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Liposomes Methods and Protocols 3rd Edition Gerard
G.M. D'Souza Digital Instant Download
Author(s): Gerard G.M. D'Souza, Hongwei Zhang
ISBN(s): 9781071629536, 1071629530
Edition: 3
File Details: PDF, 9.73 MB
Year: 2023
Language: english
Methods in
Molecular Biology 2622

Gerard G.M. D'Souza

Hongwei Zhang Editors

Liposomes
Methods and Protocols
Third Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

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 Liposomes

Methods and Protocols

Third Edition

Edited by

Gerard G. M. D'Souza and Hongwei Zhang

School of Pharmacy, MCPHS University, Boston, MA, USA
Editors
Gerard G. M. D'Souza Hongwei Zhang
School of Pharmacy School of Pharmacy
MCPHS University MCPHS University
Boston, MA, USA Boston, MA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

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Preface

The first edition of MiMB Liposomes Methods and Protocols was a two-volume edition that
compiled a diverse array of protocols for pharmaceutical nanocarriers and biological mem-
brane models. The second edition on the other hand emerged as a single-volume collection
of basic protocols that was intended to serve as a complimentary everyday companion for
scientists at all levels interested in using liposomes for drug delivery purposes. The
continued popularity of MiMB Liposomes Methods and Protocols led to this third edition
being developed. In this edition, we have sought to continue with the theme of ensuring
that fundamental protocols for the preparation and characterization of liposomes are main-
tained in a single convenient volume and ensuring that tried and tested protocols remain
accessible to established and new investigators alike. The focus remains on entry-level
protocols for the preparation of liposomes, physicochemical characterization of liposomes,
lipid analysis, drug encapsulation, surface modification, stimuli response as well as cellular
interaction, and biodistribution. Also essential to the continued health of the field of
liposomology is an up-to-date history of its evolution. To this end, Chapter 1 from the
second edition has been revised. The new version titled “From Olive Oil Emulsions to
COVID-19 Vaccines: Liposomes Came First” is highly recommended as required reading
for anyone interested in liposomology. We are extremely grateful to all the contributors to
this edition and to Prof. John Walker the MiMB series editor for his guidance in the
preparation of this edition. It is our hope that this volume will continue to serve as a
convenient reference for graduate students, postdoctoral researchers, as well as established
investigators utilizing lipid-based systems in the fields of cell and molecular biology, drug
delivery, and physical chemistry.

Boston, MA, USA Gerard G. M. D'Souza

Hongwei Zhang

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 From Olive Oil Emulsions to COVID-19 Vaccines: Liposomes

Came First . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Volkmar Weissig
2 Preparation of DRV Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Sophia G. Antimisiaris
3 Preparation of Small Unilamellar Vesicle Liposomes Using Detergent
Dialysis Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Qingyue Zhong and Hongwei Zhang
4 Thin-Film Hydration Followed by Extrusion Method
for Liposome Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Hongwei Zhang
5 Ethanol Injection Method for Liposome Preparation . . . . . . . . . . . . . . . . . . . . . . . . 65
Guangsheng Du and Xun Sun
6 Preparation of Giant Vesicles with Mixed Single-Tailed
and Double-Tailed Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Lauren A. Lowe and Anna Wang
7 Scalable Liposome Synthesis by High Aspect Ratio Microfluidic
Flow Focusing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Jung Yeon Han, Zhu Chen, and Don L. Devoe
8 Preparation of Doxorubicin Liposomes by Remote
Loading Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Jian Chen
9 Magnetic Thermosensitive Liposomes Loaded with Doxorubicin . . . . . . . . . . . . . 103
Mohamad Alawak, Alice Abu Dayyih, Ibrahim Awak,
Bernd Gutberlet, Konrad Engelhardt, and Udo Bakowsky
10 Preparation and Physical Characterization of DNA Binding
Cationic Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Vaibhav Saxena
11 Tunable pH Sensitive Lipoplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Hélène Dhotel, Michel Bessodes, and Nathalie Mignet
12 Solid Lipid Nanoparticles for Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Wei-Chung Luo and Xiuling Lu
13 Stable Discoidal Bicelles: Formulation, Characterization,
and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Ying Liu, Yan Xia, Armin Tahmasbi Rad,
Wafa Aresh, Justin M. Fang, and Mu-Ping Nieh

