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Nanoparticles for Biomedical
Applications
Fundamental Concepts, Biological Interactions and
Clinical Applications

Edited by

Eun Ji Chung
Department of Biomedical Engineering, University of Southern California
Los Angeles, CA, United States

Lorraine Leon
Department of Materials Science and Engineering, NanoScience and Technology Center
University of Central Florida Orlando, FL, United States

Carlos Rinaldi
Department of Chemical Engineering, Department of Biomedical Engineering,
University of Florida, Gainesville, FL, United States
Elsevier
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Contributors

Nicholas J. Abuid, J. Crayton Pruitt Family Department of Comprehensive Cancer Center; Department of Surgery,
Biomedical Engineering, University of Florida, Gain- Division of Vascular Surgery and Endovascular Ther-
esville, FL, United States apy; Department of Medicine, Division of Nephrology
Aitor Álvarez, Centro Singular de Investigación en and Hypertension, Keck School of Medicine, University
Química Biolóxica e Materiais Moleculares (CiQUS), of Southern California, Los Angeles, CA, United States
Departamento de Física de Partículas, Universidade de Nathan D. Donahue, Stephenson School of Biomedical
Santiago de Compostela, Santiago de Compostela, Engineering, University of Oklahoma, Norman, OK,
Spain United States
Candace Benjamin, Department of Chemistry and Bio- Natalie Dong, Department of Biomedical Engineering;
chemistry, University of Texas at Dallas, Richardson, Michelson Center for Convergent Biosciences, Los
Texas, United States Angeles, CA, United States
Agata Blasiak, N.1 Institute for Health (N.1); Department Mitsushita Doomra, NanoScience Technology Center;
of Biomedical Engineering, NUS Engineering, National Burnett School of Biomedical Science, University of
University of Singapore, Singapore Central Florida, Orlando, FL, United States
Olivia Brohlin, Department of Chemistry and Bio- Marco A. Downing, Department of Chemical Engineering,
chemistry, University of Texas at Dallas, Richardson, Herbert Wertheim College of Engineering, University
Texas, United States of Florida
Sean Burkitt, Department of Biomedical Engineering; Omolola Eniola-Adefeso, Department of Chemical Engi-
Michelson Center for Convergent Biosciences, Los neering; Department of Biomedical Engineering; Mac-
Angeles, CA, United States romolecular Science and Engineering Program,
Jos Campbell, Department of Biomedical Engineering; University of Michigan, Ann Arbor, MI, United States
Michelson Center for Convergent Biosciences, Los Allen Eyler, Department of Materials Science and Engi-
Angeles, CA, United States neering, University of Central Florida, Orlando, FL,
Carolina Carrillo-Carrión, Centro Singular de Inves- United States
tigación en Química Biolóxica e Materiais Moleculares Catherine A. Fromen, Department of Chemical and Bio-
(CiQUS), Departamento de Física de Partículas, Uni- molecular Engineering, University of Delaware,
versidade de Santiago de Compostela, Santiago de Newark, DE, United States
Compostela, Spain Eric Fuller, J. Crayton Pruitt Family Department of Bio-
Yahya Cheema, Fischell Department of Bioengineering, medical Engineering, University of Florida, Gainesville,
University of Maryland, College Park, MD, United FL, United States
States Tiffany RX. Gan, N.1 Institute for Health (N.1); Depart-
Andreina Chiu-Lam, Department of Chemical Engineer- ment of Biomedical Engineering, NUS Engineering,
ing, University of Florida, Gainesville, FL, United National University of Singapore; Department of Sur-
States gery, National University Health System, Singapore
Eun Ji Chung, Department of Biomedical Engineering; Jeremiah J. Gassensmith, Department of Chemistry and
Department of Chemical Engineering and Materials Biochemistry, University of Texas at Dallas, Richard-
Science; Eli and Edythe Broad Center for Regenerative son, Texas, United States
Medicine and Stem Cell Research; Norris

xiii
xiv Contributors

Kerim M. Gattás-Asfura, J. Crayton Pruitt Family Forrest M. Kievit, University of Nebraska, Department of
Department of Biomedical Engineering, University of Biological Systems Engineering, Lincoln, NE, United
Florida, Gainesville, FL, United States States
Mengjie Gu, Department of Pharmacology, National Jonathan Kin-Hun Lee, Department of Chemical Engi-
University of Singapore, Singapore; Cancer Science neering, University of Michigan, Ann Arbor, MI,
Institute of Singapore, National University of Singa- United States
pore, Singapore, Singapore Emily L. Kolewe, Department of Chemical and Bio-
Hao Guo, Department of Pharmacology and Pharmaceut- molecular Engineering, University of Delaware,
ical Sciences, School of Pharmacy, University of Newark, DE, United States
Southern California, Los Angeles, CA, United States Truong Thanh Lan Anh, N.1 Institute for Health (N.1);
Ankur Gupta, Department of Mechanical and Aerospace Department of Biomedical Engineering, NUS Engi-
Engineering; Princeton University, Princeton, NJ, neering, National University of Singapore, Singapore
United States Daniel J. LaShoto, J. Crayton Pruitt Family Department of
Dean Ho, N.1 Institute for Health (N.1); Department of Biomedical Engineering, University of Florida, Gain-
Biomedical Engineering, NUS Engineering; Depart- esville, FL, United States
ment of Pharmacology, Yong Loo Lin School of Tram Le, Stephenson School of Biomedical Engineering,
Medicine, National University of Singapore, Singapore University of Oklahoma, Norman, OK, United States
Leaf Huang, Division of Pharmacoengineering and Dao P. Le, Stephenson School of Biomedical Engineering,
Molecular Pharmaceutics and Center for Nano- University of Oklahoma, Norman, OK, United States
technology in Drug Delivery, Eshelman School of
Pharmacy, University of North Carolina at Chapel Hill, Joanne C. Lee, Stephenson School of Biomedical Engi-
Chapel Hill, NC, United States neering, University of Oklahoma, Norman, OK, United
States
Huang-Chiao Huang, Fischell Department of Bio-
engineering, University of Maryland, College Park, Lorraine Leon, Department of Materials Science and
MD; Marlene and Stewart Greenebaum Cancer Center, Engineering; NanoScience and Technology Center,
University of Maryland School of Medicine, Baltimore, University of Central Florida, Orlando, FL, United
MD, United States States
Collin T. Inglut, Fischell Department of Bioengineering, Barry J. Liang, Fischell Department of Bioengineering,
University of Maryland, College Park, MD, United University of Maryland, College Park, MD, United
States States
Aaron J. Sorrin, Fischell Department of Bioengineering, John Andrew MacKay, Department of Pharmacology and
University of Maryland, College Park, MD, United Pharmaceutical Sciences, School of Pharmacy;
States Department of Biomedical Engineering, Viterbi School
of Engineering; Department of Ophthalmology, Roski
Piyush K. Jain, Department of Chemical Engineering, Eye Institute, Keck School of Medicine, University of
Herbert Wertheim College of Engineering, University Southern California, Los Angeles, CA, United States
of Florida
Raquel Martínez, Centro Singular de Investigación en
Bader M. Jarai, Department of Chemical and Bio- Química Biolóxica e Materiais Moleculares (CiQUS),
molecular Engineering, University of Delaware, Departamento de Física de Partículas, Universidade de
Newark, DE, United States Santiago de Compostela, Santiago de Compostela,
Edward Kai-Hua Chow, Department of Pharmacology; Spain
Cancer Science Institute of Singapore, National Uni- Tyler Maxwell, Department of Chemistry; NanoScience
versity of Singapore, Singapore, Singapore Technology Center, University of Central Florida,
Theodore Kee, N.1 Institute for Health (N.1); Department Orlando, FL, United States
of Biomedical Engineering, NUS Engineering, National Michael McKenna, Department of Chemical Engineering,
University of Singapore, Singapore University of Washington, Seattle, WA, United States
Jeffrey Khong, N.1 Institute for Health (N.1); Department Samantha A. Meenach, Department of Biomedical and
of Biomedical Engineering, NUS Engineering, National Pharmaceutical Sciences, College of Pharmacy;
University of Singapore, Singapore Department of Chemical Engineering, College of
 Contributors xv

Engineering, University of Rhode Island, Kingston, RI, Alexia M. Poulos, J. Crayton Pruitt Family Department of
United States Biomedical Engineering, University of Florida, Gain-
Michael Mellas, Pritzker School of Molecular Engineer- esville, FL, United States
ing, University of Chicago, Chicago, IL, United States Nisha Raman, Department of Chemical and Biomolecular
Martina Migliavacca, Centro Singular de Investigación en Engineering, University of Delaware, Newark, DE,
Química Biolóxica e Materiais Moleculares (CiQUS), United States
Departamento de Física de Partículas, Universidade de Carlos Rinaldi, Department of Chemical Engineering; J.
Santiago de Compostela, Santiago de Compostela, Crayton Pruitt Family Department of Biomedical
Spain Engineering, University of Florida, Gainesville, FL,
Daniel Najafali, Fischell Department of Bioengineering, United States
University of Maryland, College Park, MD, United Angelie Rivera-Rodriguez, J. Crayton Pruitt Family
States Department of Biomedical Engineering, University of
Elizabeth Nance, Department of Chemical Engineering; Florida, Gainesville, FL, United States
Department of Radiology; Center on Human Develop- Hanieh Safari, Department of Chemical Engineering,
ment and Disability; Molecular Engineering and Sci- University of Michigan, Ann Arbor, MI, United States
ences Institute, University of Washington, Seattle, WA, Swadeshmukul Santra, Department of Chemistry, Uni-
United States versity of Central Florida, Orlando, FL, United States;
Steven M. Narum, Stephenson School of Biomedical NanoScience Technology Center, University of Central
Engineering, University of Oklahoma, Norman, OK, Florida, Orlando, FL, United States; Burnett School of
United States Biomedical Science, University of Central Florida,
María F. Navarro Poupard, Centro Singular de Inves- Orlando, FL, United States; Department of Materials
tigación en Química Biolóxica e Materiais Moleculares Science and Engineering, University of Central Florida,
(CiQUS), Departamento de Física de Partículas, Uni- Orlando, FL, United States
versidade de Santiago de Compostela, Santiago de Shehaab Savliwala, Department of Chemical Engineering,
Compostela, Spain University of Florida, Gainesville, FL, United States
Jiansheng Ng, N.1 Institute for Health (N.1); Department Kacoli Sen, Department of Chemical Engineering, Uni-
of Biomedical Engineering, NUS Engineering, National versity of Florida, Gainesville, FL, United States
University of Singapore, Singapore Nishan K. Shah, Department of Biomedical and Pharma-
Maria Gabriela Nogueira Campos, NanoScience Tech- ceutical Sciences, College of Pharmacy, University of
nology Center, University of Central Florida, Orlando, Rhode Island, Kingston, RI, United States
FL, United States; Institute of Science and Technology, Sachit Shah, Department of Materials Science and Engi-
Federal University of Alfenas, Poços de Caldas, Minas neering, University of Central Florida, Orlando, FL,
Gerais, Brazil United States
Beatriz Pelaz, Centro Singular de Investigación en Quí- Arezoo Shahrivarkevishahi, Department of Chemistry
mica Biolóxica e Materiais Moleculares (CiQUS), and Biochemistry, University of Texas at Dallas,
Departamento de Física de Partículas; Centro Singular Richardson, Texas, United States
de Investigación en Química Biolóxica e Materiais
Moleculares (CiQUS), Departamento de Química Stephen Smith, Department of Chemistry, University of
Inorgánica, Universidade de Santiago de Compostela Central Florida, Orlando, FL, United States; Nano-
Santiago de Compostela, Spain Science Technology Center, University of Central
Florida, Orlando, FL, United States
Pablo del Pino, Centro Singular de Investigación en Quí-
mica Biolóxica e Materiais Moleculares (CiQUS), Enrica Soprano, Centro Singular de Investigación en
Departamento de Física de Partículas, Universidade de Química Biolóxica e Materiais Moleculares (CiQUS),
Santiago de Compostela, Santiago de Compostela, Departamento de Física de Partículas, Universidade de
Spain Santiago de Compostela, Santiago de Compostela,
Spain
Ester Polo, Centro Singular de Investigación en Química
Biolóxica e Materiais Moleculares (CiQUS), Departa- Jillian Stabile, Fischell Department of Bioengineering,
mento de Física de Partículas, Universidade de Santiago University of Maryland, College Park, MD, United
de Compostela, Santiago de Compostela, Spain States
xvi Contributors

