified in the superna-
tant. From the difference between total drug
and the drug found in the supernatant, the
drug concentration associated with nanostruc-
tures is calculated [37,38]. Ultrafiltration can be
coupled to the process of ultracentrifugation. In
this approach, a membrane (100 kDa) is used to
separate the aqueous phase from the nanopar-
ticles. Although the free drug concentration in
this technique is determined in the ultrafiltrate,
the drug fraction associated with the nanostruc-
tures is also found by the difference between the
total and free concentrations [39]. Encapsulation
efficiency (EE%) and loading capacity (LC%) are
determined using the following equations [40]:
 100
6 1. Solid lipid nanoparticles (SLN)
Amount ðmgÞ of loaded drug determined experimentaly
LCð%Þ ¼
Theoretical amount of lipid ðmgÞ in formulation
3.2 Particle size
Size and polydispersity index are parameters
that indicate the stability of the nanoparticles.
An optimized SLN formulation should exhibit
a mean particle size less than 1 mm, together
with a small polydispersity index. Several pa-
rameters affect the particle size and polydisper-
sity, e.g., composition of the formulation
(mixture of surfactants, lipid structural proper-
ties, and also incorporated drug), methods and
conditions of the production (time, temperature,
stirring velocity, pressure, etc) [41]. There is a
relationship between the proportion of surfac-
tant/lipid and the particle size, i.e., the greater
the concentration of surfactant, the smaller the
particle size [42]. The temperature used in the
HPH technique is another parameter that inter-
feres with the particle size. In the production of
SLNs and NLCs, the lipid phase is heated up
to a temperature higher than the melting point
of the solid lipid. The final step is the cooling
down of the systems so that the lipid recrystal-
lizes to solidify the lipid droplets [43]. Homoge-
nization at low temperatures (i.e., below the
melting point of the solid lipid) favors the in-
crease of particle size, but temperatures near
the melting point improve the homogenization
and lower particle sizes can be obtained. Simi-
larly, the hot HPH procedure produces particles
with lower mean size and polydispersity than
the cold procedure [41]. Generally, the particle
size increases when lipids of long fatty acid
chains are employed. The use of mixtures of
short- and long-chain fatty acids may therefore
reduce the mean particle size and polydispersity
index of SLNs/NLCs [4,44].
 100
3.3 Zeta potential
Zeta potential is another parameter used to
evaluate the long-term stability of the particles
and its assessment is instrumental for the physi-
cochemical characterization of the nanoparticles'
dispersion. When determining the zeta potential,
it is possible to understand the interactions
between the particle and the drug. This param-
eter shows the surface electrical charge of the
particles, which is modified by changes in the
interface with the dispersing medium since there
is a dissociation of functional groups on the par-
ticle' surface and adsorption of ionic species of
the aqueous dispersion medium [39]. The mea-
surement of this parameter allows to clarify the
stability of the formulation during its storage
time. The higher the zeta potential, the higher
the electrostatic repulsion between the particles,
resulting in reduction of the risk of particles'
aggregation. According to literature, a zeta
potential higher than the 30 mV modulus gua-
rantees the stability of SLNs [45]. However, there
are SLN formulations with zeta potential below
|30 mV| that remain stable over time due to
the stereochemical stability offered by surfac-
tants [46]. Changing a formulation by varying
the concentration of its components, allows
understanding which of those whether
contribute to the stereochemical stability or to
the electrostatic stability [47]. Differential scan-
ning calorimetry, based on the study of the peaks
of SLN formulation recorded on the thermo-
gram, allows assessing if the drug is loaded
within the particles and eventually its contribu-
tion to the surface electrical charge of SLNs [48].
 3. Physicochemical properties
3.4 Particle morphology
To learn about the shape and size of nanopar-
ticles, scanning (SEM) and transmission electron
microscopy (TEM) are commonly used [50].
Atomic force microscopy (ATM) is another tech-
nique that gives information with high resolu-
tion in three dimensions at the nanometer scale
(even surface details at the atomic level), and is
also used to understand the surface morphology
of nanoparticles [39].
3.5 Differential scanning calorimetry
The physical and chemical changes of a sam-
ple can be measured as a function of its temper-
ature; differential scanning calorimetry (DSC)
quantifies the loss or heat gain resulting from
these changes. There are two types of DSC in-
struments: (1) the power compensate DSC,
which is made of two separate ovens; and (2)
the heat-flux DSC, which has only an oven that
heats up the reference and sample pans. The
sample and reference receive the heat through
the sample pan, which is placed in a disc that
is the main source of heat. Both differential
heat flow and sample temperature are moni-
tored, and the calorimetric sensitivity is main-
tained by the software linearization of the cell
calibration [52]. The endothermic processes, i.e.,
those that absorb heat, include fusion (melting),
boiling, sublimation, vaporization, desolvation,
as well as solid-solid transitions. The crucial
exothermic process, i.e., the one that releases en-
ergy, is crystallization. These analyzes can be
used to identify materials, investigate their pu-
rity, polymorphism or solvation, analyze quanti-
tative and qualitative degradation, aging, glass
transition temperature, as well as their affinity
to other substances. DSC is useful to obtain
information about the degree of crystallinity of
the lipid matrix and polymorphic behaviour. In
7
DSC analysis, a melting point depression is usu-
ally observed when transforming the bulk lipid
into lipid nanoparticles [48,52].