vii
viii Contents

14 The Post-insertion Method for the Preparation

of PEGylated Liposomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Sherif E. Emam, Nehal E. Elsadek, Taro Shimizu,
and Tatsuhiro Ishida
15 Click Chemistry for Liposome Surface Modification . . . . . . . . . . . . . . . . . . . . . . . . 173
Maria Vittoria Spanedda, Marcella De Giorgi, Béatrice Heurtault,
Antoine Kichler, Line Bourel-Bonnet, and Benoı̂t Frisch
16 Surface Modification of Liposomes Using Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . 191
Mengran Guo, Zhongshan He, Xi He, and Xiangrong Song
17 Preparation and Characterization of Trastuzumab Fab-Conjugated
Liposomes (Immunoliposomes) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
ShanShan Jin, Huimin Li, and Yuhong Xu
18 Pyrophosphorylated-Cholesterol-Modified Bone-Targeting
Liposome Formulation Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Yanzhi Liu, Zhenshan Jia, Luoyang Ma, and Dong Wang
19 Method of Simultaneous Analysis of Liposome Components
Using HPTLC/FID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Sophia Hatziantoniou and Costas Demetzos
20 High-Performance Liquid Chromatography Coupled with Tandem
Mass Spectrometry Method for the Identification and Quantification
of Lipids in Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Yujie Shi and Xiaona Li
21 DPH Probe Method for Liposome-Membrane Fluidity
Determination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Wei He
22 Imaging of Liposomes by Negative Staining Transmission Electron
Microscopy and Cryogenic Transmission Electron Microscopy . . . . . . . . . . . . . . . 245
Anand S. Ubhe
23 Visualization and Characterization of Liposomes by Atomic
Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Konrad Engelhardt, Eduard Preis, and Udo Bakowsky
24 Determination of the Subcellular Distribution of Fluorescently
Labeled Liposomes Using Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Melani A. Solomon
25 Liposome Biodistribution via Europium Complexes. . . . . . . . . . . . . . . . . . . . . . . . . 277
Nathalie Mignet and Daniel Scherman
26 Quantification of a Fluorescent Lipid DOPE-NBD by an HPLC
Method in Biological Tissue: Application to Study Liposomes’
Uptake by Human Placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Louise Fliedel, Nathalie Mignet, Thierry Fournier,
Karine Andrieux, and Khair Alhareth

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Contributors

MOHAMAD ALAWAK • Department of Pharmaceutics and Biopharmaceutics, University of

Marburg, Marburg, Germany
KHAIR ALHARETH • Université Paris Cité, CNRS, INSERM, UTCBS, Unité des Technologies
Chimiques et Biologiques pour la Santé, Paris, France
KARINE ANDRIEUX • Université Paris Cité, CNRS, INSERM, UTCBS, Unité des Technologies
Chimiques et Biologiques pour la Santé, Paris, France
SOPHIA G. ANTIMISIARIS • Laboratory of Pharmaceutical Technology, Department of
Pharmacy, University of Patras, Patras, Greece; Institute of Chemical Engineering
Sciences, Foundation for Research and Technology Hellas, FORTH/ICE-HT, Patras,
Greece
WAFA ARESH • Department of Biomedical Engineering, University of Connecticut, Storrs,
CT, USA
IBRAHIM AWAK • Department of Pharmaceutics and Biopharmaceutics, University of
Marburg, Marburg, Germany
UDO BAKOWSKY • Department of Pharmaceutics and Biopharmaceutics, University of
Marburg, Marburg, Germany
MICHEL BESSODES • Université de Paris Cité, CNRS, INSERM, UTCBS, Unité des
Technologies Chimiques et Biologiques pour la Santé, Faculté de Pharmacie, Paris, France
LINE BOUREL-BONNET • Laboratoire de Conception et Applications des Molécules Bioactives,
UMR 7199 CNRS/Université de Strasbourg, équipe 3BIO, Faculté de Pharmacie, Illkirch,
France
JIAN CHEN • School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
ZHU CHEN • Department of Mechanical Engineering, University of Maryland, College Park,
MD, USA
ALICE ABU DAYYIH • Department of Pharmaceutics and Biopharmaceutics, University of
Marburg, Marburg, Germany
MARCELLA DE GIORGI • Laboratoire de Conception et Applications des Molécules Bioactives,
UMR 7199 CNRS/Université de Strasbourg, équipe 3BIO, Faculté de Pharmacie, Illkirch,
France
COSTAS DEMETZOS • Faculty of Pharmacy, Department of Pharmaceutical Technology,
National and Kapodistrian University of Athens, Athens, Greece
DON L. DEVOE • Department of Mechanical Engineering, University of Maryland, College
Park, MD, USA
HÉLÈNE DHOTEL • Université de Paris Cité, CNRS, INSERM, UTCBS, Unité des
Technologies Chimiques et Biologiques pour la Santé, Faculté de Pharmacie, Paris, France
GUANGSHENG DU • West China School of Pharmacy, Sichuan University, Chengdu, China
NEHAL E. ELSADEK • Department of Pharmacokinetics and Biopharmaceutics, Institute of
Biomedical Sciences, Tokushima University, Tokushima, Japan
SHERIF E. EMAM • Department of Pharmacokinetics and Biopharmaceutics, Institute of
Biomedical Sciences, Tokushima University, Tokushima, Japan; Faculty of Pharmacy,
Department of Pharmaceutics and Industrial Pharmacy, Zagazig University, Zagazig,
Egypt

ix
x Contributors

KONRAD ENGELHARDT • Department of Pharmaceutics and Biopharmaceutics, University of