Cherie L. Stabler, J. Crayton Pruitt Family Department of Jonathan Wang, Biomedical Engineering, University of
Biomedical Engineering, University of Florida, Gain- Southern California, Los Angeles, CA, United States
esville, FL, United States; University of Florida Dia- Peter Wang, N.1 Institute for Health (N.1), National
betes Institute, Gainesville, FL, United States University of Singapore, Singapore; Department of
Zachary S. Stillman, Department of Chemical and Bio- Biomedical Engineering, NUS Engineering, National
molecular Engineering, University of Delaware, University of Singapore, Singapore
Newark, DE, United States Zimeng Wang, Phosphorex Inc., Hopkinton, MA, United
Sara Tabandeh, Department of Materials Science and States
Engineering, University of Central Florida, Orlando, Stefan Wilhelm, Stephenson School of Biomedical Engi-
FL, United States neering, University of Oklahoma, Norman, OK, United
Aria W. Tarudji, University of Nebraska, Department of States; Stephenson Cancer Center, Oklahoma City, OK,
Biological Systems Engineering, Lincoln, NE, United United States
States Jingru Xu, Department of Pharmacology, National Uni-
Marcus Threadcraft, College of Medicine, University of versity of Singapore, Singapore; Cancer Science Insti-
Florida, Gainesville, FL, United States tute of Singapore, National University of Singapore,
Zon Thwin, Department of Chemistry, University of Singapore, Singapore
Central Florida, Orlando, FL, United States; Nano- Wen Yang, Stephenson School of Biomedical Engineer-
Science Technology Center, University of Central ing, University of Oklahoma, Norman, OK, United
Florida, Orlando, FL, United States States
Matthew Tirrell, Pritzker School of Molecular Engineer- Cristina Zavaleta, Department of Biomedical Engineer-
ing, University of Chicago, Chicago, IL, United States ing, University of Southern California, Los Angeles,
Elisa A. Torrico Guzmán, Department of Chemical CA, United States; Michelson Center for Convergent
Engineering, College of Engineering, University of Biosciences, Los Angeles, CA, United States
Rhode Island, Kingston, RI, United States Yuan Zhang, Department of Biomedical and Pharma-
Mythreyi Unni, Department of Chemical Engineering, ceutical Sciences, College of Pharmacy, University of
University of Florida, Gainesville, FL, United States Rhode Island, Kingston, RI, United States

Swapna Vaja, Fischell Department of Bioengineering,

University of Maryland, College Park, MD, United
States
Chapter 1

A brief history of nanotechnology and

introduction to nanoparticles for
biomedical applications
Lorraine Leon1, 2, Eun Ji Chung3 and Carlos Rinaldi4, 5
1
Department of Materials Science and Engineering, University of Central Florida, Orlando, FL, United States; 2NanoScience and Technology
Center, University of Central Florida, Orlando, FL, United States; 3Department of Biomedical Engineering, University of Southern California, Los
Angeles, CA, United States; 4Department of Chemical Engineering, University of Florida, Gainesville, FL, United States; 5Department of Biomedical
Engineering, University of Florida, Gainesville, FL, United States

According to the United States National Nanotechnology Examples of nanomaterials are abundant in nature,
Initiative, nanotechnology is the understanding and control ranging from magnetotactic bacteria that use iron oxide
of matter at dimensions approximately between 1 and nanoparticles to sense magnetic fields3 to the unique colors
100 nm that enable unique size-dependant properties.1 of butterfly wings which originate from the interactions of
However, slight variations of this definition exist light with complex nanoarchitectures and not absorption
depending on the governing body, such as the European due to dyes or pigments.4 Evidence of use of nanomaterials
Commission or the International Organization for Stan- in human history can be found dating back to the fourth
dardization (ISO) Technical Committee 229, and some- century AD in the form of the “Lycurgus Cup,” which has
times sizes in the 1000 nm range are considered unusual optical properties arising from metallic nano-
nanomaterials. As this is an evolving field, the nomen- particles that make the cup appear either red or green in
clature is being constantly updated, most recently via the transmitted or reflected light, respectively.5 These unique
creation of an international working group establishing a properties of metallic nanoparticles were analyzed by
uniform descriptor system for materials on the nanoscale.2 Michael Faraday in 1857, who concluded that gold at small
This 1e100 nm size range can correspond to individual size scales produces unusual colors.6 A related observation
molecules for polymers or other macromolecules but can that the optical properties of gold at small length scales
also include higher-order assemblies into nanoparticles. would differ from bulk properties was made by Gustav Mie
For smaller, angstrom-sized atoms and molecules, this size in 1908.7 However, purposefully manipulating matter at the
range consists of small clusters or nanoparticles. Material nanoscale is a relatively recent concept, famously concep-
properties at the nanoscale are different compared with tualized by Richard Feynman in a lecture given at the
that of bulk materials, generally stemming from the char- American Physical Society in 1959 titled “There is Plenty
acteristics that are pertinent to the larger surface area to of Room at the Bottom: An Invitation to Enter a New Field
volume ratio leading to changes in chemical reactivity and of Physics”.8 This speech outlined a vision for manipu-
quantum confinement effects. For biological systems, the lating and observing things on a small scale and how this
nanometer size range can be ideal for circulating in the would revolutionize many industries as well as provide
blood stream, traversing tissues, and entering cells. This answers to many fundamental questions, particularly in the
book surveys a variety of nanoparticles and their appli- context of understanding biology. The forward vision for
cations in the biomedical arena paying close attention to the medical field was attributed to Albert Hibbs and his
fundamental concepts in nanoparticle design and synthe- concept of “swallowing the surgeon” such that diagnosis
sis, the interactions and transport of nanoparticles within could be made by tiny robots that access the interior of the
biological systems, and ultimately the use of nanoparticles body to identify the problem and repair it.8 Following this
in a clinical setting. speech, the actual term “nanotechnology” would not be

Nanoparticles for Biomedical Applications. https://doi.org/10.1016/B978-0-12-816662-8.00001-1 1

Copyright © 2020 Elsevier Inc. All rights reserved.
2 Nanoparticles for Biomedical Applications

used until 1974 by Norio Tanaguchi, where he defined to their intended cellular target without harming healthy
nanotechnology as being able to manipulate a single tissue in the early 1900s.20 This concept was later merged
nanoscale object.9 with nanotechnology into the concept of the targeted
In the early to mid 1980s, the development of the nanoparticle. The term “nanoparticle” itself began appear-
scanning tunneling microscope10 by Binning and Rohrer ing in scientific publications in 1978 and, interestingly, the
(both at IBM) and the atomic force microscope11 by sole publications in 1978 and 1979 containing the word
Binning allowed the imaging of surfaces with atomic res- nanoparticle also contained the word “Medicine” or
olution. The imaging of atoms with the scanning tunneling “Medical” (Fig. 1.1). The publication in 1978 analyzed the
microscope later led to the placement of atoms in particular in vivo distribution of 400 nm gelatin nanoparticles.
positions, famously spelling out IBM using xenon atoms in However, nanoparticle research was being conducted in the
199012 and marking the beginning of the realization of late 1960s and early 1970s using different terms such as
nanotechnology. Also in the early 1980s, K. Eric Drexler at “nanopellet” or “nanocapsule”.21 In addition, lipid-based
the Massachusetts Institute of Technology was developing nanoparticles called “liposomes” (described in Chapter
an alternative vision for nanotechnology based on bottom- 10) were discovered in the 1960s by Alec Bangham when
up nanotechnology or molecular engineering that focused attempting to image lipids using negative staining electron
heavily on biological mechanisms such as protein design.13 microscopy.22 Nanoparticle research has grown tremen-
This vision inspired by Feynman’s 1959 speech led to the dously throughout the years, and the proportion of nano-
first book on nanotechnology14 titled “Engines of Creation: particle research dedicated to the medical field is
The Coming Era of Nanotechnology” published in 1986 significant, as illustrated in Fig. 1.1. The remainder of this
and the creation of the first organization dedicated to the book will outline recent developments in the field of
development of nanotechnology, the Foresight Institute that nanoparticles for biomedical applications.
same year. In 1985, Kroto, Heath, and Smalley from Rice Chapters 2e9 and 22 describe fundamental concepts
University discovered the 60 carbon atom structure known that can be applied to a variety of nanoparticle types.
as Buckminsterfullerene,15 or colloquially the “Buckyball.” Chapter 2 describes nanoparticle integrity, protein corona,
In the 1990s, the field would continue to grow, leading and colloidal stability in biological environments. Chapter
to the creation of the first journal dedicated to the subject 3 describes different targeting mechanisms and targeting
titled “Nanotechnology,” published by IOP Publishing in ligands used for nanoparticles, while Chapter 5 analyzes
the United Kingdom and the introduction of the term how size, shape, particle rigidity, and blood hemodynamics
“Nanomedicine” in a book coauthored by Drexler. The affect targeting. Chapter 4 describes passive targeting
scientific advancements in the field continued to flourish, techniques specifically in the context of leaky vasculature
including different types of nanoparticles described in this in cancer applications. Chapter 6 describes different
book, such as the discovery of the carbon nanotube in methods to administer nanoparticles, such as parenteral,
199116 (described in Chapter 14) or the discovery of the pulmonary, nasal, oral, transdermal, and ocular. Chapter 7
polyelectrolyte complex micelle (polyion complex micelle) describes the different surface, en-route, and cellular bar-
by Kataoka in 199517 (described in Chapter 20). riers that nanoparticles face once entering the body, and the
Eventually, the enthusiasm and promise of nanotech- design challenges introduced by these barriers. Chapter 8
nology would begin permeating the political sphere, lead- describes how to perform and interpret pharmacokinetic
ing to the creation of the Interagency Working Group on studies of nanoparticles to facilitate their approval in the
Nanotechnology in 1998, which catalyzed the National clinic. Chapter 9 describes common methods of character-
Nanotechnology Initiative in the United States in 2000.18 izing nanoparticles such as electron microscopy and light-
Similar initiatives would begin around the globe, such as scattering techniques. Chapter 22 discusses how the
the Canadian National Institute for Nanotechnology formed emerging field of artificial intelligence can play a role in
in 2001. These surges in funding would bring many new optimizing combinatorial nanoparticle therapies for
developments to the field, particularly in the context of oncology and infectious diseases.
medicine. The United States National Institute of Health Chapters 10e21 describe different types of nano-
National Cancer Institute launched an alliance for nano- particles and their applications in a biomedical context.
technology in cancer in 2004, establishing multiinstitu- Chapter 10 describes the design and fabrication of different
tional collaboration, research, training, and characterization liposomes. Chapter 11 describes proteinaceous cages,
centers. These Centers of Cancer Nanotechnology Excel- called virus-like particles. Chapter 12 discusses gold
lence are scheduled to lose funding in 2020, claiming not nanoparticles and their application in photothermal therapy,
lack of interest in the field but the transition of nanotech- surgery, and imaging. Chapter 13 discusses magnetic
nology from an emerging field to a more established field.19 nanoparticles and their clinical and emerging applications.
Nobel laureate Paul Ehrlich introduced the concept of Chapter 14 describes carbon-based nanoparticles such as
targeted therapy or “magic bullets” that can go specifically fullerenes, nanotubes, and nanodiamonds. Chapter 15
 A brief history of nanotechnology and introduction to nanoparticles Chapter | 1 3

FIGURE 1.1 Publications containing the word “Nanoparticle” (Blue) and publications containing the word “Nanoparticle” and “Medicine” or “Medical”
(Orange) between the years of 1978 and 2018. Analysis done using Web of Science. Please note the logarithmic y-axis.

describes quantum dots and their applications in sensing, References

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Overall, this book is a collection of different topics that 8. Feynman RP. There’s plenty of Room at the bottom. Engineering and
are fundamental to the design and application of nano- Science; 1960. p. 22e36.
particles in biomedicine and that are written by experts in 9. Mulvaney P. Nanoscience vs nanotechnologyddefining the field.
the nanomedicine field. This book is intended to provide a ACS Nano 2015;9:2215e7.
broad understanding of the field of nanoparticles and 10. Binnig G, Rohrer H. Scanning tunneling microscopydfrom birth to
nanomedicine for newcomers and established biomedical adolescence. Rev Mod Phys 1987;59:615.
11. Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev
researchers. It is our intention that this book will also be
Lett 1986;56:930e3.
suitable as a textbook for advanced coursework in the field
12. Eigler DM, Schweizer E. Positioning single atoms with a scanning
of nanoparticles for biomedical applications. tunnelling microscope. Nature 1990;344:524e6.
4 Nanoparticles for Biomedical Applications

13. Drexler KE. Molecular engineering: an approach to the development 18. National Research Council. Small wonders, endless frontiers: a re-
of general capabilities for molecular manipulation. Proc Natl Acad view of the National Nanotechnology Initiative. 2002.
Sci USA 1981;78:5275e8. 19. Service R. U.S. cancer institute cancels nanotech research centers.
14. Eric KD. Engines of creation: the coming era of nanotechnology. Science 2019.
1986. 20. Strebhardt K, Ullrich A. Paul Ehrlich’s magic bullet concept: 100
15. Kroto HW, Heath JR, O’Brien SC, Curl RF, Smalley RE. C 60 : years of progress. Nat Rev Cancer 2008;8:473e80.
buckminsterfullerene. Nature 1985;318:162. 21. KREUTER J. Nanoparticlesda historical perspective. Int J Pharm
16. Iijima S. Helical microtubules of graphitic carbon. Nature 2007;331:1e10.
1991;354:56. 22. Bangham AD, Horne RW. Negative staining of phospholipids and
17. Harada A, Kataoka K. Formation of polyion complex micelles in an their structural modification by surface-active agents as observed in
aqueous milieu from a pair of oppositely-charged block copolymers the electron microscope. J Mol Biol 1964;8:660e8.
with poly (ethylene glycol) segments. Macromolecules
1995;28:5294e9.
Chapter 2