3.6 Stability of formulations and release
profile
The stability of the loaded drug within lipid
nanoparticles is dependent on the chemical
composition of the lipid matrix (e.g., type of
lipid, surfactant) and production procedure.
Upon storage, triglycerides undergo polymor-
phic changes that may result in drug expulsion
from the lipid nanoparticles [53]. Surfactants
also play a relevant role on the crystallization
behavior of the lipid nanoparticles, i.e.,
whether the recrystallization of the particles
occurs onto their surface or within the lipid
core [54]. The long-term stability of lipid nano-
particles over storage is therefore dependent
on the lipid and surfactant composition of the
particles and of the production procedure
[55]. The presence of imperfections in the lipid
matrix offers higher capacity to accommodate
drug molecules, improving the loading capac-
ity of the particles [19,56]. To achieve a modi-
fied release profile of the loaded drug, the
particles should however retain the drug dur-
ing storage until their administration. To
increase the encapsulation efficiency, the use
of mixed lipids (having fatty acids with
different liquid and solid chain lengths) is usu-
ally recommended. These blends create small
liquid reservoirs inside the particles e as hap-
pens in the NLCs e delaying the polymorphic
changes over storage [18, 53, 57]. The encapsu-
lation efficiency of lipophilic drugs is also
higher in SLNs and NLCs than hydrophilic
drugs. To load these latter in lipid matrices,
other strategies are needed such as the devel-
opment of insoluble conjugates between the
8 1. Solid lipid nanoparticles (SLN)
lipid and the drug by a covalent bonding [58,
59]. Hydrophilic drugs might show higher
risk to be partitioned to the aqueous phase dur-
ing the production of SLNs and create a drug-
enriched shell model. Indeed, upon cooling of
the dispersions, the lipid solidifies first, crystal-
lizes and forms the cores in which the hydro-
philic drug will be precipitating onto their
surface. This type of SLNs (drug-enriched shell
model) does not exhibit a modified release pro-
file but rather a fast release attributed to the
presence of drug onto the surface of the
SLNs. For drugs that solidify first (e.g., of
melting point higher than that of the solid
lipid) or for lipophilic drugs, a drug enriched
core model can be produced, which exhibits a
modified release profile [17]. Typical methods
used for assessment of the in vitro release are
the dialysis and the static or dynamic Franz
diffusion methods. The assays can be designed
so that isotonicity and pH value can be
adjusted to mimic the intended administration
route, and can also include the effect of protein
adsorption, plasma compatibility, whole blood
compatibility and sterilisation. It is known that
in a physiological environment, proteins can
bind the surfaces of nanoparticles, forming a
nanoparticleeprotein complex that influences
the biological response. Nanoparticles can be
incubated with bulk serum, plasma and also
with solutions of individual proteins, in order
to evaluate which physical properties are
responsible for the protein binding onto their
surface [60], such as the type of polymer used
to stabilize the particles [61,62]. Freeze-drying
may also be used to enhance the long-term
stability of SLNs and NLCs. Lipid
nanoparticles can be transformed in a dry
product to improve their physicochemical
stability [63].
4. Administration routes and drug
bioavailability
Pharmaceutical nanotechnology comes up as a
strategy to improve the bioavailability of poorly-
water soluble drugs, enhancing their therapeutic
effectiveness and reducing the risk of adverse re-
actions [64e67]. SLNs have been extensively
exploited as an interesting approach to improve
the drug’s bioavailability in particular those of
class II and IV of the biopharmaceutical classifica-
tion system (BCS) [64, 67]. Indeed, due to their
lipid composition, they may act as absorption en-
hancers [67]. On the other hand, surfactants sur-
rounding the particles besides ensuring their
steric stability in aqueous dispersion, they induce
specific surface-chemical properties and may also
modulate the biopharmaceutical profile. For the
selection of the best surfactant, several parameters
have to be taken into account e.g., hydrophilic-
lipophilic balance (HLB) values, their effect on
the lipid polymorphism and on the particle size.
The HLB values for the stabilization of oil-in-
water dispersions vary between 8e18 [68]. The
right choice of the surfactant minimizes the
risk of production of particles’ aggregates which
may compromise the stability of the dispersion
in vitro and its performance in vivo [45].
4.1 Topical and dermal routes
The administration of drugs through the skin
may contribute to increase the drug’s bioavail-
ability as it overcomes the first-pass meta-
bolism. This administration route reduces the
in inter/intra-patient variation and increases
patient compliance. However, it is also associ-
ated with interactions of drug and/or excipients
with skin that may cause irritation [69]. The
 4. Administration routes and drug bioavailability
loading of drugs within lipid nanoparticles can
minimize the risk of skin irritation and aller-
genic reactions, by preventing direct contact
between the drug and the skin and by control-
ling the drug release through the skin
[14,70e72]. Besides, as they are composed of
biocompatible and physiological lipids of
GRAS status, SLNs and NLCs exhibit low risk
of acute and/or chronic toxicity [10]. Most of
the surfactants used in the production of SLNs
and NLCs are already used in topical pharma-
ceutical or cosmetic formulations. To further
reduce the risk of irritation, the surfactants
should be non-ionic, while polyethoxylated sur-
factants should also be avoided
[14,42,49,73e75]. There is evidence that most
of the PEG-free surfactants have shown to stabi-
lize lipid nanoparticles without the need of
cosurfactants. In addition, when compared to
the PEG-containing counterparts, the PEG-free
surfactants have been shown to require less con-
centration of surfactant to obtain small and uni-
form particle size [76]. The methods used to
evaluate the skin irritation include typical in
vitro test methods based on the reconstructed
human epidermis (RhE) and also in vivo animal
tests. In vivo experiments are more useful but
their cost, tight regulation, and ethical issues to-
wards the promotion of reduction, reuse, and
recycling imply the need to develop improved
in vitro tests [77]. New strategies to decrease
skin irritation are based on the use of controlled
release systems, i.e., creating a gradual drug de-
livery that prevents the accumulation of high
concentrations of drug in the skin, which are
usually responsible for this skin irritation, and
also increase drug deposition in the piloseba-
ceous unit, which reduces the dose frequency
and risk of adverse events [78]. Lipid nanopar-
ticles have additional advantages as they can
prevent and even reduce skin irritation by the
reinforcement and repair of the stratum
9
corneum lipid film. Indeed, these nanoparticles
create a protective lipid film onto the skin upon
their topical application, promoting skin hydra-
tion [76].