Marburg, Marburg, Germany
JUSTIN M. FANG • Department of Biomedical Engineering, University of Connecticut, Storrs,
CT, USA
LOUISE FLIEDEL • Université Paris Cité, CNRS, INSERM, UTCBS, Unité des Technologies
Chimiques et Biologiques pour la Santé, Paris, France; Université Paris Cité, INSERM,
Pathophysiology and Pharmacotoxicology of the Human Placenta, Pre and Postnatal
Microbiota Unit (3PHM), Paris, France
THIERRY FOURNIER • Université Paris Cité, INSERM, Pathophysiology and
Pharmacotoxicology of the Human Placenta, Pre and Postnatal Microbiota Unit (3PHM),
Paris, France
BENOÎT FRISCH • Laboratoire de Conception et Applications des Molécules Bioactives, UMR
7199 CNRS/Université de Strasbourg, équipe 3BIO, Faculté de Pharmacie, Illkirch, France
MENGRAN GUO • Department of Critical Care Medicine, State Key Laboratory of Biotherapy
and Cancer Center, West China Hospital, Sichuan University, Chengdu, China
BERND GUTBERLET • Department of Pharmaceutics and Biopharmaceutics, University of
Marburg, Marburg, Germany
JUNG YEON HAN • Department of Mechanical Engineering, University of Maryland, College
Park, MD, USA
SOPHIA HATZIANTONIOU • Department of Pharmacy, Laboratory of Pharmaceutical
Technology, School of Health Sciences, University of Patras, Rion, Greece
WEI HE • School of Pharmacy, China Pharmaceutical University, Nanjing, China
XI HE • Department of Critical Care Medicine, State Key Laboratory of Biotherapy and
Cancer Center, West China Hospital, Sichuan University, Chengdu, China
ZHONGSHAN HE • Department of Critical Care Medicine, State Key Laboratory of
Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China
BÉATRICE HEURTAULT • Laboratoire de Conception et Applications des Molécules Bioactives,
UMR 7199 CNRS/Université de Strasbourg, équipe 3BIO, Faculté de Pharmacie, Illkirch,
France
TATSUHIRO ISHIDA • Department of Pharmacokinetics and Biopharmaceutics, Institute of
Biomedical Sciences, Tokushima University, Tokushima, Japan
ZHENSHAN JIA • Department of Pharmaceutical Sciences, College of Pharmacy, University of
Nebraska Medical Center, Omaha, NE, USA
SHANSHAN JIN • Hangzhou Highfiled Bipharmaceuticals Inc., Hangzhou, China
ANTOINE KICHLER • Laboratoire de Conception et Applications des Molécules Bioactives,
UMR 7199 CNRS/Université de Strasbourg, équipe 3BIO, Faculté de Pharmacie, Illkirch,
France
HUIMIN LI • Yunnan Key Laboratory of Screening and Research on Anti-pathogen Plant
Resources in Western Yunnan, Dali University, Dali, China
XIAONA LI • Department of Pharmacy, Peking University Third Hospital, Beijing, People’s
Republic of China
YANZHI LIU • Department of Pharmaceutical Sciences, College of Pharmacy, University of
Nebraska Medical Center, Omaha, NE, USA; Zhanjiang Central Hospital, Guangdong
Medical University, Zhanjiang, PR China
YING LIU • Department of Chemical and Biomolecular Engineering, University of
Connecticut, Storrs, CT, USA
 Contributors xi

LAUREN A. LOWE • School of Chemistry, UNSW Sydney, Sydney, NSW, Australia; Australian
Centre for Astrobiology, UNSW Sydney, Sydney, NSW, Australia
XIULING LU • School of Pharmacy, University of Connecticut, Storrs, CT, USA
WEI-CHUNG LUO • School of Pharmacy, University of Connecticut, Storrs, CT, USA
LUOYANG MA • Zhanjiang Central Hospital, Guangdong Medical University, Zhanjiang,
PR China
NATHALIE MIGNET • Université de Paris Cité, CNRS, INSERM, UTCBS, Unité des
Technologies Chimiques et Biologiques pour la Santé, Faculté de Pharmacie, Paris, France
MU-PING NIEH • Department of Chemical and Biomolecular Engineering, University of
Connecticut, Storrs, CT, USA; Department of Biomedical Engineering, University of
Connecticut, Storrs, CT, USA; Polymer Program, Institute of Materials Science, University
of Connecticut, Storrs, CT, USA
EDUARD PREIS • Department of Pharmaceutics and Biopharmaceutics, University of
Marburg, Marburg, Germany
ARMIN TAHMASBI RAD • Department of Biomedical Engineering, University of Connecticut,
Storrs, CT, USA
VAIBHAV SAXENA • Pharmaceutical Development, Sage Therapeutics, Cambridge, MA, USA
DANIEL SCHERMAN • Université Paris Cité, CNRS, INSERM, UTCBS, Unité des
Technologies Chimiques et Biologiques pour la Santé, Faculté de Pharmacie, Paris, France
YUJIE SHI • School of Pharmaceutical Sciences, Peking University, Beijing, People’s Republic
of China
TARO SHIMIZU • Department of Pharmacokinetics and Biopharmaceutics, Institute of
Biomedical Sciences, Tokushima University, Tokushima, Japan
MELANI A. SOLOMON • Catalent Cell and Gene Therapy, Gaithersburg, MD, USA
XIANGRONG SONG • Department of Critical Care Medicine, State Key Laboratory of
Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China
MARIA VITTORIA SPANEDDA • Laboratoire de Conception et Applications des Molécules
Bioactives, UMR 7199 CNRS/Université de Strasbourg, équipe 3BIO, Faculté de
Pharmacie, Illkirch, France
XUN SUN • West China School of Pharmacy, Sichuan University, Chengdu, China
ANAND S. UBHE • AbbVie, Irvine, CA, USA
ANNA WANG • School of Chemistry, UNSW Sydney, Sydney, NSW, Australia; Australian
Centre for Astrobiology, UNSW Sydney, Sydney, NSW, Australia
DONG WANG • Department of Pharmaceutical Sciences, College of Pharmacy, University of
Nebraska Medical Center, Omaha, NE, USA
VOLKMAR WEISSIG • Midwestern University College of Pharmacy Glendale, Department of
Pharmaceutical Sciences, Glendale, AZ, USA
YAN XIA • Department of Chemical and Biomolecular Engineering, University of
Connecticut, Storrs, CT, USA
YUHONG XU • Yunnan Key Laboratory of Screening and Research on Anti-pathogen
Plant Resources in Western Yunnan, Dali University, Dali, China
HONGWEI ZHANG • Department of Pharmaceutical Sciences, School of Pharmacy-Boston,
MCPHS University, Boston, MA, USA
QINGYUE ZHONG • Department of Pharmaceutical Sciences, School of Pharmacy-Boston,
MCPHS University, Boston, MA, USA
 Chapter 1