Nanoparticle behavior and stability in

biological environments
Raquel Martı́nez1, Marı́a F. Navarro Poupard1, Aitor Álvarez1, Enrica Soprano1, Martina Migliavacca1,
Carolina Carrillo-Carrión1, Ester Polo1, Beatriz Pelaz1, 2 and Pablo del Pino1
1
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Física de Partículas, Universidade de
Santiago de Compostela, Santiago de Compostela, Spain; 2Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS),
Departamento de Química Inorgánica, Universidade de Santiago de Compostela Santiago de Compostela, Spain

2.1 Introduction On the other hand, NP degradation (as in breaking

down NPs to smaller building blocks such as atoms, clus-
Since the initial progress in the field of interfacing inorganic ters, and molecules) may become useful for drug release
nanoparticles (NPs) with biological settings (biomolecules, applications and/or to avoid long-term accumulation inside
biofluids, cells, and animal models), our knowledge about how living subjects such as cells or animals, thereby positively
the individual physicochemical properties of NPs become impacting on the NP biocompatibility.15 We should also
affected by such bionanointerfacing has greatly evolved.1,2 acknowledge, in any case, that some NP-based applications
Numerous reports have addressed, with varying degrees of benefit of biomolecule-driven aggregation and/or self-
comprehensiveness, the influence of NP parameters such as assembly processes,16 mostly in the biosensing area,
size,3 shape,4 charge,5 colloidal stability,6 corrosion,7 stiff- including colorimetric sensors, surface-enhanced Raman
ness,8 and so forth, on interactions with molecules, living cells, scattering, and magnetic relaxation switching.
and animal models.9,10 The motivation is twofold: first, engi- In this chapter, NP behavior and stability in the context
neered nanomaterials have raised great expectations for health, of biology and medicine will be addressed (see Fig. 2.1).
including applications as contrast agents, drug nanocarriers, Toward this end, first, we will provide the most important
and mediators for hyperthermia and photodynamic therapy2; concepts and considerations to the fabrication and charac-
second, nanomaterials may be also released unintentionally, terization of NPs for intended use in biological milieu;
for instance, through mechanical wear or chemical powder secondly, we will briefly expose and discuss several case
waste, and therefore, they are subject of health concerns.9 examples of relevance in the field of bionanotechnology.
The typical sizes of NPs lay between molecules and bulk Herein, for the sake of brevity, we will exclusively
materials, that is, within the size range of organelles and approach engineered NPs obtained by bottom-up fabrica-
proteins, with which they share one important feature: ag- tion methods. We believe that such NPs are the most
gregation and loss of colloidal stability negatively affect their adequate models to deal with the topic in question, owing
function. In the case of proteins, aggregation typically leads to to their homogeneity in terms of physicochemical proper-
critical conformational changes such as amyloidosis,11,12 ties, thereby allowing for correlating model NPs with their
whereas in the case of NPs, it may result in precipitation, behavior in biological settings.
reshaping, corrosion, and most importantly, loss of the
intended physicochemical properties, such as optoelectronic,
2.2 General considerations on the
magnetic, or catalytic capabilities.13 Ultimately, preserving
the original size, shape, structure, composition, and state of fabrication of nanoparticles
aggregation of NPs when they are immersed in biological In the following section, we briefly introduce some general
environments, at least for a specific time required to achieve concepts and approaches, which may be useful to design
the NP-dependent intended use, is essential to maintain the different NPs with robust biostability. For the reader interested
NP’s function and avoid potential unwanted effects.14 in further details, we recommend a highly comprehensive

Nanoparticles for Biomedical Applications. https://doi.org/10.1016/B978-0-12-816662-8.00002-3 5

Copyright © 2020 Elsevier Inc. All rights reserved.
6 Nanoparticles for Biomedical Applications

FIGURE 2.1 Nanoparticle (NP) behavior and stability in

biological settings are governed by different properties, such
as protein corona, functionalization, and NP surface in-
teractions with the surrounding media.

protocol article17 and a handful of reviews,9,10,14,18,19 recently metal or metal oxide precursors are dispersed in a solvent
published by some of us (among others). (polar, nonpolar, or mixtures); (2) temperature of the sol-
vent and/or the addition of a reducing agent dictates when
2.2.1 Synthesis of inorganic nanoparticles nuclei (precursors of the NPs) start to grow; (3) the addition
of surfactants, linkers or capping molecules, influences
The synthesis of NPs has greatly developed in the past shape and size; (4) the absolute and relative concentration
two decades. Currently, fine control over the size, shape, of reactants, as well as heating ramps during the NP
structure, and composition of the final products can be growth, influence the final size and shape. Ultimately, after
readily achieved by different well-established wet- their synthesis and purification, usually by centrifugal
chemistry synthetic methods (both in aqueous and in precipitation, one obtains a colloidal dispersion of NPs
organic solvents); importantly, such methods are highly coated by a shell (e.g., purely organic, silica, MOF), which
reproducible in any advanced chemistry laboratory.20 provides the NPs with steric or ionic hindrance and thereby
It is well known that the size, shape, composition, structure with colloidal stability; otherwise, NPs would aggregate or
(that is, coreeshell, element doping, and so forth), and mono- collapse into the corresponding bulk materials.
dispersity dictate the physicochemical properties of NPs. These
factors are particularly critical when referring to plasmonic,
2.2.2 Surface modification (functionalization)
magnetic, photoemissive, and catalytic NPs.13 In the case of
plasmonic NPs (e.g., gold, silver, copper, and/or aluminum), Independently of whether the NPs are produced in aqueous
such factors determine the position of the localized surface media or in organic solvents, the stabilizing shell after the
plasmon resonance (LSPR) bands, photothermal capabilities, synthesis is in most of the cases insufficient to stabilize the
and near- and far-field scattering processes, among others16; in NP in biological media, in which high ionic strengths,
the case of magnetic NPs (e.g., magnetite, maghemite, doped polarity, pH values, and the coexistence of polyelectrolytes,
ferrites, and iron), the characteristics of their hysteresis loop biomolecules, and biomacromolecules in high concentra-
(that is, coercive and saturation fields, and magnetization) will tion may trigger NP precipitation, NP reshaping, and in
be dictated by such factors21; for photoemissive NPs some cases, NP degradation. Thus, further chemical surface
(e.g., quantum dots [QDs], up- or downconversion NPs, or engineering (in the following referred to as functionaliza-
nanoperovskites), the photoemission intensity and wavelength, tion) is typically required to warrant colloidal stability in
and their quantum yield (QY) are influenced by such morpho/ such media.19 After the synthetic and purification pro-
structural characteristics22; lastly, the performance of hetero- cesses, the produced NPs do not present pristine surfaces
geneous nanocatalysts (e.g., palladium, copper, gold, ruthe- (that is, they are not “naked”); they may be considered
nium, or platinum) is influenced by crystal structure, valence coreeshell inorganiceorganic nanostructures. Briefly, the
state, and coating of their surfaces.23 stabilizing shell may provide two types of stabilization
For the vast majority of wet-chemical synthetic routes to mechanisms: (1) electrostatic repulsion by charged species
produce NPs, some common aspects may be outlined: (1) (for instance, citric acid, compounds containing carboxylic
 Nanoparticle behavior and stability in biological environments Chapter | 2 7

or amine groups, polyelectrolytes, or polymers) and/or (2) NP surface hydration. Let us consider polymer-coated NPs;
steric hindrance (e.g., polyether derivatives, proteins, in general, polymer coatings collapse onto the NPs surface
gelatin, MOF-based shells, silica shells). Thus, the original after sample drying onto substrates used for transmission
surfactant (as coating after the synthesis and purification electron microscopy (TEM) or scanning electron micro-
steps) is typically exchanged or modified to enhance their scopy. Thus, extracting useful information about the
colloidal stability in biological settings. One should keep in colloidal state of NPs from electron microscopy data is
mind that biological relevant pH values range from the challenging, when not impossible, due to NP aggregation
extracellular neutral environment (pH w7) to the acidic during the sample preparation. One can rarely find exam-
lysosomes (pH w4.5); thus, if the NPs are stabilized by ples in which TEM (using negative staining to resolve the
charged groups, these should remain charged during oper- morphology of the organic shell) and DLS data can be
ation at biological pH values. In Table 2.1, a selection of directly compared; among these rare examples, we have
common NP stabilizers is provided together with a selec- recently reported on a 2D library of PEGylated Au NPs,8
tion of the corresponding references for each case. which was used to correlate some of their basic physico-
chemical properties (such as size, z-potential, hydrophilic-
ity, elasticity, and catalytic activity) with their interaction
2.3 Characterization of colloidal with cells (see Fig. 2.2). In this example, diameters
stability extracted from negative staining TEM micrographs of the
In general, engineered NPs for bioapplications are expected coreeshell Au-NP/PEG shells were similar to the hydro-
to behave like macromolecules dispersed in solution; that dynamic diameters as obtained from the number distribu-
is, they should not sediment or aggregate or disintegrate or tion with DLS.
reshape when they are exposed to biological settings.57 Laser Doppler anemometry (LDA), typically performed
Therefore, a proper characterization of engineered NPs in in aqueous solutions with a known pH, provides informa-
different biological media should be mandatory, if one aims tion about how a particle moves under the influence of an
to understand the interaction and functioning of such NPs applied electric field; therefore, the z-potential is also
in the presence of biomolecules, or inside living cells or referred to as electrokinetic potential. Although the inter-
animals. To this aim, several characterization techniques pretation of LDA data is more complex,61 stated in simple
may be used in parallel. In the following, we briefly discuss terms, z-potential values are normally related to the net
the most common ones, typically available (if not all, most charge of an NP in solution under those specific conditions.
of them) in any laboratory devoted to nanobiotechnology. Commonly, values above
30 mV have been considered as
requirement for colloidal stability. Nevertheless, this last
assumption is not a universal truth. The isoelectric point of
2.3.1 Dynamic light scattering and laser the NPs can be also determined by LDA, performing
Doppler anemometry titration experiments in which the case examples are
recorded against pH.
Particle analyzers such as the Malvern’s Zetasizer family and The combination of DLS and LDA allows for extracting
the Horiba’s nanoPartica family, among others, allow for valuable information regarding the colloidal stability of
determining the hydrodynamic diameter dh and zeta potential NPs in complex media. For instance, as a routine charac-
(typically denoted z-potential) of NPs dispersed in solution. As terization, the evolution of the hydrodynamic diameter (dh)
an alternative to dynamic light scattering (DLS) analyzers in and z-potential of NPs over time in relevant biological
which the extracted dh is based on time-dependent scattering media such as phosphate buffer saline (PBS), cell media
intensity fluctuations (correlogram), nanoparticle tracking an- with or without fetal bovine serum (FBS), and antibiotics is
alyzers (NTAs, such as the ZetaView or the Nanosight families) becoming more and more usual (see Section 7).
directly provide information about dh from individual NPs; that
is, although both techniques measure Brownian motion
2.3.2 UV-Vis spectroscopy
(diffusion constant) in solution, NTA raw data provide number-
weighted dh distributions, whereas DLS experimentally The absorbance spectra of solutions of NPs provide
provides intensity-weighted distributions, which after decon- meaningful insights about their colloidal state. This fast,
volution can be expressed in number-weighted size distribu- ultrasensitive, affordable, and reliable technique is typi-
tions. For the interested reader, other less common techniques cally used to characterize plasmonic NPs, as changes in
for particle size analysis can be used, including analytical ul- their LSPR bands are directly related to changes in the
tracentrifugation, differential centrifugal sedimentation, or dispersion media (i.e., polarity) and more importantly, to
asymmetric-flow field-flow fractionation (AF4).58e60 the NPs aggregation state induced, for instance, by ionic
The number-weighted hydrodynamic size typically ex- screening (e.g., salts present in the medium) or by the
ceeds the size obtained by electron microscopy due to the presence of proteins, charged polymers, or other
8 Nanoparticles for Biomedical Applications

TABLE 2.1 Common materials used to form robust coatings around nanoparticles (NPs), aiming enhanced
colloidal stability.