4.2 Ocular delivery
For the treatment of eye diseases, direct ocular
instillation is the most accepted approach by pa-
tients. Lipid nanoparticles have also gained inter-
est as drug carriers for this administration route,
due to their biocompatibility with the ocular tis-
sues, mucoadhesiveness and modified-release
profile [79e81]. Conventional ophthalmic solu-
tions have low precorneal retention time. Lipid
nanoparticles increase the retention time of ocular
drugs, improving their bioavailability [82]. To be
suitable for ocular instillation, lipid nanoparticles
should be of small particle size to avoid blurred
vision and discomfort [83]. To avoid damage of
the corneal tissues, inflammation and immuno-
logic reactions, the formulations also need to
exhibit sterility, isotonicity and pH between
7e9. Toxicity of the formulations that could alter
the corneal epithelial integrity or disrupt the tis-
sue, resulting in deficient drug delivery into eye
(which is not their aim) also need to be consid-
ered [84]. The Draize rabbit eye test is routinely
used to evaluate eye irritation. This test was
developed with the aim to predict human eye irri-
tation of pharmaceutic and cosmetic products. Its
drawbacks are the ethical issues associated with
the use of animals, its costs and the number of
variables of the test [85].
4.3 Oral administration
Oral delivery is painless and is easy for self-
administration. This administration route has
high patient compliance and is appropriate for
outpatients. All these advantages make it the
most accepted drug administration route [86].
22 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds
Drug transporters are the proteins that are
present in many organs (liver, lung, kidney,
intestine, brain, skin, blood vessels, and others).
Naturally, the proteins play important roles for
traffic between organs and elimination of drugs
and foreign compounds. Their function some-
how is beneficial, but they may also display
deleterious effects that do not allow drugs to
enter the target organ to show their effects.
In the BBB, drug transporters and drug metabo-
lizing enzymes are also present to control the
access to the brain and local concentration of
endobiotics and xenobiotics.
Efflux pumps demonstrate resistance to cyto-
toxic drugs, hence also act as a barrier for drug
delivery. By their nature, efflux pumps are trans-
port proteins involved in the extrusion of toxic
substrates from cells into the external environ-
ment. These membrane proteins function as a
pump that can decrease the intracellular accu-
mulation of drug, leading to the ineffective
drug therapy. For better drug delivery, the key
to understanding how these pumps operate
involves the determination of the structures of
representative pumps and the elucidation of
the conformational changes that accompany
drug translocation.
4. Nanocarrier: a strategy to overcome
biological barriers
A primary reason for drug failing to demon-
strate its effect is the biology underlying the mo-
lecular-, cellular-, and tissue-level barriers,
which makes the delivery process extremely
complex. Therefore, to bypass the limitations
regarding the biology of conventional drug
delivery, it might be best to improve on the
concepts and approaches that are efficient and
effective [37,38].
One way to overcome barrier challenges
could be formation of effective drug delivery.
Nanoparticles, due to their smaller size and
various other properties like surface coating,
type of matrix used, and timely delivery, can
help to deliver a drug at a target site. The suc-
cessful outcome of the nanocarrier to help the
bioactive compounds show their effect upon
reaching the target site of action is also deter-
mined by the in vivo behavior of the nanocarrier,
in particular in the phase of biological membrane
passage to reach the systemic circulation, during
traveling in the circulation to reach the target site
and after reaching the target.
Various nanoparticles are being studied for
delivery of synthetic as well as bioactive drugs.
In this section, different drug delivery systems
explored for bioactive drug delivery are dis-
cussed (Table 2.1).
Different types of nanocarrier systems are
developed based on the characteristic of the
bioactive compounds as well as the aim of their
delivery target. Table 2.2 presents various nano-
systems along with their method of preparation.
4.1 Lipid-based nanocarrier systems
Considering the complex in vivo barrier sys-
tem in which the drugs are protected to be able
to survive for therapeutic presentation, various
strategies in the area of formulation and delivery
either to target the drug at specific site or to con-
trol the drug release have recently been reported
and described [31e36]. The former focuses more
on the use of the excipients, the composition, and
the manufacturing process, while the later em-
phasizes the drug-carrier constructs. The
following discussion reviews the typically used
drug delivery systems in bioactive delivery that
are effective in overcoming the biological bar-
riers, thereby improving the drug therapeutic in-
dex. In this category, we selected liposomes,
nanoemulsions, and lipid nanoparticles; other
forms of nanoparticles are still under research
and have not yet reached the market like the
above ones, hence we decided to discuss only
the relevant ones.