From Olive Oil Emulsions to COVID-19 Vaccines: Liposomes

Came First
Volkmar Weissig

Abstract
It has been a long journey from Pliny the Elder (23–79 AD) to the FDA approval of the first injectable
nanomedicine in 1997. A journey powered by intellectual curiosity, which began with sprinkling olive oil on
seawater and culminated in playing around with smears of egg lecithin on microscopic slides. This brief
review highlights how a few pairs of gifted hands attached to highly motivated brains have turned a curious
discovery made under a microscopic lens into novel nanotherapeutics including liposome-based anti-cancer
drugs and potent liposomal vaccines given to millions.

Key words Liposomes, Phospholipids, Biological membranes, Drug delivery, Membrane models,
Vaccines, COVID-19

1 Pliny the Elder, Benjamin Franklin, Singer, and Nicolson

Following the first observations of living cells under a simple light

microscope by Anton van Leeuwenhoek in the late 1600s, the
question ultimately arose as to what holds such microscopic struc-
tures all together. The history of trying to understand biological
membranes [1] can arguably be traced back to Pliny the Elder
(23–79 AD) who noted that “. . .sea water is made smooth by
olive oil, and so they sprinkle oil on their faces because it calms
the rough element. . .” [1, 2]. Being aware of Pliny’s observation or
not, about 1700 years later in 1774, Benjamin Franklin sprinkled
oil droplets on water in a pond in Clapham Common and saw the
oil spreading on the water surface until it was “smooth as a looking
glass.” He subsequently published his observation in the Philosoph-
ical Transactions of the Royal Society [1, 2]. The same experiment
was repeated again, i.e., for the third time in the span of almost
2000 years in 1890 by Lord Raleigh who was able to measure the
area to which a defined volume of oil would expand and in addition
also to calculate the thickness of the oil film [1, 2]. Approximately

Gerard G. M. D'Souza and Hongwei Zhang (eds.), Liposomes: Methods and Protocols,
Methods in Molecular Biology, vol. 2622, https://doi.org/10.1007/978-1-0716-2954-3_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

1
2 Volkmar Weissig

during the same time Lord Raleigh was investigating oil films,
Charles Ernest Overton, a young doctoral student at the University
of Zurich, found that the chemical nature of a substance, i.e., oily
versus watery, determines whether it passes through the membrane
into a cell which lead him to the conclusion that there are simila-
rities between cell membranes and lipids such as olive oil [1, 2]. In
1917, Langmuir described cell membranes as a layer of lipids one
molecule thick [3]. Eight years later, Gorter and Grendel con-
cluded from their studies that “the phospholipid molecules that
formed the cell membrane were arranged in two layers to form a
lipid bilayer” [4], and Danielli along with Robertson in 1935
proposed a model wherein bilayer of lipids were sequestered
between two monolayers of unfolded proteins [5]. The currently
still accepted fluid mosaic model was finally proposed by Singer and
Nicolson in 1972 [6, 7].