NP stabilizerx Original surfactantA Inorganic core Diameter/length (nm) References

8,24,25
PEG Citrate Au (spherical) 13e28
26,27
CTAB Au (rods) 33e100
28
Sulfate Au (triangular plates) 100e160
29,30
SDS Ag (spherical) 4
31,32
PMA Dodecanethiol Au (spherical) 2e6
33,34
PEG/dodecylamine Au (spherical/rod) 20e100
33
PEG/dodecylamine Ag (triangular nanoplates) 70
30
SDS Ag (spherical) 4
21,35e37
Oleylamine/dodecylamine Iron oxides (spherical) 4e90
36,38,39
TOPO QDs (CdSe, CdS, etc.) 2e7
40,41
Oleic acid Upconverting NPs (spherical) 15e30
6
Oleic acid ZnO (spherical) 7
Citrate Au (spherical) 13e28 34
PVP
CTAB Au (rods) 33e100
42
Oleate Upconverting NPs (spherical) 20
43
LbL (polyelectrolytes) Citrate Au (spherical) 20e100
43
CTAB Au (rods) 500
44
pDA Citrate Au (spherical) 50
45
PEG Au (rods) 44
46
Citrate Iron oxide (spherical) 19e240
47,48
Proteins (BSA, HRP, etc.) Citrate Au (spherical) 15e19
49
Fe3O4 (spherical) n.d.
50
CTAB Au (rods) 100
51
Silica APS Au (sphere) 15
52
CTAB Au (rods) 50
53
Oleic acid Iron oxide (spherical) 5
42
PVP Upconverting NPs (spherical) 20
7,54,55
MOFs (ZIF-8 based) CTAB Au (nanostars) 120
56
PVP Au (spherical) 13e30
56
PVP Ag (cubic) 160
31
MUA TOAB Au (spherical) 3e7
29,30
SDS Ag (spherical) 4
x
PEG, polyethylene glycol; PMA, poly[isobutyleneealtemaleic anhydride]egraftedodecyl; PVP, polyvinylpyrrolidone; LbL, layer-by-layer; pDA, poly-
dopamine; BSA, bovine serum albumin; HRP, horseradish peroxidase; MOFs, metal organic frameworks; ZIF-8, zeolitic imidazolate framework; MUA,
mercaptoundecanoic acid.
A
CTAB, cetyltrimethylammonium bromide; SDS, sodium S-dodecylthiosulfate; PEG, polyethylene glycol; TOPO, tri-n-octylphosphine oxide; APS, (3-
aminopropyl)trimethoxysilane; PVP, polyvinylpyrrolidone; TOAB, tetraoctylammonium bromide.
 Nanoparticle behavior and stability in biological environments Chapter | 2 9

FIGURE 2.2 (A) PEGylated Au nanoparticles (NPs), showing different properties in vacuum and in solution. dC and dCS refer to the diameters of the Au
cit
cores and of the cores with the PEG shell (the coreeshell system), respectively, as determined by transmission electron microscopy (TEM). dhðNÞ and
PEG
dhðNÞ refer to the hydrodynamic diameters as obtained from the number distribution with DLS of the originally citric acid stabilized Au NPs before
PEGylation (that is, PEG coating) and of the PEGylated NPs, respectively. (B) Negative staining TEM images of two types of PEGylated NPs are shown,
in which dC increases, whereas dCS is kept constant at ca. 38 nm. (C) Negative staining TEM images of two types of PEGylated NPs are shown, in which
dCS increases, whereas dC is kept constant at ca. 23 nm. Scale

bar: 50 nm. (D) Different variables related to the size of the PEGylated Au NPs. (E)
Heatmap of the proportion of PEG in the NP size RTEM PEG . Adapted with permission from del Pino, P. et al. Basic physicochemical properties of
polyethylene glycol coated gold nanoparticles that determine their interaction with cells. Angew Chem Int Ed 2016;55:5483e5487.

biomolecules.62 Also, as in solutions of nucleic acids, functionalization.17,64 In general, highly concentrated so-
dyes, or in general, any UV-Vis active molecule, absor- lutions of NPs are placed in the gel’s lane entry; the
bance at NP-type specific wavelengths can be used to porosity of the gel is controlled by the percentage of the
determine NP concentration in solution (using the corre- agarose (typically in the range of 1%e2% in buffer).
sponding calibration curves).8 Moreover, this technique Upon a voltage application, the NPs will move to the
can be used with nonplasmonic NPs since aggregation cathode or the anode depending on their charge; negative
induces an absorbance broad peak in the NIR due to and positive ones will migrate to the anode and cathode,
scattering produced by the NP aggregates.63 respectively. That said, only colloidally stable NPs will be
able to move inside the gel. This is due to the required use
2.3.3 Gel electrophoresis of rich electrolyte buffers (e.g., tris/borate/EDTAdTBE
0.5x), in which the presence of salts induces aggregation
This technique based on the use of a polymeric matrix, of nonstable NPs in the lane entry; such aggregates will,
typically agarose, allows to get valuable information in general, not migrate inside the shell or produce
about the colloidal stability of the NPs and even of the smearing. Notice that in case of monodisperse, colloidally
surface modifications introduced after stable NPs, one expects narrow migration bands (the
10 Nanoparticles for Biomedical Applications

2.4 Concentration determination: dose

matters
The concentration determination (i.e., atomic species and/
or NPs as individual species) is another critical parameter
when working with NPs, for which analytical techniques
such as inductively coupled plasma mass spectrometry
(ICP-MS) and/or NTA are typically used. As already
described, NPs are hybrid systems built of an inorganic
core stabilized by an organic shell. Ideally, the specific
composition and molecular formula of the NPs, that is, the
precise number of ligands and number of inorganic atoms
forming the inorganic core should be known. However,
this information has been solely accurately reported for
small particles or clusters, such as Au-NPs with core
diameter w3 nm, stabilized by chains of the synthetic
amino acid tiopronin.67 Yet the complexity in determining
the specific composition of NPs increases with both the
FIGURE 2.3 Electrophoretic mobility of 5 nm Au/100b HS-ssDNA
NP size and the number of ligands.
conjugates (3% gel). The first lane (left to the right) corresponds to 5- This parameter, frequently omitted, is of vital impor-
nm particles (single band). When w1 equivalent of DNA is added to tance to interpret colloidal stability results since NP
the Au particles (second lane), discrete bands appear (namely 0, 1, 2, 3,.). corrosion (that is, leaking of atoms and/or surfactants)
When the DNA amount is doubled (third lane), the intensity of the discrete directly induces biological effects, including cytotoxicity,
bands changes and additional retarded bands appear (4, 5). Because of the
discrete character, each band can be directly assigned to a unique number
oxidative stress, genotoxicity, and immunotoxicity.68,69
of DNA strands per particle. Reproduced with permission from Zanchet, D, As an example, considering NPs with the same inorganic
Micheel, C, Parak, WJ, Gerion, D, Alivisatos, AP. Electrophoretic isola- core composition, the active surface is directly related to
tion of discrete Au nanocrystal/DNA conjugates. Nano Lett the square radius. So, when comparing NPs with radius r,
2001;1:32e35. 2r, or 4r, the surface and, therefore, the atoms located on
the surface for the latter two samples will be 4 and 16
narrower the band, the higher the degree of NP mono- times the ones located onto the surface for the smaller NP
dispersity). Polydisperse or nonhomogeneously function- (r). The number of surface atoms is critical when the NPs
alized NPs will produce smearing or distinct bands. For exhibit catalytic properties or if they can suffer from
instance, gel electrophoresis has been used to determine degradation processes triggered on their surface. Similar
the number of ligands per NP (see Fig. 2.3). examples can be provided considering the NPs volume
and, thus, the number of NPs that are contained within the
same mass of material.
2.3.4 Size exclusion chromatography
One of the most extended ways of expressing NP
Size chromatograms of NPs provide valuable information dosage is milligrams of material per milliliter of solution
about both the NP size of the sample and their colloidal (mg/mL). However, the conversion of the mg/mL con-
stability. Moreover, the comparison of their elution time centrations to molarities (mol of NPs per L) provides a
with that of proteins or polymers with a known size can be more easily comparable metric, allowing for a more
used to determine their hydrodynamic radius.17,66 Alter- rational comparison of the results. Using this nomencla-
natively, size exclusion columns (e.g., PD-10 desalting ture, NPs may be considered as molecules. The approxi-
columns packed with Sephadex G-25 resin) allows for mate determination of the inorganic NP molecular weights
rapid buffer exchange, desalting, and removal of small is relatively straightforward by considering their di-
contaminants (salts, dyes, surfactants, radioactive labels) mensions (volume of a single NP) and the density of the
from NP solutions using gravity flow; that is, NPs will not bulk material from what they are made of. Then, providing
be retained in the Sephadex resin, whereas nonattached different metrics of the dosage used would help to extract
molecules will be delayed, thereby allowing for purification more comprehensive data from the analysis and to
of NP products. compare results obtained by different authors in different
For more information about this and other comple- laboratories and using different methodologies. For more
mentary techniques for characterization of NP colloidal detailed information about this, the reader is referred to
stability, see the protocol article by Hühn et al.17 previous work.30,70
 Nanoparticle behavior and stability in biological environments Chapter | 2 11

2.5 Determination of the protein equilibrium, and therefore the corona identity.71 Only few
corona techniques are able to study the in situ corona without a
previous purification step, such as fluorescence correlation
The formation of a layer of adsorbed proteins onto the spectroscopy (FCS),71,77 flow cytometry,78 diffusion-
surface of NPs exposed to protein containing media is ordered nuclear magnetic resonance (NMR) spectros-
among the most crucial factors influencing the interaction copy,79 nanoparticle tracking analysis (NTA),80 and
of NPs with living matter, and therefore, it may determine isothermal titration calorimetry.27,72 However, such
the biological fate of NPs as well as their colloidal prop- studies are still rare. Importantly, to extract quantifiable
erties (see Fig. 2.4). This protein layer named protein data (e.g., dissociation constants, dh) from these tech-
corona has been subject of intense research during the last niques, studies are usually restricted to model proteins
decade,71 originally initiated by the group of K. (e.g., albumin, transferrin, fibrinogen, immunoglobulins,
Dawson.72e74 When the complexity of the biological fluid low-density lipoproteins). To our knowledge, examples of
is higher than just proteins, that is, any biological fluid or noneoptics-based methods for exploring the corona for-
supplemented cell media, other molecules beside proteins mation in situ are virtually nonexisting; one exception
such as sugars, or lipids, can also be adsorbed onto the NPs. was recently reported by Carril et al., in which NPs are
In those cases, the term biomolecular corona can be used labeled with 19F and their diffusion coefficient measured
instead of protein corona.75 using 19F diffusion-ordered NMR spectroscopy. 19F
A deeper knowledge about the identity of the adsorbed diffusion NMR measurements of hydrodynamic radii
proteins, their abundance, and their orientation should shed allowed for in situ characterization of NPs in complex
some light in understanding the mechanisms by which the environments by quantification of protein adsorption to
NPs are recognized by cells, both in vivo and in vitro.76 the surface of NPs, as determined by increase in hydro-
The capability of the proteins to interact with a particular dynamic radius (see Fig. 2.5).79
NP model is determined by their affinity constant. How- As shown above in Fig. 2.5, by evaluating the size of
ever, the complexity of the media, in which a huge amount the NPs upon the dispersion in protein containing media,
of nonbounded proteins among other compounds are information regarding the protein corona formation can also
available and ready to interact with the NPs, leads to a be obtained. Of course, the size of the NPs should be small
dynamic behavior of the corona, where the composition is a enough to be able to distinguish the protein-coated NPs
result of a competition equilibria between the affinities of from the noncoated ones and from the measurement error.
the media components. Of course, this dynamic nature The size increment for particles bigger than 50 nm caused
hinders its straightforward characterization. by the formation of a protein layer will be roughly in the
Another challenging issue is that the actual isolation of same order of magnitude of measurement errors from DLS,
the NPs with the corresponding corona (for instance, by NTA, FCS, or NMR. Therefore, such studies are limited to
centrifugation, SEC, electrophoresis) alters this relatively small NPs. Notice, however, that in the ultrasmall

FIGURE 2.4 (A) Formation of the protein corona is sensitive to the dispersion of the nanoparticles (NPs) in solution, for example, via steric effects;
dissociation equilibrium coefficient KD is given by the ratio of the off- to on-rates for protein binding to the NPs. (B) If dried NPs (in this case, gold NPs of
ca. dc ¼ 15 nm in core diameter) are observed to form agglomerates in transmission electron microscopy imaging (scale bars are 100 nm in both mi-
crographs), they are usually also agglomerated in solution. (C) Agglomeration in solution can be observed with UV/Vis absorption spectroscopy, in which
the absorption A is plotted versus the wavelength l. Reproduced with permission from Del Pino, P. et al. Protein corona formation around nanoparticles -
from the past to the future. Mater Horizons 2014; 1:301e313 (2014) with permission from The Royal Society of Chemistry.
12 Nanoparticles for Biomedical Applications

general, after circulation for varying time periods in living

animals, NPs are extracted for protein corona analysis.