 4. Nanocarrier: a strategy to overcome biological barriers 23
TABLE 2.1 Bioactive-loaded nanocarrier systems and the potential medical promise.

Challenges in Added value of

Class Category Compound Desired functionality formulation nanoemulsions

Flavonoid Flavanols EGCG, catechin, Free-radical scavenging, Catechins generally - Increased

epicatechin anticancer, decreasing exhibit high water storage
cholesterol level in solubility. Partition in the stability of
blood, preventing lipophilic core is easily
arterial sclerosis, improved by adding degradable
thrombosis, heart 1-dodecanol catechins
attacks, reducing
fat and sugar uptake
Flavonols Quercetin, Free-radical scavenging, The scarce solubility of - Improved
kaempferol, fighting the effects of most flavonols in oil phase thermo- and
myricetin aging and inflammation, requires the addition of photostability
downregulating or amphiphilic molecules in - Enhanced
suppressing the lipophilic core delivery
inflammatory pathways
and functions

Flavones Apigenin, Antimutagenic, Supersaturated flavones - Improved

luteolin, rutin, antiinvasive, and in oil phases easily form physical
tangeretin antiproliferative agent crystals, requiring the stability
addition of compounds, - Bioaccessibility
such as soy protein
isolates, to slow down the
crystallization process
Flavanones Naringenin, Antiinflammatory, Flavanones exhibit poor - Increased
hesperidin anticarcinogenic, water solubility. Oil and release of
hepatoprotective, and emulsifier are added to compounds
antilipid peroxidation ensure maximum loading - Improved the
and stability in the bioavailability
nanoemulsion
Isoflavones Daidzein, Protection against Incorporation in oil Improved the
genistein, hormone-related droplets should be permeation
puerarin disorders, menopausal promoted by suitable through epidermal
symptoms, heart disease, emulsifiers (i.e., lecithin) layers
and osteoporosis
Nonflavonoids Hydroxybenzoic Gallic acid, Antiproliferative and Complexation with Increased
acids ellagic acid, antioxidant phospholipids is bioaccessibility,
p-hydroxybenzoic required to increase reduced
acid physical stability in interaction with
nanoemulsions the food matrix
Hydroxycinnamic Cinnamic acid, Free-radical scavenging, The solubility of some Improved
acids coumaric acid, preventing cell damage phenolic acids is very biological activity,
ferulic acid, by ultraviolet light, low in aqueous permeability, the
caffeic acid antiaging, decreasing solutions, but they can absence of
blood glucose easily be adsorbed at cytotoxic effect
oil/water interface of
nanoemulsions

(Continued)
Another random document with
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 Fig. 7.—Harem court in the palace of Sargon at Khorsabad; compiled from
Thomas.
Observe that the courts of the harem give access to three main
groups of chambers, and that those groups have no direct
communication with each other. Each of the three has its own
separate entrance. Observe also that the three bed chambers we
have mentioned have no entrances but those from the inner court;
that they are all richly decorated, and that nothing in their shape or
arrangement admits of the idea that they were for the use of
attendants or others in an inferior station—-oriental custom having at
all times caused such persons to sleep on carpets, mats, or
mattresses, spread on the paved floors at night and put away in
cupboards during the day—and you will allow that the conclusion to
which those who have studied the plan of Sargon’s harem have
arrived, is, at least, a very probable one. Sargon had three queens,
who inhabited the three suites of apartments; each had assigned to
her use one of the state bedrooms we have described, but only
occupied it when called upon to receive her royal spouse.[26] On
other nights she slept in her own apartments among her eunuchs
and female domestics. These apartments comprised a kind of large
saloon open to the sky, but sheltered at one end by a semi-dome (T,
X, and especially Z, where the interior is in a better state of
preservation). Stretched upon the cushions with which the daïs at
this end of the room was strewn, the sultana, if we may use such a
term, like those of modern Turkey, could enjoy the performances of
musicians, singers, and dancers, she could receive visits and kill her
time in the dreamy fashion so dear to Orientals. We have already
given (Vol. I. Fig. 55,) a restoration in perspective of the semi-dome
which, according to Thomas, covered the further ends of these
reception halls.[27]
Suppose this part of the palace restored to its original condition; it
would be quite ready to receive the harem of any Persian or Turkish
prince. The same precautions against escape or intrusion, the same
careful isolation of rival claimants for the master’s favours, would still
be taken. With its indolent and passionate inmates a jealousy that
hesitates at no crime by which a rival can be removed, is common
enough, and among: the numerous slaves a willing instrument for
the execution of any vengeful project is easily found. The moral, like
the physical conditions, have changed but little, and the oriental
architect has still to adopt the precautions found necessary thirty
centuries ago.