2 The Penny Dropped: Membranes Came First

Among those landmarks of biomembrane history, a serendipitous

observation made by Alec Bangham during the early 1960s
deserves undoubtedly a special place. In the early 1960s, biological
membranes were increasingly being visualized through the use of
electron microscopy. Following appropriate staining, membranes
showed up in electron micrographs as two dark bands separated by
a light region [8] which David Robertson interpreted correctly as
two opposed phospholipid monolayers [9]. According to Alec
Bangham “the concept of the bimolecular sheet of phospholipids
surrounding cells and cell organelles became compulsive, and soon
anyone who had an oscilloscope and enough patience was making
black lipid membranes (BLM)” [10]. Preparing a BLM must have
been quite challenging and apparently also requires some level of
experience [11]. Moreover, long-term experiments using BLM are
almost impossible due to the very limited life time of around 1 h or
less for any given BLM. Even nowadays, work is still continuing to
enhance the stability of BLMs in order to extend their life span
[12, 13]. Therefore, the design and development of a stable and
easy to prepare model for a biological membrane was highly called
for, something Alec Bangham might have been aware of but cer-
tainly did not plan on doing. During that time, i.e., the very early
1960s, Bangham was interested in studying the role biological
membranes, in particular phospholipids play in the process of
blood clotting. While doing so, he in his own words “played
around with smears of egg lecithin on microscope slides and
became fascinated by the way in which they reacted with water to
 From Olive Oil Emulsions to COVID-19 Vaccines: Liposomes Came First 3

form mobile fronds1 of delicate and intricate structure”

[10]. Around 1962 Alex Bangham got easy access to a newly
acquired electron microscope because its operator, Bob Horne,
was his sailing friend [10]. During the very first session at the
microscope, when looking at Bangham’s lipid dispersions in nega-
tively stained samples, they saw “unmistakable vesicles all over the
place” [10]. In Bangham’s own words “the penny dropped, phos-
pholipids in aqueous negatively stained samples were spontaneously
forming closed membrane systems” [10]. As much logical as it
appears to us today, the simple observation that phospholipids
when exposed to water spontaneously self-assemble into vesicular
structures surrounded by membranes has profound philosophical
implications, something which generally seems to be somewhat
underappreciated in the literature dealing with Bangham’s body
of work. As obvious as it now seems, there is no life without
membranes. “Membranes Came First” was the title of a talk Alec
Bangham gave at the beginning of the 1970s at Bristol University
proposing that “something like liposomes must have been available
to house the first forms of cellular life” [14]. This talk and a
subsequent sabbatical in Bangham’s lab inspired David Deamer
who soon found that phospholipid molecules could actually be
synthesized under simulated prebiotic conditions [15]. Moreover,
membrane forming amphiphilic molecules could also be found in
the meteorite which crashed down in Australia in September
1969 [16].

3 Multilamellar Smectic Mesophases

To prove this self-assembly behavior of hydrated phospholipids,

however, Bangham and Horne had to work two more years and
“at no time did (they) imagine that (their) model system would
prove to be anything like as useful as the BLM” [10]. They pub-
lished their findings in 1964 in the Journal of Molecular Biology
[17], referring to what they saw under the microscope as “multi-
lamellar smectic mesophases.” Shortly thereafter Alec Bangham
provided evidence for the ability of lipid bilayers to maintain con-
centration gradients of ions, which were disrupted upon adding
detergents to this system [18, 19]. According to David Deamer,
“this evidence, along with the planar bilayer models being devel-
oped at the same time, established that lipid bilayers are the primary
permeability barrier of all cell membranes. It was the membrane
equivalent of finding the double helix structure of DNA. . .”
[14]. Unfortunately, only one of these two groundbreaking dis-
coveries in life sciences was honored with the Nobel Prize. An early

1
Frond: A large leaf (palm, fern) with many divisions (Merriam Webster)
4 Volkmar Weissig

visitor in Bangham’s lab was Gerald Weissmann [14] who proposed

to call Bangham’s smectic mesophases “liposomes” which are
defined as microscopic vesicles composed of one or more lipid
bilayers [20]. Upon Weissmann’s return to the USA, he “pro-
ceeded to evangelize (liposomes) prodigiously” [10]. Another
early guest in Bangham’s lab during that time was Demetrios
Papahadjopoulos who intended to study the role of phospholipids
in the process of blood coagulation. But “he never had a chance,
and in no time at all was publishing papers [21, 22] on
liposomes” [10].
In the following years, Alec Bangham kept demonstrating the
usefulness of liposomes as a membrane model for studying funda-
mental membrane properties such as permeation but also adhesion
and fusion [23–25]. During the following four decades, biological
membrane models have grown in complexity and functionality
[26], but nevertheless, liposomes are, besides supported bilayers,
membrane nanodiscs and hybrid membranes, still an indisputably
important tool for membrane biophysicists and biochemists. Ever
since Bangham’s early studies, liposomes remain an essential and
basic model for the study of any biological, biochemical, biophysi-
cal, pharmacological, or pharmaceutical phenomenon which in one
way or another involves phospholipid membranes. Just to give a few
examples, liposomes are being used to study drug transfer and drug
uptake [27], cytochrome p450-dependent drug metabolism [28],
cellular osmosensors [29], as well as mechanosensitive ion channels
[30]. The amino acid transport through membranes [31], the
interactions of soluble proteins with membranes [32], and proper-
ties of integral membrane proteins [33] have been investigated
employing liposomes as a membrane model or as a model of lyso-
somes by incorporating lysozyme into these vesicles [34]. Using
liposomes, actin polymerization [35], lipid domains [36], respira-
tory cytochromes [37], antioxidant activities [38–40], colloidal
aggregation [41], membrane translocation [42], protein folding
[43], vesicular transport [44], and lipid organization in biological
membranes [45, 46] have been investigated. The list could go on.
In addition to using liposomes as a membrane model, Alec
Bangham extended the use of liposomes to the study of the mecha-
nism of action of drugs such as local anesthetics [47]. In 1989, on
the occasion of his paper about the “Diffusion of univalent ions
across the lamellae of swollen phospholipids” [18] being high-
lighted by Current Contents as “This Week’s Citation Classic,”
Bangham summarized “. . .liposomes proved not only to be a
most useful and revealing model of the passive areas of a cell
membrane. . . but also a pharmacological punching bag, notably
with regard to the action of anesthetics, a favored field of my own
before I retired” [10]. Bangham’s key finding about anesthetics,
published together with Sheena Johnson and Keith Miller [48],
was the fact that liposomes become permeable to ionic solutes in
From Olive Oil Emulsions to COVID-19 Vaccines: Liposomes Came First 5