2.6 Stability in biologically relevant

media
The stability of engineered NPs measured in biologically
relevant media, such as high ionic strength buffers, protein-
supplemented media, cell media, or plasma, provides a more
accurate scenario than measurement in plain water; such
measurements illustrate the actual features of the NP disper-
sion that will dictate their interaction with cells or living or-
ganisms. In the literature, thousands of different protocols to
decorate the NP surface are available. However, not all of them
are suitable for all the NPs. For example, using ligands that
contain reactive groups that will preferentially react with a
specific material, for example, gold and thiolated molecules,
ensures a strong ligandeNP interaction that will enhance the
NP stability. Yet, this strong interaction might not be enough if
the ligand length is too short compared with the NP size or if
the ligand hydrophilicity is low. So, a rational selection of the
elements that compose our nanomaterial is required. Further-
more, perfectly stable NPs in water or in low salt content
aqueous solutions can suffer from an impaired stability once
they are in a harsher, more complex environment, as the bio-
logical media are. The charge losses induced by screening
processes86,87 and the unspecific protein adsorption, as dis-
FIGURE 2.5 A) Size increase in the presence of human serum albumin cussed above, are two of the major inducers of NP aggregation.
(HSA) for three types of 19F-labeled nanoparticles (NPs). (B) Hydrody- We should also acknowledge that protein adsorption (i.e.,
namic radii rh
standard deviation (from at least three measurements) as
measured in situ (i.e., under equilibrium with excess proteins present in
protein corona) may also stabilize NPs in biological media.
solution) for the three types of NPs in the presence of increasing con- That said, NP aggregation is not the only process that affect the
centrations cHSA of HSA in PBS, and the corresponding fit based on the NP stability in these media; for instance, leaking of metal ions
Hill’s model for the case of NP-F/NH2@PMA, which was the only NP due to redox reactions, the production of reactive oxidative
type that underwent an increase of size due to protein adsorption. In the species, and processes of ligand replacement can also occur,
case of NP-F/NH2 and NP-F/COOH, no protein adsorption in terms of no
significant change in hydrodynamic radius was observed. (C) Size mea-
among others. These processes that directly affect NP integrity
surements in different media. Hydrodynamic radii rh
standard deviation will be discussed in Section 7. Regarding the NP aggregation
(from at least two measurements) as measured for the three types of NPs: state, obviously, having completely aggregated NPs that are
in water, aqueous buffer (HEPES or PBS), in the presence of HSA (under not colloidally stable and rapidly precipitate, differs from
saturation conditions), in isolated plasma, and in blood. Reproduced with partial aggregation. The first aggregation type will produce big
permission from Carril, M. et al. In situ detection of the protein corona in
complex environments. Nat Commun 2017;8:1542.
NP groupings that might even be visible with the naked eye.
Those aggregates will quickly sediment, and if big enough,
they might not be even internalized by cells. Needless to say,
NP size range (1e3 nm in diameter), the identity of the such aggregation will dramatically affect the properties of the
protein corona may rapidly fluctuate81; that is, size (in NPs that are related to their size and shape. The fact that the
combination with surface identity) may be used to nearly NPs are dramatically, irreversibly aggregated can lead to
eliminate long-lived interactions between NPs and the wrong conclusions and therefore to misinterpret their cell
biomolecular environment. regulation capabilities; specially, if the aggregation of the NP is
Last in this section, we should acknowledge that in vitro not considered. More important than the aggregation of the
studies concerning protein corona formation have failed to NPs that can be detected just at plain sight is the partial ag-
minimally reproduce commonly observed in vivo gregation, which produces aggregates that can only be
phenomena.82e85 Therefore, recent articles have shifted the observed using techniques such as DLS. For plasmonic NPs,
focus toward in vivo protein corona formation, for which, in the formation of such aggregates as discussed above can be
 Nanoparticle behavior and stability in biological environments Chapter | 2 13

detected easily by a change in the color solution; however, behavior of NPs in biological settings. That is, the stability
many other NPs do not exhibit this property. Partial aggrega- of the NPs in complex media is the result of the contribu-
tion in solutions of TiO2, ZnO, silica, or iron oxide NPs will not tion of several factors as already mentioned. Despite great
be visible without a proper analysis, for instance, by DLS.6,88 efforts achieved during the last decade to correlate protein
The routine characterization of the aggregation state in corona formation with NP physicochemical properties,
media such as PBS, HEPES, or other biological relevant many unresolved fundamental questions remain.96 The
buffers at the biological pH range, protein containing unspecific adsorption of proteins is driven by several
media such PBS with BSA or FBS, and cell media with entangled NP features such as the NP net charge,97 the
and without supplements (i.e., FBS, antibiotics, and hydrophobicity/hydrophilicity of the NPs,8 the local surface
essential amino acids), among others, should be consid- charge (for instance, the distribution of the charged groups
ered as a fundamental physicochemical characterization of in zwitterionic polymers),98 the functional groups or li-
nanomaterials. gands attached to the NP surface,93 and the colloidal sta-
bility.59 Other parameters are defined by the proteins
themselves such as the protein concentration, the protein
2.7 Correlating basic physicochemical affinity, or the protein conformation99; finally, the media
properties with nanoparticle also contribute with parameters such as the pH, the salt
behavior in biological settings concentration, or the temperature.100,101
We want to acknowledge that building a designed
For a long time, an ideal nanocarrier has been considered as fusion protein corona onto NPs (what the authors called
one that among other properties should be able to avoid protein corona shield nanoparticle) has been recently re-
unspecific protein adsorption. Evading the formation of the ported to minimize interactions with serum proteins,
corona provides the nanocarrier with longer circulation thereby preventing the clearance of NPs by macrophages,
times, thereby enhancing the success rate to achieve effi- while ensuring systematic targeting functions in vitro and
cient targeting.85 Toward this end, PEGylation of NPs is in vivo.102
the most common strategy. This approach was described in Lastly in this section, we want to emphasize that the
the 1950s and 1960s for PEGylated surfaces.89,90 This general term NP refers to an enormously heterogeneous
ability of PEG chains to prevent protein adsorption has group of materials, as the term protein refers to an enor-
been widely explored in vitro and in vivo; it is well known mously heterogeneous group of macromolecules composed
that PEG shells prevent the opsonization of NPs.91 How- of amino acids. Therefore, as in proteins, we cannot expect
ever, the PEGylation of NPs modifies more than just one to extract universal rules from examples focused on
physicochemical property; the hydrodynamic size is also correlating some NP physicochemical properties with NP
increased, the surface charge is reduced, and normally, the behavior in biological settings. As in current proteomics
colloidal stability is enhanced.77 On this note, we reported studies, we may advance toward more comprehensive
that PEGylated NPs with longer PEG chains presented studies (large-scale study of NPs), with the general aim of
higher hydrophobicities than NPs PEGylated with shorter building a “nanomics” analogous to proteomics.
chains.8 Not only does the PEG length play a role, but also
the grafting density controls the stealth capabilities of PEG
shells.92,93 We also demonstrated that, in most of the cases,
2.8 Degradation in biological settings
the PEGylation of NPs is not fully capable of preventing Unspecific protein adsorption is among the most relevant
the unspecific adsorption of proteins.77 Indeed, Schöttler processes that NPs suffer in biological settings; however, it
et al. reported that protein adsorption is required to create is not the only one. The components of biological media
the stealth effect of PEGylated nanocarriers.94 include dissolved oxygen and other potential oxidative
Usually, the differences in protein adsorption have been agents that can affect the integrity of the NP core, leading to
associated to changes in the surface charge, NP curvature, ion leaking and thus NP corrosion. Manifold articles have
size, or shape.93 For instance, it has been reported that ul- reported on the induced toxicity of NPs due to these pro-
trasmall NPs (diameter < 3 nm) may not acquire a resilient cesses. In the case of silver NPs, the ion leaking process has
protein corona.81 However, the difficulties to relate the been extensively used as antibacterial103 and to induce
results obtained in different studies, when varying the NP controlled toxicity in cancer cells.104 In the case of Cd-
model, have led to the researchers to carry out more sys- containing QDs, release of Cd ions upon QD corrosion in
tematic studies. On this note, we have prepared a series of biological settings, and its toxic effects, has been well
NPs libraries in which an individual physicochemical studied in the past two decades.105 In general, the uncon-
property is modified, whereas others are kept con- trolled NP degradation upon biological corrosion is un-
stant.8,77,95 These studies have clarified that any parameter wanted. NPs can also trigger region of species production
in an exclusive basis is not solely responsible for the final in biological settings,106 which has been used as an
14 Nanoparticles for Biomedical Applications

FIGURE 2.6 (A) Scheme of the experimental setup. Cells are loaded with nanoparticles (NPs), which are composed out of three fluorescence-labeled
components: QDs, PMA-ATTO, and HSA-Cy7. The NP degradation over time and the subsequent exocytosis of the different components are the
processes under study. (B) Time-dependent decrease in intracellular fluorescence due to NPs, which had been internalized by HeLa cells, as obtained by
flow cytometry analysis. Two types of NPs were studied: (A) HSA-Cy7 adsorbed to QDs, i.e., PMA-ATTO-QD@(HSA-Cy7)ads, (B) HSA-Cy7 cross-
linked to QDs, i.e., PMA-ATTO-QD@(HSA-Cy7)cov, and (C) control samples (PMA-QD, PMA-ATTO, HSA-Cy7). Data were derived by cells
grown under (1) normal conditions or (2) inhibiting conditions. The graphs represent the normalized intensity values per cell, calculated as I [%] ¼ I(t)
[a.u.]/I(t ¼ 0) [a.u.], of each label as a function of follow-up times t. Error bars correspond to the standard deviation (SD) from three independent ex-
periments. Reproduced with permission from Carrillo-Carrion, C. et al. Triple-labeling of polymer-coated quantum dots and adsorbed proteins for
tracing their fate in cell cultures. ACS Nano 2019;13:4631e4639.

advantage, for instance, in photodynamic therapy. How- to an acidic environment and enzyme digestion processes.
ever, this is totally unwanted in most situations at extra- and We cannot forget that enzymes are also present in cell
intracellular levels. media and the nature and abundance of these enzymes will
Recently, the need to better understand the biological vary with the cellular type and the tissue location. For
fate of NPs in vivo has helped to understand that upon cell instance, the enzymatic composition in lungs is different
internalization, NPs may be affected by different degrada- than that of the spleen, liver, and so forth. These differences
tion processes. It is worth underlining that understanding are very important in tumors, which may be used as a perk
such degradation process is even more critical in the case of for enzyme-controlled drug release.107
working with intrinsically toxic elements, as for example, Kreyling et al. reported the in vitro and in vivo enzy-
Cd-containing QDs. In general, after internalization in cells matic degradation of polymer-coated gold NPs.32 In this
by endocytic processes, NPs will end up in the endo/lyso- example, the degradation in vivo induced different accu-
somal system of cells, and therefore, they will be submitted mulation and excretion pathways of the inorganic NPs
 Nanoparticle behavior and stability in biological environments Chapter | 2 15

FIGURE 2.7 Dual enzymatic

reaction-assisted gemcitabine (GEM)
nanovectors were used to achieve
multistage tumor cell targeting and
efficient drug release during the
GEM-transporting process. (A)
Schematic illustration of the prepa-
ration of nanovectors and their
enzyme-sensitive behavior. (B)
Schematic illustration of the nano-
vectors delivering GEM to pancreatic
cancer cells. Reproduced with
permission from Han, H. et al. Dual
enzymatic reaction-assisted gemcita-
bine delivery systems for pro-
grammed pancreatic cancer therapy.
ACS Nano 2017;11:1281e1291.

cores and the corresponding polymer coatings (i.e., organic partly transported with the underlying polymer-coated QDs
shells). This was induced by the degradation of amide into cells, which is imperative if the attached protein is
bonds, which was an essential part of this particular poly- working as a targeting unit as well as in the case of ther-
meric shell. Some potential enzymes were identified apeutic proteins aimed to be delivered inside cells. More-
in vitro, and a similar enzymatic degradation was hypoth- over, upon desorption of proteins, those initially adsorbed
esized in vivo. The results reported by Kreyling et al. to the QDs remained longer inside cells compared with free
specifically highlight the loss of integrity of the bio- proteins. Part of the polymer shell was released from the
molecules attached to the NP surface, such as antibodies, QDs by enzymatic degradation, probably by degradation of
carbohydrates, and peptides, which typically are aimed to amide bonds as found previously by Kreyling et al.,32 but
carry out targeting functions. On the same note, Zhu et al. importantly, such polymer shell degradation was on a
explored a library of enzymes for degradation of the slower time scale than protein desorption. The significance
chemical bonds formed by click chemistry, which were of this work is to remark that when designing an engineered
used to attach fluorophores (reporters) to a polymeric shell NP for a specific intended use, one should keep in mind
acting as NP coating.108 two facts: (1) NPs do not remain a constant entity once they
Similarly, Parak et al. studied the in vitro NP integrity interact with living matter and (2) different NP components
upon internalization by cells of polymer-coated CdSe/ZnS can have different pharmacokinetics and biodistribution.
QDs modified with a model protein corona (i.e., an outer Understanding how enzymatic NP degradation pro-
shell of HSA protein molecules, either adsorbed or chem- cesses work has allowed for the development of more
ically linked to the surface of the polymer-coated QD).109 efficient nanocarriers. Han et al. reported a nanovector for
In this work, a combination of flow cytometry and confocal pancreas cancer targeting (see Fig. 2.7).107 This vector was
microscopy was proposed as methodological approach for modified with a shell that degrades sequentially to finally
analyzing individually the exocytosis of the different parts induce the gemcitabine (GEM) release inside the tumoral
of the NP, and such data were correlated with the degra- pancreatic cells. A stealth PEG layer was added to the
dation of the different components inside living cells (see nanocarrier to increase their blood circulation time. How-
Fig. 2.6). For that, the NP was triple fluorescence labeled: ever, this PEG shell was attached through a metal-
the QD core having intrinsic fluorescence, the polymer loproteinase 9 (MM-9) cleavable bond to an RGD peptide.
shell labeled with a fluorescent organic dye, and the HSA Thus, NPs that were in the tumoral region lost the PEG
labeled with another dye. Results indicated that HSA was shell, allowing the peptide to drive the NPs inside the
16 Nanoparticles for Biomedical Applications

tumoral cells, where cathepsin B degrades the peptide bond 9. Rivera-Gil P, et al. The challenge to relate the physicochemical
by which GEM was attached to the nanocarrier. properties of colloidal nanoparticles to their cytotoxicity. Acc Chem
Res 2013;46:743e9.
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Chapter 3

Active targeting and transport

Aria W. Tarudji and Forrest M. Kievit
University of Nebraska, Department of Biological Systems Engineering, Lincoln, NE, United States