We find another example of this pre-existence of modern
arrangements in the vast extent of the palace offices. These consist
of a series of chambers to the south-west of the court marked A, and
of a whole quarter, larger than the harem, which lies in the south-
eastern corner of the mound, and includes several wide quadrangles
(B, C´, C, D, D´, F, G, &c.).[28] We could not describe this part of the
plan in detail without giving it more space than we can spare. We
must be content with telling our readers that by careful study, of their
dispositions and of the objects found in them during the excavations,
M. Place has succeeded in determining, sometimes with absolute
certainty, sometimes with very great probability, the destination of
nearly every group of chambers in this part of the palace. The south-
west side of the great court was occupied by stores; the rooms were
filled with jars, with enamelled bricks, with things made of iron and
copper, with provisions and various utensils for the use of the palace,
and with the plunder taken from conquered countries; it was, in tact,
what would now be called the khazneh or treasury. The warehouses
did not communicate with each other; they had but one door, that
leading into the great court. But opening out of each there was a
small inner room, which served perhaps as the residence of a store-
keeper.
At the opposite side of the court lay what Place calls the active
section of the offices (la partie active des dépendances), the rooms
where all those domestic labours were carried on without which the
luxurious life of the royal dwelling would have come to a standstill.
Kitchens and bakehouses were easily recognized by the contents of
the clay vases found in them; bronze rings let into the wall betrayed
the stables—in the East of our own day, horses and camels are
picketed to similar rings. Close to the stables a long gallery, in which
a large number of chariots and sets of harness could be conveniently
arranged, has been recognized as a coach-house. There are but few
rooms in which some glimpse of their probable destination has not
been caught. In two small chambers between courts A and B, the
flooring stones are pierced with round holes leading to square
sewers, which, in their turn, join a large brick-vaulted drain. The use
of such a contrivance is obvious.[29]
We may fairly suppose that the rooms in which no special
indication of their purpose was found, were mostly servants’
lodgings. They are, as a rule, of very small size.
On the other hand, courts were ample and passages wide. Plenty
of space was required for the circulation of the domestics who
supplied the tables of the seraglio and harem, for exercising horses,
and for washing chariots. If, after the explorations of Place, any
doubts could remain as to the purpose of this quarter of the palace,
they would be removed by the Assyrian texts. Upon the terra-cotta
prism on which Sennacherib, after narrating his campaigns,
describes the restoration of his palace, he says, “the kings, my
predecessors, constructed the office court for baggage, for
exercising horses, for the storing of utensils.” Esarhaddon speaks, in
another inscription, of “the part built by the kings, his predecessors,
for holding baggage, for lodging horses, camels, dromedaries and
chariots.”[30]
We have now made the tour of the palace, and we find ourselves
again before the propylæum whence we set out. This propylæum
must have been one of the finest creations of Assyrian architecture.
It had no fewer than ten winged bulls of different sizes, some
parallel, others perpendicular, to the direction of the wall. There were
six in the central doorway, which was, in all probability, reserved for
the king and his suite. A pair of smaller colossi flanked each of the
two side doors, through which passed, no doubt between files of
guards, the ceaseless crowd of visitors, soldiers, and domestics. The
conception of this façade, with its high substructure, and the
ascending: lines of a double flight of steps connecting it with the
town below, is really grand, and the size of the court into which it led,
not much less than two acres and a half, was worthy of such an
approach.
The huge dimensions of this court are to be explained, not only
by the desire for imposing size, but also by the important part it
played in the economy of the palace. By its means the three main
divisions, the seraglio, the harem, and the khan, were put into
communication with each other. When there were no particular
reasons for making a détour, it was crossed by any one desiring to
go from one part to another. It was a kind of general rendezvous and
common passage, and its great size was no more than necessary for
the convenient circulation of servants with provisions for the royal
tables, of military detachments, of workmen going to their work, of
the harem ladies taking the air in palanquins escorted by eunuchs,
and of royal processions, in which the king himself took part.
As to whether or no any part of the platform was laid out in
gardens, or the courts planted with trees and flowers, we do not
know. Of course the excavations would tell us nothing on that point,
but evidence is not wanting that the masters for whom all this
architectural splendour was created were not without a love for
shady groves, and that they were fond of having trees in the
neighbourhood of their dwellings. The hanging gardens of Babylon
have been famous for more than twenty centuries. The bas-reliefs
tell us that the Assyrians had an inclination towards the same kind of
luxury. On a sculptured fragment from Kouyundjik we find a range of
trees crowning a terrace supported by a row of pointed arches (Vol.
I., Fig. 42); another slab, from the same palace of Sennacherib,
shows us trees upheld by a colonnade (Fig. 8). If Sargon established
in any part of his palace a garden like that hinted at in the sculptured
scene in which Assurbanipal is shown at table with his wife (Vol. I.,
Fig. 27), it must have been in the north-western angle of the
platform, near the temple and staged tower. In this corner of the
mound there is plenty of open space, and being farther from the
principal entrances of the palace, it is more quiet and retired than
any other part of the royal dwelling. Here then, if anywhere, we may
imagine terraces covered with vegetable earth, in which the vine, the
fig, the pomegranate and the tall pyramid of the cypress, could
flourish and cast their grateful shadows. The existence of such
gardens is, however, so uncertain, that we have given them no place
in our attempts at restoration.
 Fig. 8.—A hanging garden; from
Layard.