the presence of general anesthetics, implying these drugs somehow

seem to insert themselves into phospholipid membranes or at least
disturb their integrity. Ten years later, Keith Miller commented:
“To a field whose most powerful model nearly seven decades ago
had been a jar of olive oil, the liposome’s arrival was a liberating
force” [14, 49]. Remarkably, Alec Bangham’s “creative instincts”
[14] became further apparent when he suggested to use liposomes
composed mainly of dipalmitoylphosphatidylcholine (DPPC) for
the treatment of infantile respiratory distress syndrome. He was
hoping that such lipid formulation could easily be delivered to the
airways of newborns and replenish the occasionally insufficient
amount of DPPC in the lungs of newborns. Notably, Alec Bang-
ham called such a lipid mixture “ALEC” which stands for “artificial
lung expanding compound” [14]. A paper summarizing a clinical
trial to evaluate ALEC states that “artificial surfactant (ALEC)
given to premature babies at birth significantly reduces their mor-
tality . . . and should prove a valuable addition to treatment”
[14, 50].
One year before his death, in his very last publication at the age
of 88 as the sole author (!), Alec Bangham sums up the impact of his
serendipitous observation from the beginning of the 1960s on his
scientific career by writing “it was the odd pattern of a well-drawn
drop of blood that initiated my curiosity and eased my career away
from morbid anatomy to that of the physical chemistry of cell
surfaces” [51]. But not only did this odd pattern of a well-drawn
drop of blood change Alec Bangham’s career and not only made his
peculiar observation of multilamellar smectic mesophases on a
microscopic slide biological membranes experimentally easily acces-
sible, his serendipitous discovery was to be followed later by an
entirely new sub-discipline in pharmaceutical science and technol-
ogy. His observation that the lamellar structures formed by phos-
pholipids exposed to aqueous buffers are able to sequester small
buffer molecules has led to the development of the entire colloidal
(nowadays called nano) drug delivery concept. It started by the
realization that liposomes, in addition to entrapping salts present in
the buffer solutions used to prepare liposomes, could also entrap
other molecules, for instance, pharmacologically active agents.
During that time, Brenda Ryman at the Royal Free Hospital
School of Medicine in London (UK) was working on glycogen
storage disease [52–54], in search of a system for the delivery of
glycosidase (a glycogen degrading enzyme) to hepatocytes. At that
time, Gregory Gregoriadis, based at the Albert Einstein College of
Medicine in New York, was working on a newly discovered [55, 56]
property of desialylated glycoproteins to home to the hepatocytes
after i.v. injection, thus enabling the transport of glycosidase chem-
ically linked to a desialylated glycoprotein to home to the liver.
Because of their common scientific interests, Ryman invited Gre-
goriadis to carry out postdoctoral work in her laboratory. The plan
6 Volkmar Weissig

was to test both, desialylated peptides linked to glycosidase and

liposomes, a hitherto untested system used by the Head of the
department (Jack Lucy) as a model for cell membranes. It was
eventually decided to start with liposomes which have never been
used in animal work nor had they ever been proposed as a drug
delivery system. Gregory Gregoriadis himself described his entry
into the at that time untested field of liposomes and its technology
in a poetic fashion [57] (reprinted with permission from Elsevier):
Albert Einstein, The Bronx, New York.
Gentiles and Jews (impartial to pork),
Academic discussions and ifs and buts
Ceroloplasmin and test tubes, decapitated rats.
Exciting sequel of a previous discovery.
One could deduce from the total recovery
Of counts injected by the parenteral route
Into the blood stream or the pad of the foot:
Desialylated proteins home to the liver!
Will drugs be targeted? Post-doctoral fever. . .
November of sixty-nine, still in New York,
expiring visa, in search of work.
Abandoned reagents of forgotten adventure,
unfinished note books. An issue of Nature,
classified advertisement, turn of fate
Dear Madam of London, U.K.
I am flying to join you, if I may.