3.1 Introduction barrier (e.g., bloodebrain barrier [BBB] and bloodetestes

barrier).1e7 This has been shown in various elegant studies
In the biomedical field, nanoparticles (NPs) are mainly used with tumor targeting where total tumor accumulation was
in drug delivery, imaging, and theranosticsdthe ability of not affected by the presence of a targeting agent on the
NPs to act as both a therapeutic and diagnostic tool. One of surface of the NP, but the targeting agent did enhance
the most well-known examples of nanomaterials in medical distribution throughout the tumor as well as intracellular
use is Doxil, a liposomal NP that carries doxorubicin, a delivery.8e11 Moreover, since actively targeted NPs do not
standard chemotherapeutic agent used for breast and other solely depend on EPR, actively targeted NPs allow tar-
types of cancers. Doxil was developed to take advantage of geting of hematological malignancies,12,13 small metastatic
the leakiness of blood vessels in tumors that allow it to tumors,14 neurodegenerative disorders,15e17 and other dis-
permeate into the cancer tissue before releasing the drug as eases that do not show the EPR effect.
well as being retained in the tumor because of the lack of The nanosize of NPs (typically 5e200 nm) results in an
lymphatic drainage. This so-called enhanced permeability extraordinarily high surface-area-to-volume ratio, which
and retention (EPR) effect that is utilized by many developed can lead to upward of a square meter of surface area per
NP systems is considered as passive targeting approach (see milligram of NP. The high surface-area-to-volume ratio
Chapter 4). On the other hand, active targeting relies on the allows for relatively large quantities of active targeting li-
specific binding of targeting ligands on the surface of the gands to be conjugated or adsorbed onto the NP surface.
NPs to target cell receptors. Some of the typical active tar- Targeting ligands in close proximity to one another on the
geting ligands used are antibodies, proteins, peptides, NPs surface can lead to a higher binding affinity to target
aptamers, small molecules, and natural polymers. Each cells through the multivalent effect, where the binding
ligand contains advantages and disadvantages in altering the strength between NPs and targeted tissue is stronger than
physicochemical properties of the NPs because of ligand size individual ligands to the tissue.18,19 The concentration of
and charge, binding affinity and avidity, specificity, conju- ligands mounted on the NPs affects the total affinity
gation mechanism, and cost of production. binding of ligands. For example, Poon et al. showed that
Active targeting is often used to increase the delivery there is a decrease in the dissociation constant, KD, indi-
rate, accumulation, and retention of the NPs inside the cating an increase in binding affinity, when there are more
targeted tissue, which allows better drug release and more folates on the surface of the NPs.19 Additionally, Hong
effective drug delivery. However, active targeting should et al. found a similar multivalent effect using generation-5
not be thought of as actively seeking out target tissue as dendrimers conjugated with folic acid, where an increase of
with a heat-seeking missile. Rather, actively targeted NPs up to 15,200-fold was observed when compared with the
still fully distribute throughout the body but only accu- KD of free folic acid binding.20 However, there appears to
mulate and are retained more efficiently in target tissues as be a limit to this multivalent effect. As ligand concentration
with walking along a beach scattered with landmines where on the surface of the NP is too concentrated, steric binding
the landmine only goes off when contacted. More impor- interference begins to prevent binding of targeting ligand to
tantly, active targeting increases the selectivity of inter- antigen, resulting in reduced binding affinities.19
nalization and endocytosis by specific cells, improves target Despite extensive research with actively targeted NPs,
tissue distribution, and increases the permeability of bio- there has not been any active targeting NP system used
logical membranes by targeting an active transporter of the clinically. Some have shown safety and efficacy in phase 1

Nanoparticles for Biomedical Applications. https://doi.org/10.1016/B978-0-12-816662-8.00003-5 19

Copyright © 2020 Elsevier Inc. All rights reserved.
20 Nanoparticles for Biomedical Applications

and phase 2 clinical trials, but none have passed large, oxygen or nitrogen atom with a hydrogen atom, which is
multicenter phase 3 clinical trials.21 In this chapter, we formed by the polarity difference between oxygen or nitro-
describe the physical phenomena behind active targeting gen and hydrogen. The hydrogen bond usually occurs at a
mechanisms and place them within the context of actively distance of 0.15e0.5 nm.23 However, in aqueous solutions,
targeted NPs. We provide examples of how these different the hydrogen bond between targeting and targeted molecules
strategies are used for active targeting of NPs to various issignificantly weakened as both form hydrogen bonds with
diseases. the surrounding solvent. The ionic bond, on the other hand,
is stronger in the ability to interact and attract an oppositely
charged atom. An ionic bond works across a much larger
3.2 The strength of molecular distance and can reach up to 10 nm apart. The hydrophobic
interactions effect significantly helps the targeting ability as water mol-
Binding affinity is the strength of a molecule to bind with a ecules in aqueous solutions interact with hydrophobic mol-
targeted counterpart molecule. Every molecule has a binding ecules less than with water or other hydrophilic molecules.
affinity with other molecules as can be observed with many The interaction range of hydrophobic molecules is less than
docking simulation studies in a two-molecule system. 10 nm and decays exponentially with distance. The high
However, in the real world, these two molecules are sur- surface-area-to-volume ratio of NPs also generates a high
rounded by other large and small molecules and a high surface energy, around 7.6e454 J/m2 depending on the size,
concentration of water (w55 M). Therefore, the binding shape, and material of the NP, which can lead to nonspecific
affinity of an active targeting agent to a targeted molecule binding to nontarget molecules. Therefore, any targeting
must be stronger than the binding affinity with nonspecific agent must have a binding energy that is greater than the
molecules. The interaction between active targeting and surface energy of the NP to provide successful active tar-
targeted molecules can be through various chemical forces, geting. Depending on the NP, the surface energy can be
including the hydrophobic effect, ionic bond, hydrogen substantial, so the NP must be adequately coated with a
bond, and van der Waals interactions (Table 3.1). Van der biocompatible polymer such as polyethylene glycol (PEG)
Waals forces are the weakest among the chemical in- to reduce this surface energy and allow for active targeting.
teractions, having a binding energy of 4 kJ/mol, just above The binding between the targeting and the targeted
the 2.6 kJ/mol of average kinetic energy of a molecule in molecules is initialized when the molecules are around
solution at 37
C.22 Even though the cumulative strength of 10 nm apart. The ionic interaction brings the molecules
van der Waals forces increases when there are more atoms to closer until the secondary bonds, which act in the shorter
interact, the van der Waals forces are easily overcome by the range, such as hydrophobic interactions, hydrogen bonds,
other forces and bonds. Moreover, the small interacting and van der Waals forces, pull them together. Even though
surface area between a targeting and targeted molecule re- the primary ionic bond is strong at long-range interactions,
duces the van der Waals forces significantly. For example, both primary and secondary bonds are essential in keeping
the binding site of an antibody is usually only over several both molecules together because the total binding energy of
amino acids, or 0.4e8 nm.2Van der Waals forces also work primary and secondary bonds is usually much higher than
across a short range of approximately 0.5 nm because of the the binding energy of the primary bond alone.23 On the
exponential decay as the distance increases. Furthermore, other hand, the same attraction mechanisms are also formed
van der Waals forces become repulsive closer than 0.4 nm with other molecules, which often cause nonspecific bind-
apart. The second weakest bond energy is the hydrogen ing and NP surface energyegenerated protein corona
bond. Hydrogen bonds occur in an interaction between an around the NPs that can potentially overwhelm specific

TABLE 3.1 Various types of binding energy between two molecules in a solution.

Bond Energy (kJ/mol) Length of interaction (nm)

Ion-dipole 20 10
Hydrogen 20 0.15e0.5
Hydrophobic <40 10
Van der Waals 4 0.4e0.6
Average molecular kinetic energy in solution 2.6
Nanoparticle surface energy 7.6e454
 Active targeting and transport Chapter | 3 21

TABLE 3.2 Effect of nanoparticle physicochemical

properties on surface binding energy.

Effect on corona coverage and

Property thickness
Size Inversely proportional
Aspect ratio Directly proportional
Zeta potential Directly (both þ and ) proportional
Hydrophobicity Directly proportional
Surface Directly proportional
roughness

higher surface area per mass of NP and thus show greater

corona formation as compared with larger NPs.25
Nonspherical NPs have a higher surface-area-to-volume
ratio than spherical NPs so they show more corona
formation compared with that of spherical NPs.26 Further-
more, NPs with a rough surface have a greater surface-area-
to-volume ratio as compared with NPs with a smooth
surface and thus show more corona formation and thickness
as compared withsmooth NPs. A higher degree of hydro-
phobicity on the surface of an NP also affects the corona
formation because of the relatively strong binding energy
associated with hydrophobic binding in aqueous solution.25
The surface charge of an NP also affects corona formation
through ionic interactions that attract and binds opsonins to
FIGURE 3.1 Opsonization of SiO2-PEG-transferrin nanoparticlesurface
blocking the binding with (A) free transferrin receptor and (B) transferrin
the surface of NPs.27 Corona formation also negatively
receptor on cell surface.24 Credit: Reproduced with permission from Anna affects the physicochemical properties of the NPs and leads
S et al.: Transferrin-functionalized nanoparticles lose their targeting ca- to a reduction in blood circulation half-life. Furthermore,
pabilities when a biomolecule corona adsorbs on the surface, Nat Nano- the corona will often cover the targeting agents on the
technol 8:137e143, 2013. surface of the NP so they can no longer bind with their
target molecules, reducing the effectiveness of the NPs
ligandeantigen interactions (Fig. 3.1).24 Therefore, the in vivo.24 PEG is also used to reduce opsonin binding on
combined specificity of targeting agents and the ability of the NPs by both reducing the surface energy of the NP and
NPs to prevent corona formation are both crucial. through steric hindrance. As a result, PEG was found to
Blood plasma contains a large amount of proteins, increase the half-life of circulating NPs inside the blood.28
biomolecules, cells, and salt, which interact with one
another and readily adsorb on the surface of NPs to form 3.3 Targeting agents
opsonins, which are recognized by the immune system for
removal. The opsonins on the surface of NPs form the Targeting agents are one of the main components in
corona, where the thickness is directly proportional to the actively targeted NPs. Most ligands utilize 3D structure,
surface energy of the NP. The surface energy of a bare, charge, and hydrophobicity, which complement its targeted
uncoated NP is affected by physicochemical parameters molecules to increase the binding specificity. Various li-
such as size, shape, surface charge, and hydrophobicity, as gands are utilized in actively targeted NPs with a wide
summarized in Table 3.2. The surface-area-to-volume ratio range of molecular weights, targeting specificities, and
of an NP is one of the main factors affecting corona for- manufacturing costs (Table 3.3). Each ligand has its own
mation on the surface of NPs because surface energy is advantages and disadvantages for its use in actively tar-
directly proportional with surface area. Smaller NPs have geted NPs.
22 Nanoparticles for Biomedical Applications

TABLE 3.3 Comparison of molecular weight, binding affinity between ligands and targeted molecules, half-life
in blood circulation, targeting specificity, and manufacturing cost between different targeting agents that are
commonly used in actively targeted nanoparticles.

Targeting agents Molecular weight (kDa) Binding affinity range (KD) Blood half-life Specificity Cost
Antibody (IgG) 150 107e1013 M 15e30 days þþþ $$$
5 11
F(ab’)2 110 10 e10 M Several days þþþ $$$$
5 11
F(ab) 50 10 e10 M 30 min þþþ $$
5 11
scFV 28 10 -10 M 10e30 min þþþ $$
Aptamers 5e50 107e1011 M 5e10 min þþþ $$
Peptides 1.5e50 109e1012 M 4e15 min þþþ $$
7 15
Protein 80 10 e10 M 10e20 days þþ $$
3 5
Carbohydrate 0.2e100 10 e10 M Minutes to hours þþ $$
Vitamin 0.3e1.4 109e1011 M Hours to days þþ $

þ: the targeting specificity of ligands relative to one another (þ ¼ not specific, þþ ¼ moderately specific, þþþ ¼ very specific).
$: the estimated production costs of targeting ligands determined from various commercial sources ($ ¼ <USD0.25/mmol, $$ ¼ USD0.25e5/mmol,
$$$ ¼ USD5e10/mmol, $$$$¼ > USD10/mmol).