For the service of such a building a liberal supply of water was
necessary. Whence did it come? and how was it stored? I have been
amazed to find that most of those who have studied the Assyrian
palaces have never asked themselves these questions.[31] One
might have expected to find the building provided, as is usual in hot
countries, with spacious cisterns that could be easily filled during the
rainy season; but neither at Khorsabad, Kouyundjik, nor Nimroud,
have the slightest traces of any such tanks been found. With the
materials at their disposal it would, perhaps, have been too difficult
for the Assyrian builders to make them water-tight. Neither have any
wells been discovered. Their depth must have been too great for
common use. We must remember that the height of the mound has
to be added to the distance below the ordinary surface of the country
at which watery strata would be tapped. It is, on the whole, probable
that the supply for the palace inmates was carried up in earthenware
jars, and that the service occupied a string of women, horses, and
donkeys, passing and repassing between the river, or rather the
canal, that carried the waters of the Khausser to the very foot of the
mound, and the palace, from morning until night.[32]

We have now concluded our study of the arrangements of an

Assyrian palace, and we may safely affirm that those arrangements
were not invented, all standing, by the architect of Sargon. They
were suggested partly by the nature of the materials used, partly by
the necessities to be met. The plan of an Assyrian palace must have
grown in scale and consistence with the power of the Assyrian kings.
As their resources became greater, and their engineers more skilled,
increased convenience and a richer decoration was demanded from
their architects. We have dwelt at length upon Khorsabad, because it
affords the completest and best preserved example of a type often
repeated in the course of ten or twelve centuries. In some respects,
in its constructive processes and the taste of its decorations, for
instance, the Assyrian palace resembled the other buildings of the
country; its chief originality consisted in the number of its rooms and
the principles on which they were distributed.
The method followed in the combination of these countless
apartments is, as M. Place has said, “almost naïve in its
simplicity.”[33] The plan is divided into as many separate
parallelograms as there were departments to be accommodated;
these rectangles are so arranged that they touch each other either at
an angle or by the length of a side, but they never penetrate one into
the other, and they never command one another. They are
contiguous, or nearly so, but always independent. Thus the palace
contains three main divisions, the seraglio, the harem, and the khan.
Each of these is a rectangle, and each lies upon one side of the
great common square marked A on our plan. The same principle
holds good in the minor subdivisions. These consist of smaller
rectangles, also opening upon uncovered courts, and without any
lateral communication with each other. Examine the plan and you will
see the system carried out as rigidly in the seraglio as in the harem.
Thus the various sections of the palace are at once isolated and
close together, so that their occupants could live their lives and
perform their duties in the most perfect independence.
The methodical spirit by which these combinations were
governed was all the more necessary in a building where no
superposition of one story upon another was possible. The whole
palace was one vast ground floor. To arrange on one level more than
thirty courtyards and more than two hundred halls and chambers, to
provide convenient means of access from one to the other, to keep
accessory parts in due subordination, to give each room its most
fitting place in the whole—such was the problem put before the
Assyrian constructor. Profiting by a long experience he solved it with
the utmost judgment, and proved himself to be wanting neither in
forethought, skill, nor inventive power.

§ 3. Other Palaces of Mesopotamia.

The type of palace we have studied at Khorsabad, is, like the

staged towers, a development from Chaldæan structures whose
leading lines were established many centuries before the princes of
Calah and Nineveh began to raise their sumptuous houses. The
sites of the ancient cities of Lower Chaldæa inclose buildings that
seem to date from a very remote epoch, buildings in which we may
recognize the first sketch, as it were, for the magnificent dwellings of
Sargon and Sennacherib.
The most important of these buildings, and the most interesting,
is the ruin at Warka, which Loftus calls Wuswas (Fig. 172, Vol. I.,
letter B on the plan).[34] Unfortunately his explorations were very
partial and his description is very summary, while his plan of the ruin
only gives a small part of it (Fig. 9). There is, however, enough to
show the general character of the structure. The latter stood upon a
rectangular mound about 660 feet long and 500 wide. In spite of the
enormous accumulation of rubbish, Loftus succeeded in making out
an open door in the outer wall, and several chambers of different
sizes communicating with a large court. There was the same
thickness of wall and the same absence of symmetry as at
Khorsabad; the openings were not in the middle of the rooms. In the
long wall, decorated with panels and grooves, which still stands
among the ruins to a height of about twenty-four feet and a length of
about 172 feet, the posterior façade, through which there was no
means of ingress and egress, may be recognized. We have already
copied Loftus’s reproduction of this façade for the sake of its
decoration (Fig. 100, Vol. I.).

Fig. 9.—Plan of a palace at Warka;

from Loftus.
The building at Sirtella (Tello) in which M. de Sarzec discovered
such curious statues, was less extensive; it was only about 175 feet
long by 102 wide. The faces of the parallelogram were slightly
convex, giving to the building something of the general form of a
terra-cotta tub (Fig. 150, Vol I.). Here the excavations were pushed
far enough to give us a better idea of the general arrangement than
we can get at Warka. A great central court, about which numerous
square and oblong apartments are arranged, has been cleared;
there is a separate quarter, which may be the harem; at one angle of
the court the massive stages of a zigguratt may be recognized. The
walls are entirely of burnt brick. They are decorated only on the
principal façade, where the ornaments belong to the same class as
those of Wuswas—semi-columns mixed with grooves in which the
elevation of a stepped battlement is reproduced horizontally.