Based on their initial work on the liposomal encapsulation of

amyloglucosidase, Gregory Gregoriadis together with the “Madam
of London”, i.e., Brenda Ryman proposed in 1971 liposomes as
carriers of enzymes or drugs as a new approach for the treatment of
diseases, including storage diseases [58]. With an entrapment effi-
ciency of between 4% and 10%, they succeeded in entrapping
Aspergillus niger amyloglucosidase (EC 3.2.1.3) together with
131
I-labelled albumin into liposomes composed of phosphatidyl-
choline, cholesterol, and dicetyl phosphate. Considering they had
prepared liposomes via probe sonication with an MSE 60 W soni-
cator [59], the encapsulation efficiency of proteins with up to 10%
was very good even by today’s standards. Thirteen years later, in
1984, he published together with Chris Kirby a protocol for “high
yield drug entrapment in liposomes” [60] which they called
dehydration–rehydration vesicles (DRVs). Over the following
30 years, the list of potentially therapeutic agents which have
been encapsulated into DRV’s has grown tremendously [61]. The
type of liposomes as made by Gregoriadis and Ryman via probe
sonication of a lipid suspension was years later named Small Uni-
lamellar Vesicles (SUVs), which have become known for their
generally low encapsulation efficiency.
Back to 1971, Gregoriadis and Ryman injected the amyloglu-
cosidase and labeled albumin containing liposomes intravenously
into rats. About 60% of all liposomes were eliminated from the
circulation within 10 min. Remarkably, using [3H]-cholesterol
From Olive Oil Emulsions to COVID-19 Vaccines: Liposomes Came First 7

labeled and 131I-albumin-loaded liposomes, they found that circu-

lating liposomes seemed to stay intact; no measurable leakage of
131
I-albumin was found. Most of the radioactivity was recovered in
the liver and to some extent in the spleen, with a maximum of 56%
of the injected dose after the first 15 min. This rapid clearance of
i.v. injected liposomes via organs of the RES system, as firstly
described by Gregoriadis and Ryman in 1971, proved to become
the major and almost insurmountable hurdle for getting liposome-
based drugs into the clinic. Subsequently, during the middle and
late 1980s, the extreme short half-life of liposomes in circulation
contributed to a general waning of the original enthusiasm asso-
ciated with liposomes as a potentially universal drug carrier except
for when the liver and spleen are the target tissues. This barrier
toward the clinical application of liposomes fell in 1990, when
Alexander Klibanov, who obtained his Ph.D. degree in Vladimir
Torchilin’s lab in Moscow and at that time worked as a Postdoc in
Leaf Huang’s lab then in Nashville, Tennessee, grafted polyethyl-
ene glycol (PEG) chains onto the surface of liposomes in order to
significantly prolong their circulation time [62]. Such flexible long
PEG chains literally shield liposomes from interacting with opso-
nins, thereby significantly slowing down their clearance from circu-
lation via the RES system [63, 64] In addition, what is extremely
important from the pharmaceutical manufacturer’s view of point,
these PEG chains also minimize vesicle to vesicle interactions
thereby reducing vesicle aggregation, which in turn dramatically
improves the stability of such liposomal formulations [65]. Only
5 years later, in 1995, the FDA (USA) approved the first injectable
liposomal drug, Doxil, which are doxorubicin-loaded liposomes
the surface of which is protected by a layer of PEG chains
[66]. But again back to 1971, after subcellular fractionation of
the liver following i.v. injection of radiolabeled and enzyme-loaded
liposomes into rats, Gregoriadis and Ryman recovered most of the
liposomes and their content in the mitochondrial-lysosomal frac-
tion as well as the cytosol of both, liver parenchymal and Kupffer
cells. In other words, liposomes indeed revealed themselves as “a
promising vehicle for the direction of enzymes and drugs to the
liver and spleen” [58]. Following this very first biodistribution
study of liposomes containing radiolabeled cargo (131I-albumin
and [3H] cholesterol), Gregoriadis traced i.v. injected liposomes
based on enzyme activity [67]. He encapsulated yeast beta-
fructofuranosidase (invertase) or, in control experiments,131I-la-
beled albumin into liposomes composed of phosphatidylcholine,
cholesterol, and phosphatidic acid. Liposomal encapsulation of
invertase almost completely shielded the enzyme from substrate
added to the liposome suspension, i.e., the beta-fructofuranosidase
activity in the liposomal preparations was latent. Following injec-
tion, the beta-fructofuranosidase activity in blood retained its
latency, but the activity declined to 50% of the injected dose in
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 Anthropology - Workbook
Third 2021 - Research Center