3.3.1 Antibody/antibody fragment or cell. Therefore, the orientation of Ab attachment to NPs

is important to ensure functionality.
Antibodies (Abs) are one of the earliest and most
NPs with Fc regions facing outward can be recognized
commonly used ligands for actively targeted NPs. Abs are a
and phagocytosed by leukocytes. One possibility to over-
specific type of protein produced in B cells that bind to
come this limitation is by removing the Fc region from the
target antigens as part of the body’s adaptive immune
Ab. Whole Ab can be cleaved with pepsin and papain to
system. Here, we consider Abs separate from proteins since
produce F(ab’)2 (110 kDa) and Fab (50 kDa), respectively.
Abs are widely used in NP targeting studies because of
F(ab’)2 can also be cleaved by dithiothreitol to generate
their high specificity, strong affinity, and a wide variety of
Fab’ (55 kDa). Since the variable region is the most crucial
available targets.
part in binding with an antigen, the cleaving of Ab does not
There are five major classes of antibodies or immuno-
eliminate the binding ability of Ab fragments. Moreover,
globulins (Ig) that are produced in mammals: IgA, IgD,
work has been done with a single chain of the variable
IgE, IgG, and IgM, all made up of combinations of heavy
fragment (scFv, 25 kDa) to only keep the variable region
chains (50 kDa or 440 amino acids each) and light chains
while having much smaller molecules than the whole Ab or
(25 kDa or 220 amino acids each).29e33 The most common
Ab fragments (Fig. 3.2).33,35,36
Ig used in active targeting is IgG.31,32 IgG is the dominant
F(ab’)2 and full Abs (KD ¼ 6.9  109 M) have bind-
class of human Ig and consists of two identical light chains
ing affinities up to 100 times stronger than the binding
and two identical heavy chains that are held together by
affinities of Fab and scFv (KD ¼ 6  107 M and
several disulfide bonds between cysteines to form a
5.9  107 M, respectively).37 F(ab’)2 and Abs have two
150 kDa molecule (Table 3.3). The heavy and light chains
binding regions, whereas Fab and scFv only have one
each contain three complementary-determining regions,
binding region; thus, the lack of a multivalent effect may
located at the N-termini of Abs. These complementary-
account for some of this loss in binding affinity or increase
determining regions have the most variable amino acid
in KD. This was shown empirically by utilizing high-speed
combinations to complement the specific target antigen and
atomic force microscopy. Preiner et al.38 found that whole
also provide a very strong avidity in the range of
Abs showed bipedal stochastic walking on a surface of
107e1013 M (Table 3.3).34 The C-termini of Abs contain
regularly spaced epitopes until the Abs found two antigens,
a fragment crystallizable region (Fc), which has a high
within a 6e12 nm range, which perfectly aligned with both
affinity toward leukocytes. The structure of Abs allows the
binding regions of the Ab. Conversely, monovalent Fab
Abs to recognize and bind to the antigen of pathogen or
fragments might rotate on the same antigen until it found
cell, whereas the Fc region remains accessible for leukocyte
the same orientation and stand still. The multivalent effect
binding and subsequent phagocytosis of the target pathogen
might need to be considered when designing the density of
 Active targeting and transport Chapter | 3 23

FIGURE 3.2 An illustration showing the shape and dimension of Ab and Ab fragments from both front and side views.

Abs and Ab fragments on the NP surface because subop- An amide bond is a stable linkage formed by the
timal distance and geometric constraint destabilize multiple condensation reaction of a carboxylic acid and a pri-
binding.38e41 mary amine, usually through a carbodiimide reaction
The specificity and high affinity of Abs to bind with anti- (Table 3.4). Carbodiimide reactions often utilize 1-ethyl-
gens combined with the ability of NPs as a delivery vehicle and 3-(3-dimethylaminopropyl)carbodiimide (EDC) to form
imaging agent have brought many opportunities and break- an intermediate ester bond before being substituted by a
throughs in biomedicine. Therefore, the conjugation of Abs primary amine group. An amide bond can also be
onto the surface of NPs has been widely studied, such as Ab- formed by activating primary alcohol groups on Abs
NP conjugation strategy, the density of Ab on NPs (serine, threonine) or NPs with p-toluenesulfonyl chlo-
surface,39e41 and controlled orientation of Ab on the surface.42 ride to become amine reactive. An advantage of the
Abs possess a large number of functional groups that can amide bond is that no modification on the Ab is
be conjugated to NPs. These include carboxylic acid groups required, reducing the risk of loss in reactivity and
(glutamic and aspartic acids), amine groups (lysine, aspara- denaturation. One of the limitations is the inability to
gine, and glutamine), and thiol groups (cysteine). However, control the orientation of the Ab on NP because of the
the spread of these amino acids along the Abs results in an distribution of carboxylic acid and amine groups
uncontrollable orientation of conjugated Ab on NP. NPs throughout the Ab. The Ab might link to more than one
containing a random orientation of Abs resulted in a 10-fold NP, to more than one site of the same NP, or even
reduction in antigen-binding affinity compared with NPs linkage to NP on the variable region, which can block
where the Ab-binding regions all faced outward.43 Therefore, Ab binding to targeted molecules. Therefore, the ratio
various conjugation strategies for Ab attachment on NPs result of reactants must be carefully controlled.
in different abilities to control orientation as outlined below.
l Schiff base reaction:
3.3.2 Several common covalent-binding A reaction between an aldehyde and primary amine
reactions forms an imine bond, a Schiff base. Typically, NPs are
activated with aldehyde groups such as by reaction with
l Carboxylic acid and primary amine reaction: excess glutaraldehyde. Another way of binding Ab to NP
 24
Nanoparticles for Biomedical Applications
TABLE 3.4 Commonly used linkage, functional group, and chemical reactions to bind antibodies (Abs) with nanoparticles (NPs), as well as the binding
stability and Abs orientation on the surface of the NPs.

Type of Commonly used

bond Linkage Conjugated functional group reaction Linkage stability Abs orientation
Covalent Amide bond Primary amineecarboxylic acid Carbodiimide reaction Stable Uncontrolled
Imine bond Hydrazide/hydrazineealdehyde/ Schiff base reaction Labile under acid condition Can be
ketone controlled
Thioether bond Thiolemaleimide Michael reaction Stable Uncontrolled
Disulfide bond Thiolethiol Labile under reducing Uncontrolled
conditions
Dative bond Thiolemetal Stable Uncontrolled
Triazole ring Azideealkyne Click chemistry Stable Uncontrolled
Noncovalent Biotineavidin Biotineavidin Stable Can be
Biotinestreptavidin controlled
Protein A and G Protein A/GeAb Fc region Labile under competing Abs Controlled

Ionic bond (physioadsorption) Labile under competing Uncontrolled

opsonins

Nucleotide-binding site Nucleotide-binding siteeindole Ultraviolet free radical Controlled

eindole
 Active targeting and transport Chapter | 3 25

with Schiff base is to utilize hydrazide or hydrazine func- an alkyne functional group (Table 3.4). Click reactions to
tional group (ReNHeNH2) with aldehyde or ketone to conjugate Abs to NPs have been shown to be fivefold to
form a hydrazone bond (Table 3.4). eightfold more efficient than amide reactions.47,48 Never-
While these conjugation reactions do not ensure a well- theless, Ab orientation on the NPs is still difficult to control.
oriented Ab, the mild oxidation of vicinal hydroxyl groups
with sodium periodate on carbohydrate residues to become
aldehyde groups can result in oriented Ab conjugation.44 3.3.3 Several common non-covalent bindings
This takes advantage of the fact that carbohydrate residues
l Avidinebiotin:
on Abs are specifically located on the Fc region. As an
example, an oxidized Ab was conjugated to a hydrazide The specific binding between avidin and biotin is one of
group of a bifunctional PEG that also contains a dithiolar- the strongest known non-covalent bonds (KD w 1015 M)
omatic group. The dithiolaromatic group was then used to (Table 3.4). Avidin is a glycoprotein with four subunits,
bind to the surface of a gold NP through a covalent bond which can bind to up to four biotins. An advantage of this
between sulfur and gold atom.44 reaction is that avidin can be introduced directly into the Ab
backbone in the Fc region through genetic engineering of
l Thiol reaction:
hybridomas, in vitro cell cultures that produce Abs. The
Although thiols are present on Abs, they are involved resulting fusion protein can then be attached to biotinylated
in intramolecular disulfide bridges within the native Ab. NPs with well-controlled orientation and stoichiometry.
Hence, these disulfide bridges must first be reduced to
l Proteins A and G:
make thiols available for reaction. Conversely, free thiols
can be introduced onto Abs by reaction with amine Protein A and protein G are proteins utilized by
groups, such as with using Traut’s reagent. Free thiols on Staphylococcus aureus and Streptococcus C40, respec-
Abs can be used to conjugate to NPs through disulfide or tively. Both protein A and protein G are able to bind and
thioether bonds (Table 3.4). Typically, disulfide bonds are neutralize Ab from immune recognition, as they specif-
not used because they are difficult to control and often ically bind to the Fc region of IgG. This has provided the
lead to cross-linking between NPs. On the other hand, opportunity to utilize these proteins to control Ab orienta-
thioethers can be formed with maleimide-activated NPs as tion on NPs (Table 3.4). Up to 95% of protein A on the
well as through Michael addition reactions with aldehydes surface of cyanoacrylic NPs was found to bind with Ab
on the NP, with efficiency up to 95%e99%.45 Abs with during the conjugation reaction.49 However, in vivo studies
activated thiol groups can also create a covalent bond to were disappointing where Ab conjugated NPs accumulated
metal NPs (e.g., gold NPs), which is often called a dative mostly in the liver and spleen. The adsorption of excess
bond where the bond was formed through electron from protein in blood serum might compete with protein A,
the metal atom shared to the thiol group (Table 3.4). For which can break the binding between Ab and protein A.50
example, Abs were reduced with tris(2-carboxyethyl)
l Physioadsorption:
phosphine, a weak reducing agent that allows more spe-
cific disulfide bond reduction on Ab, which are then Adsorption of Abs on the NP surface was one of the
available for dative binding to the gold NPs.46 However, earliest studied attachment techniques. Ab-bound
the reduction method might be too harsh to the Ab that it phospholipid-based NPs were prepared by merely adding
might damage the Ab tertiary structure and reduce the the ligand to the NPs solution. If the phospholipid charge
binding activity of Ab significantly when compared was neutral, 4%e40% of the IgG bound to the liposomal
withthe carbodiimide reaction and ionic interaction.46 NP. When anionic phospholipids were used, the binding of
Similar to the formation of an amide bond or a Schiff base, Ab to NP was about 50% higher than that of neutral
the orientation of Abs on the NPs is also difficult to phospholipids.51 These results suggest that Ab adsorption
control using these thiol reactions. on liposomal NPs depends on the hydrophobicity, as well
as the ionic charge of the liposome NP (Table 3.4). Ab-
l Click chemistries:
bound liposomal NPs increased the antigen binding by
Click chemistries are a set of reactions that are highly 30%e50% compared with free Ab.51 However, adsorbed
specific, stereospecific, efficient, easily performed, reactive Abs might be exchanged by stronger binding opsonins in
in easily removed solvents and eliminate the need for the blood, potentially decreasing the effectiveness of the
chromatography to remove byproducts. These reactions Ab-NP.
might require a catalyst, a substance involved in a chemical
l Others:
reaction that increases the reaction rate but is not consumed
in the overall reaction. Copper is a typical catalyst used in A small indole molecule can be entrapped into the
click chemistry between an azide functional group (N3) and nucleotide-binding site of Ab (KD ¼ 1e8 mM) with the
26 Nanoparticles for Biomedical Applications

help of ultraviolet light (Table 3.4). The nucleotide-binding through 8e20 panning steps from a large pool of aptamers
site allows specific site and orientation of Ab on the NP (1013e1015 aptamer sequences) until only several high-
surface.52 Utilizing the nucleotide-binding site of Ab affinity aptamers toward targeted molecule are obtained at
increased NP binding and sensitivity toward antigen the end of the cycles.
significantly as compared with a carbodiimide reaction, Similar to Abs, aptamers can bind to various antigens,
ionic interaction, and thiol bond.46 such as inorganic molecules, small organic molecules,
Despite the advantages of Abs as a targeting ligand, its proteins, peptides, carbohydrates, antibiotics, whole cells,
large size can significantly increase the hydrodynamic and even organisms, with high specificity and selectivity.
size of small NPs. For example, 12.5 nm NPs increase in The interactions between aptamers and antigens are usually
size to 25 nm after being conjugated with Abs.46 The based on electrostatic interactions, hydrophilic interactions,
production cost of Ab is also very expensive because of and complementary shapes. The multivalent effect can also
the need for live animal hosts to produce titers. be exploited with aptamer-modified NPs to increase bind-
Mammalian cell culture can be used as a potentially ing affinity. This was shown using a dimer aptamer
cheaper method but requires significant numbers of cells composed of two aptamers (15-mer and 29-mer thrombin
to generate useable quantities of Ab. F(ab’)2 is derived aptamers linked by a flexible linker), which bind two
from whole Ab with further purification, which makes the different sites on thrombin. The dimer aptamer decreased
production cost of F(ab’)2 even more expensive than the the KD 10-fold when compared with the KD of the 15-mer
production cost of the whole Ab. Even though Fab can be thrombin aptamer itself.58 A similar effect was found with
derived from whole Ab, similar to F(ab)’2 production, Fab multiple different aptamer sequences on a single NP.
and scFv can also be produced from bacteria, which grow Multitargeted NPs were able to accumulate on the targeted
much faster and in higher densities than mammalian cell molecule at a higher level than NPs modified with a single
culture and can reduce the production cost of Fab and aptamer sequence.59
scFv. Another advantage of aptamers is that they can be
engineered to have multiple regions with different func-
tions such as to promote entrapment inside an NP. For
3.3.4 Aptamers example, phosphorothioate-modified DNA bases were
Beyond their use for storing and expressing the genetic used to give a hydrophobic characteristic to the aptamers
code, nucleic acids (DNA and RNA) form two- and three- while a tumor targeting sequence was kept hydrophilic.60
dimensional structures, which are more stable than linear This allowed the active targeting domain to be exposed on
oligonucleotides. Oligonucleotides are well-known for their the surface of the NP while entrapping the hydrophobic
double-stranded helical structures and their single-stranded domain inside the NP during NP synthesis. The entrapped
hairpin structures. However, these are not the only three- aptamer was able to improve accumulation of the targeted
dimensional structures of oligonucleotides. For example, NPs on lung cancer cells grown subcutaneously in nude
a thrombin-binding aptamer is a G-quadruplex structure mice.
formed by the stacks of planar guanine tetrads (Fig. 3.3), One of the limitations of aptamers is the high serum
where molecular structure of the thrombin-binding aptamer nuclease activity in blood that can quickly degrade nu-
was determined by NMR spectroscopy and X-ray crystal- cleotides or aptamersdunmodified nucleotides have sta-
lography.55 The natural use of oligonucleotides to bind with bility half-lives of as short as 2 min in blood serum
other molecules is very limited; however, random ar- (Table 3.3).61 These very short half-lives can be overcome
rangements and panning of oligonucleotides have led to the by chemical modifications of the aptamer backbone such
evolution of oligonucleotide-based targeting ligands. The as capping the 30 -terminus to prevent 30 -exonuclease ac-
randomly arranged oligonucleotide sequences are called tivity, which is much higher than 50 -exonuclease activity
aptamers, which means: to fit.56 in serum. Another strategy is to create enantiomer nucle-
An early and widely used method to produce aptamers otide backbones (L-ribose and L-deoxyribose), which also
is through SELEX (Systematic Evolution of Ligands by reduces nuclease degradation.62 Despite the adaptability
Exponential enrichment), which was developed in 1990.57 of aptamers, their binding is typically limited toward hy-
In the same year, an independent study found a specific drophobic and negativelycharged antigens. Aptamers are
three-dimensional structure of RNA that was able to bind very hydrophilic, and the phosphate backbone is nega-
with a small molecule.56 SELEX produces aptamers of tively charged; therefore, the difference between
20e100 oligonucleotides, with molecular weights of hydrophobicityehydrophilicity characteristics of antigen
5e50 kDa (Table 3.3). The process of SELEX usually goes and aptamers weakens the binding because of the
 Active targeting and transport Chapter | 3 27