In none of the ruins of habitations found in this district by the
English explorers, were the chambers other than rectangular. Taylor
cleared a few halls in two buildings at Mugheir (Fig. 10) and Abou-
Sharein (Figs. 11 and 12) respectively. Both of these stood on
artificial mounds, and it is difficult to believe that they were private
dwellings. The walls of several rooms at Mugheir seemed to have
been decorated with glazed bricks; at Abou-Sharein there was
nothing but roughly painted stucco. In one chamber the figure of a
man with a bird on his fist might yet be distinguished.
Fig. 10.—Plan of chambers at Mugheir;
from Taylor.
Fig. 11.—Plan of chambers at Abou-Sharein; from Taylor.
 Fig. 12.—Plan of chambers at Abou-Sharein; from Taylor.
It is in Babylon that we ought to have found the masterpieces of
this architecture, in that capital of Nebuchadnezzar where the
Chaldæan genius, just before it finally lost its autonomy, made the
supreme effort that resulted in the buildings attributed by the
travelled Greeks to their famous Semiramis. We have no reason to
disbelieve Ctesias when he says that there were two palaces in
Babylon, one on the left and another on the right bank of the
Euphrates. “Semiramis,” says Diodorus, following his usual guide,
“built a double residence for herself, close to the river and on both
sides of the bridge, whence she might at one and the same time
enjoy the view over the whole city, and, so to speak, keep the keys
of the most important parts of the capital in her own power. As the
Euphrates runs southward through Babylon, one of these palaces
faced the rising, the other the setting, sun. Round the palace that
faced westwards, she built a wall sixty stades in circumference,
&c.”[35]
 The larger and more richly decorated of the two palaces was that
on the left bank.[36] Its opposite neighbour has vanished and left no
trace. The Euphrates has been gradually encroaching on its right
bank ever since the days of antiquity, and has long ago disunited
and carried away the last stones and bricks of the western palace.
The eastern palace is on the other hand still represented by one of
the great mounds that dominate the plain; this mound is called the
Kasr, or castle (Fig. 183, vol. i.). Its circumference is now not far
short of a mile.[37] Its form is that of an oblong parallelogram, with its
longest side next the river and parallel to it. The flanks of the mound
have, however, been so deeply seamed by searchers for treasure
and building materials that no vestige of its arrangements is now to
be traced. The bricks employed in the building all bore the name of
Nebuchadnezzar.
South of the Kasr there is another mound, rising about one
hundred feet above the plain and very irregular in shape. This is Tel-
Amran-ibn-Ali, or Tell-Amran, (Fig. 183, vol. i.). It is agreed that this
contains all that remains of the hanging gardens, a conjecture that is
confirmed by the numerous tombs dating from the Seleucid, the
Parthian, and the Sassanid periods, which have been found in its
flanks whenever any excavation has been attempted.[38] Tell-Amram
seems to have been a far more popular depository for corpses than
either Babil, the Kasr, or the Birs-Nimroud, a preference which is
easily explained. Whether we believe, with Diodorus, that the
gardens were supported by great stone architraves, or with Strabo,
that they stood upon several stories of vaults, we may understand
that in either case their substructure offered long galleries which,
when the gardens were no longer kept up and the whole building
was abandoned to itself, were readily turned into burial places.[39]
The palace and temple mounds did not offer the same facilities.
They were solid, and graves would have had to be cut in them
before a corpse could be buried in their substance. The Kasr was a
ready-made catacomb into which any number of coffins could be
thrust with the smallest expenditure of trouble.
 Excavations in the Kasr and at Tell-Amran might bring many
precious objects to light, but we can hardly think that any room or
other part of a building in such good preservation as many of those
in the Assyrian palaces would be recovered. To the latter, then, we
shall have again to turn to complete our study of the civil architecture
of Mesopotamia.
If we have placed the edifices from which the English explorers
have drawn so many precious monuments in the second line, it is
not only because their exploration is incomplete, but also because
they do not lend themselves to our purpose quite so readily as that
cleared by MM. Botta and Place. At Khorsabad there have never
been any buildings but those of Sargon; city and palace were built at
a single operation, and those who undertake their study do not run
any risk of confusion between the work of different generations. The
plan we have discussed so minutely is really that elaborated by the
Assyrian architect to whom Sargon committed the direction of the
work. We can hardly say the same of the ruins explored by Mr.
Layard and his successors. The mounds of Nimroud and Kouyundjik
saw one royal dwelling succeed another, and the architects who
were employed upon them hardly had their hands free. They had, to
a certain extent, to reckon with buildings already in existence. These
may sometimes have prevented them from extending their works as
far as they wished in one direction or another, or even compelled
them now and then to vary the levels of their floors; so that it is not
always easy for a modern explorer to know exactly how he stands
among the ruins of their creations, or to clearly distinguish the work
of one date from that of another.[40]
It was at Nimroud that this perplexity was chiefly felt, until the
decipherment of the inscriptions came to enable different periods
and princes to be easily distinguished. This name of Nimroud,
handed down by the ancient traditions collected in Genesis, has
been given to a mound which rises about six leagues to the south of
Mossoul, on the left bank of the Tigris, and both by its form and
elevation attracts the attention of every traveller that descends the
stream. The river is now at some distance from the ruins, but as our
map shows (Fig. 1), it is easy to trace its ancient bed, which was
close to the foot of the mound. The latter is an elongated
parallelogram, about 1,300 yards in one direction, and 750 in the
other (see Vol. I., Fig. 145). Above its weather-beaten sides, and the
flat expanse at their summit, stood, before the excavations began,
the apex of the conical mound in which Layard found the lower
stories of a staged tower (Fig. 13). Calah seems to have been the
first capital of the Assyrian Empire and even to have preserved some
considerable importance after the Sargonids had transported the
seat of government to Nineveh, and built their most sumptuous
buildings in the latter city. Nearly every king of any importance, down
to the very last years of the monarchy, left the mark of his hand upon
Nimroud.[41]
Of all the royal buildings at Calah that which has been most
methodically and thoroughly cleared is the oldest of all, the north-
western palace, or palace of Assurnazirpal (885–860). It has not
been entirely laid open, but the most richly decorated parts,
corresponding to the seraglio at Khorsabad, have been cleared. The
adjoining plan (Fig. 14) shows arrangements quite similar to those of
Sargon’s palace. A large court is surrounded on three sides by as
many rectangular groups of apartments, each group forming a
separate suite, with its own entrances to the court.