Prepared by: Prof. Jones

Date: July 28, 2025

Abstract 1: Interdisciplinary approaches

Learning Objective 1: Learning outcomes and objectives
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Theoretical framework and methodology
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Learning Objective 3: Learning outcomes and objectives
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 4: Experimental procedures and results
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 5: Study tips and learning strategies
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Case studies and real-world applications
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 6: Diagram/Chart/Graph]
Definition: Critical analysis and evaluation
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Interdisciplinary approaches
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Important: Study tips and learning strategies
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Ethical considerations and implications
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Module 2: Current trends and future directions
Remember: Critical analysis and evaluation
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Learning outcomes and objectives
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Assessment criteria and rubrics
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 13: Historical development and evolution
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Literature review and discussion
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Research findings and conclusions
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Assessment criteria and rubrics
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Comparative analysis and synthesis
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 18: Diagram/Chart/Graph]
Remember: Assessment criteria and rubrics
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Current trends and future directions
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Summary 3: Case studies and real-world applications
Definition: Assessment criteria and rubrics
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Experimental procedures and results
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 22: Diagram/Chart/Graph]
Note: Research findings and conclusions
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 23: Diagram/Chart/Graph]
Note: Key terms and definitions
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Practical applications and examples
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Experimental procedures and results
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Study tips and learning strategies
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 27: Current trends and future directions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Historical development and evolution
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Comparative analysis and synthesis
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Topic 4: Historical development and evolution
Note: Theoretical framework and methodology
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Critical analysis and evaluation
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Best practices and recommendations
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 33: Assessment criteria and rubrics
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Assessment criteria and rubrics
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Ethical considerations and implications
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Fundamental concepts and principles
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Assessment criteria and rubrics
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 38: Case studies and real-world applications
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 39: Case studies and real-world applications
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Results 5: Fundamental concepts and principles
Example 40: Problem-solving strategies and techniques
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Assessment criteria and rubrics
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 42: Historical development and evolution
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 43: Problem-solving strategies and techniques
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Critical analysis and evaluation
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 45: Diagram/Chart/Graph]
Key Concept: Learning outcomes and objectives
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 46: Diagram/Chart/Graph]
Important: Best practices and recommendations
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Definition: Comparative analysis and synthesis
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 48: Practical applications and examples
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 49: Diagram/Chart/Graph]
Note: Theoretical framework and methodology
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Conclusion 6: Key terms and definitions
Note: Case studies and real-world applications
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 51: Diagram/Chart/Graph]
Definition: Interdisciplinary approaches
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Research findings and conclusions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Literature review and discussion
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Experimental procedures and results
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Fundamental concepts and principles
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Comparative analysis and synthesis
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Experimental procedures and results
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 58: Learning outcomes and objectives
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Case studies and real-world applications
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 60: Diagram/Chart/Graph]
Unit 7: Research findings and conclusions
Definition: Practical applications and examples
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Problem-solving strategies and techniques
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Comparative analysis and synthesis
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Problem-solving strategies and techniques
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Practical applications and examples
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 65: Diagram/Chart/Graph]
Practice Problem 65: Study tips and learning strategies
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Problem-solving strategies and techniques
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 67: Critical analysis and evaluation
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Ethical considerations and implications
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 69: Literature review and discussion
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Test 8: Statistical analysis and interpretation
Example 70: Practical applications and examples
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 71: Learning outcomes and objectives
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Current trends and future directions
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 73: Diagram/Chart/Graph]
Note: Problem-solving strategies and techniques
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Practical applications and examples
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 75: Literature review and discussion
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Experimental procedures and results
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Comparative analysis and synthesis
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Historical development and evolution
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Statistical analysis and interpretation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice 9: Literature review and discussion
Important: Fundamental concepts and principles
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Learning outcomes and objectives
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Comparative analysis and synthesis
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Practical applications and examples
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Ethical considerations and implications
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Comparative analysis and synthesis
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Historical development and evolution
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 87: Interdisciplinary approaches
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 88: Historical development and evolution
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Critical analysis and evaluation
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 90: Diagram/Chart/Graph]
Summary 10: Literature review and discussion
Example 90: Theoretical framework and methodology
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Key terms and definitions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 92: Diagram/Chart/Graph]
Example 92: Learning outcomes and objectives
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 93: Critical analysis and evaluation
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Critical analysis and evaluation
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Critical analysis and evaluation
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Theoretical framework and methodology
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 97: Case studies and real-world applications
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 98: Practical applications and examples
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 99: Study tips and learning strategies
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Quiz 11: Practical applications and examples
Remember: Fundamental concepts and principles
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Best practices and recommendations
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Theoretical framework and methodology
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 103: Current trends and future directions
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 104: Diagram/Chart/Graph]
Example 104: Theoretical framework and methodology
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Practical applications and examples
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Learning outcomes and objectives
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 107: Learning outcomes and objectives
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 108: Study tips and learning strategies
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 109: Diagram/Chart/Graph]
Practice Problem 109: Case studies and real-world applications
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Lesson 12: Practical applications and examples
Example 110: Critical analysis and evaluation
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Theoretical framework and methodology
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Experimental procedures and results
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Interdisciplinary approaches
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Practical applications and examples
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Experimental procedures and results
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 116: Diagram/Chart/Graph]
Practice Problem 116: Ethical considerations and implications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Research findings and conclusions
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Fundamental concepts and principles
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 119: Diagram/Chart/Graph]
Note: Current trends and future directions
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Topic 13: Critical analysis and evaluation
Important: Problem-solving strategies and techniques
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 121: Diagram/Chart/Graph]
Key Concept: Experimental procedures and results
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 122: Experimental procedures and results
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 123: Diagram/Chart/Graph]
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