 Fig. 13.—General view of Nimroud; from Layard.
The chief entrance faces the north. Two great doorways flanked
by winged and human-headed lions, give access to a long gallery (4
on plan). At the western end of this gallery there is a small platform
or daïs raised several steps above the rest of the floor. Upon this, no
doubt, the king’s throne was placed on those reception days when
subjects and vassals crowded to his feet. Some idea of what such a
reception must have been may be gained from an Indian Durbar, or
from the Sultan of Turkey’s annual review of all his great
functionaries of state at the feast of Courban-Baïram. I witnessed the
latter ceremony in the Old Seraglio in 1857, and when those great
officers, like the mollahs and sheiks of the dervishes, who had
preserved the turban and floating robes of the East, bent to the feet
of Abd-al-Medjid, I was irresistibly reminded of the pompous
ceremonials sculptured on the walls of Nineveh and Persepolis.
 Fig. 14.—-Plan of the north-western palace at Nimroud;
from Layard.
The walls of this saloon were entirely lined in their lower parts
with reliefs representing the king surrounded by his chief officers,
offering prayers to the god of his people and doing homage for the
destruction of his enemies and for successful hunts (Fig. 15). The
figures in these reliefs are larger than life. A doorway flanked by two
bulls leads into another saloon (2 on plan) rather shorter and
narrower than the first. In this the ornamentation is less varied. The
limestone slabs are carved with eagle-headed genii in pairs,
separated by the sacred tree (Vol. I., Fig. 8). The inner wall of this
saloon is pierced with a fine doorway leading into the central court
(1), while in one corner there is a narrower opening into a third long
hall (6), which runs along the eastern side of the court. It was in this
latter room that the finest sculptures, those that may perhaps be
considered the masterpieces of the Assyrian artists, were found.
Behind this saloon there was another, rather longer, but not quite so
wide (7); then five chambers, completing the palace on this side. To
the south of the great court there were two large halls (3 and 5)
similar in arrangement to those already mentioned but less richly
decorated, and several smaller rooms opening some into the halls,
others into the passages on the west of the court. As to whether the
latter was inclosed or not on the west by buildings like those on the
other three sides we cannot now be certain, as on that side the
mound has been much broken away by the floods of the Tigris,
which once bathed its foot. There is nothing to forbid the hypothesis
of a grand staircase on this side leading up from the river bank.[42]
In the central and south-western palaces, built by Shalmaneser II.
and his grandson Vulnirari III. the excavations have not been carried
far enough to allow the plans to be restored. The explorers have
been content to carry off inscriptions and fragments of sculpture in
stone, ivory, and metal.[43]
The south-western palace, or palace of Esarhaddon, has been
the scene of explorations sufficiently prolonged to give us some idea
of its general arrangements (Fig. 16). A curious circumstance was
noticed by the English explorers. While the works of Assurbanipal
bore the strongest marks of care and skill, those of Esarhaddon
showed signs of having been carried out with a haste that amounted
to precipitation, and his palace was never finished. Nearly all the
alabaster slabs were taken from older buildings.[44] Most of these
were fixed with their original carved surfaces against the wall, but a
few were turned the proper way. Doubtless, had time served, these
would have been smoothed down and reworked. Nothing was
finished, however, but the bulls and sphinxes at the doors (Vol. I. Fig.
85) and a few reliefs in their immediate neighbourhood.[45]
Esarhaddon died, no doubt, before the completion of the work, which
was never continued.
Fig. 15.—Assurnazirpal offering a libation to the gods after his victory over a wild
bull. British Museum. Drawn by Saint-Elme Gautier.
And yet his architect was by no means lacking in ambition. Upon
the southern face of the building he intended to build the largest hall,
which, so far as we know, was ever attempted in an Assyrian palace.
This saloon would have been about 170 feet long by 63 feet wide. As
soon as the walls were raised he saw that he could not roof it in.
Neither barrel vault nor timber ceiling could have so great a span. He
determined to get over the difficulty by erecting a central wall down
the major axis of the room, upon which either timber beams or the
springers of a double vault could rest. This wall was pierced by
several openings, and was stopped some distance short of the two
end walls. It divided the saloon into four different rooms (marked 1,
2, 3, 4 on our plan) each of which was by no means small. Even with
this modification the magnificence of the original plan did not entirely
disappear. The two colossal lions opposite the door were very wide
apart, and all the openings between the various subdivisions were
large enough to allow the eye to range freely over the whole saloon,
and to grasp the first thought of the architect in its entirety.