FIGURE 3.3 (A) Size comparison between Ab and thrombin aptamer53 and (B) chemical structure of thrombin-binding aptamer.54 Credit: Reproduced
with permission from Jennifer FL et al.: Aptamer therapeutics advance, Curr Opin Chem Biol 10:282e289, 2006; and Baobin C et al.: Stability and
bioactivity of thrombin binding aptamers modified with D-/l-isothymidine in the loop regions, Org Biomol Chem 12:8866e8876, 2014.

hydrophobic force preventing hydrogen bonding to occur. to NP in a similar conjugation technique as Ab conjugation
Likewise, if the antigen is negatively charged, there will to NP. The conjugation site and the availability of the active
be a repulsive ionic interaction between the antigen and site for binding with antigen are also important when
aptamer. considering peptide conjugation strategies. On the other
hand, peptides are similar to nucleic acid aptamers as most
3.3.5 Proteins peptides are easily synthesized in the lab and bind with a
specific target. Peptide sequences can be discovered through
Similar to Abs, proteins also contain functional groups on
multiround panning processes using phage display technol-
their N- and C-termini, as well as the amino acid building
ogy.67 Peptides can also be engineered to have similar active
blocks. Transferrin has been extensively used as a targeting binding sites with antibodies and proteins. The binding af-
protein on the surface of NPs because of its ability to target
finities of peptides are comparable withthe binding affinities
the transferrin receptor, which is upregulated in many types
of antibody fragments, which are in the nanomolar range.
of cancer cells. One of the advantages using proteins is the
Peptides can be designed with multiple binding sites to take
higher molecular weight compared withthe other ligands
advantage of the multivalent effect, which increases the
(e.g., MW of transferrin is 80 kDa), which significantly
binding affinity of the peptides to the antigens (Fig. 3.4).66
increase the circulation time of the protein in the blood
Arginineeglycineeaspartate (RGD) tripeptide and its
(Table 3.3). One of the most common conjugations of
derivatives are well-known for their ability to bind to
transferrin to NPs is through carbodiimides reaction to form integrins, the binding proteins of cells with the extracellular
an amide bond. For example, transferrin conjugated to
matrix. Integrins are often upregulated in solid tumors;
PEGeliposomal NPs wasused to target disseminated
hence many tumor-targeted NPs have relied on RGD-based
gastric cancer cells through an intraperitoneal injection.63
peptides as a targeting agent.68e72 To increase binding af-
The transferrin-targeted NPs had higher uptake and pene-
finity and specificity to a5b1 integrin, which is overex-
tration into solid tumor tissue, which resulted in a higher
pressed on breast, prostate, rectal, and colon cancers, a
survival rate in a human xenograft mouse model as
multivalent peptide containing GRGDSP and PHSRN
compared with control NPs without transferrin.63 In another
subunits was developed.73,74 From the crystal structure,
study, transferrin-targeted NPs were found to penetrate GRGDSP and PHSRN peptides are able to bind on the
hepatic and cervical tumor spheroids much more than the
same face of fibronectin 3.5 nm apart.75 RGD domain of
free drug and non-targeted NPs.64
the GRGDSP is a well-known component to interact with
However, the competition between proteins on NPs and
fibronectin, whereas PHSRN peptides arethe synergy re-
the excess protein in the blood plasma might reduce the
gion that increases the avidity between targeting peptides
targeting ability of protein targeting NPs toward the protein
and fibronectin. This multivalent peptide was attached to
receptors.64,65
the surface of a liposomal/micellar NP carrying doxoru-
bicin to improve delivery into colon cancer cells. As a
3.3.6 Peptides result, the multivalent peptide sequence was able to
Peptides can be thought of as very small proteins, as they improve the delivery rate as compared with the NPs tar-
consist of a short chain of amino acids and can be conjugated geted with GRGDSP alone.74
28 Nanoparticles for Biomedical Applications

targeting various tumor types in mouse models for intra-

operative imaging11,83,84 as well as drug delivery.82,85,86
Several common functional groups on carbohydrates
that can be readily conjugated are hydroxyl (alcohol)
groups, ethers, aldehydes, and ketones. Some carbohy-
drates also contain carboxylic groups, such as in HA.

3.3.8 Vitamins
Vitamins are molecules, which are essential for the body
but are not part of minerals, essential fatty acids, and
FIGURE 3.4 Two binding sites of a polypeptides chain of GRGDSP and
essential amino acids. Vitamins can be hydrophobic
PSHRN increase the binding specificity and avidity toward fibronectin III
on the repeats 9 and 10, compared with the individual binding sites. (vitamin A, D, E, and K) or hydrophilic (vitamin B and C).
Inspired by Kokkoli E.66 Almost every tissue has vitamin receptors, but some tumors
and specific tissues often overexpressed vitamin receptors,
However, peptides have several disadvantages such as which allows for vitamins to be used in actively targeted
cleaving and degradation of peptides exposed on the sur- NPs.
face of NPs by serum protease in the blood plasma, which Two of the most utilized vitamins in actively targeted
reduce the effectiveness of peptide targeting. Fortunately, NPs are folate (vitamin B9) and vitamin B12. Folate is
the stability of peptides in blood plasma can be improved required for biosynthesis of purines and pyrimidines. Even
by adding cysteines to form disulfide bonds generating a though the folate receptor availability on the normal cells is
more stable cyclic peptide, blocking the C- and N-terminus, limited, folate receptors are commonly overexpressed on
utilizing D-amino acids backbones, or using synthetic the surface of activated myeloid cells and tumor cells, such
amino acids that are incompatible with proteases.76e78 The as ovarian, lung, breast, and brain cancers.87,88 On the other
small size of peptides also makes them prone to kidney hand, vitamin B12 is often used in active targeting for in-
clearance (MW ¼ 1.5e50 kDa) (Table 3.3), which can be testine absorption, as vitamin B12 promotes receptor-
overcome by conjugating peptides to NPs. mediated endocytosis to go through the intestine endothe-
lial cells into the blood vessel.89e92
Vitamin B12 is much larger (1355 Da) than other vi-
3.3.7 Carbohydrates
tamins (e.g., folate is 441 Da) (Table 3.3). Therefore,
Carbohydrates consist of carbon, hydrogen, and oxygen, vitamin B12 needs a specific pathway to be absorbed in the
and sometimes phosphorus, sulfur, and nitrogen atoms. intestine. In the small intestine, vitamin B12 interacts with
There are three types of carbohydrates: monosaccharides, intrinsic factor protein, which helps to bind to intrinsic
disaccharides, and polysaccharides. Monosaccharides are factor receptor and is absorbed through the intestinal wall.
the simplest form of carbohydrates, such as glucose, fruc- Folate, on the other hand, has a carboxylic acid group
tose, and galactose, and are usually the building blocks of that is readily conjugated with an amine group and other
the more complex carbohydrates. Disaccharides comprise functional groups. The conjugation of folate on the car-
two monosaccharides, such as sucrose (glucose and fruc- boxylic acid also does not affect the KD of folate to the
tose), lactose (glucose and galactose), and maltose (glucose folate receptor, as the terminal carboxylic group is more
and glucose). Polysaccharides and oligosaccharides are a reactive for conjugation. There are two folate receptors,
chain of sugars with three or more monosaccharides and FR-a (KD w 1011) and FR-b (KD w 109) (Table 3.3).93
can be linear (such as with amylose) or branched (such as FR-a is overexpressed on 40% of human cancers and has
with glycogen). Therefore, the molecular weight and blood limited expression in normal cells. On the other hand, FR-b
circulation half-life of carbohydrates widely varied, with is upregulated in activated myeloid cells, such as 70% of
molecular weights of 0.2e100 kDa and blood circulation acute myeloid leukemia cells.94,95 Folate is also stable, is
half-lives of minutes to hours (Table 3.3). inexpensive, promotes rapid internalization into tumor
Many cancer cells overexpress the CD44 cellsurface cells, is readily conjugated, and is nonimmunogenic.
marker, which binds to hyaluronic acid (HA), a glycos- Vitamins as targeting agents offer high specificity to
aminoglycan component of the extracellular matrix. HA their respective receptors (KD ¼ 109e1011 M),93 non-
has garnered significant attention as it can be conjugated to immunogenicity, as well as ease of production leading to
the NP surface as a targeting agent similar with other very low costs (Table 3.3). However, vitamin-modified NPs
ligands,79e81 and it can also be utilized as the HA-based NP have similar disadvantages as protein-targeting NPs, in
building block reducing the need for secondary which excess vitamins in the body might compete with
conjugations.11,82e86 HA-based NPs have been used for vitamin-modified NPs to bind with the specific
 Active targeting and transport Chapter | 3 29

receptor,96e99 reducing the targeting ability. Moreover, the uptake into these cells.14 Active Ab-mediated targeting of NPs
expression of vitamin receptors (e.g., folate receptor) on the to tumors can be further enhanced when NPs are loaded with
surface of multiple cell types, such as activated myeloid chemotherapeutic agents. Chemotherapy loading into the
cells and tumor cells, might cause off-targeting using active targeted NPs reduced the immune response against
vitamin-targeting NPs. these PEG-coated NPs, allowing for increased tumor delivery
compared with the active targeted NPs without chemotherapy
loading.112 The reduction in the immune response was a result
3.4 Active targeting strategies for of the cytotoxicity of the chemotherapy agent to both adaptive
various diseases and innate immune cells, which blocked any subsequent im-
NPs have been widely studied over the past few decades in the mune response.112
biomedical field. However, most studies have focused on Leukemia is a hematological malignancy that does not
cancer, which has led to the formation of a substantial body of form a solid tumor despite some evidence of EPR observed
literature on understanding the effects of physicochemical, in bone marrow103; hence, there is no EPR effect that NPs
molecular, and active targeting properties of NPs in tumor can exploit. Therefore, actively targeted NPs might be ideal
tissues and the body. Fortunately, much of this literature, such in targeting leukemia cells to reduce the toxicity of sys-
as ligand conjugation techniques, physicochemical properties temic therapies. On acute myeloid leukemia, epidermal
of NPs that increase blood circulation half-life, and strategies growth factor receptor (EGFR) is often overexpressed,
that improve tissue penetration, can be translated to use in which can readily be targeted by an anti-EGFR Ab,
other diseases. Even though cancer is still one of the most aptamer, or peptide. In one study, anti-EGFR Ab was
focused research areas of NPs, the utility of NPs in non- biotinylated to allow for attachment to NeutrAvidin-
neoplastic diseases has begun to gain attention. This includes modified mesoporous silica NPs (MSNs).13 Active tar-
the use of the active targeting ability of NPs to increase geted NPs were able to specifically bind to and be inter-
transmembrane penetration, accumulation in non-EPR dis- nalized by EGFR-expressing leukemia cell both in vitro
eases, and more selective and specific therapeutics delivery and in an ex ovo model. Following this, chemotherapy-
and imaging. Here, focus is given to studies that have pre- loaded, anti-EGFR Ab-targeted MSNs showed active
clinical mechanistic data. targeting-mediated cell kill in leukemia cells both in vitro
and in a mouse model, which improved survival.113
The GE11 peptide, an EGF mimetic, has been conju-
3.4.1 Cancer gated to various NPs for active targeting of EGFR-
Cancer is an unregulated cell growth usually caused by mu- overexpressing tumors.114e117 However, the internaliza-
tations in cell growth signaling and receptor regulatory pro- tion mechanism for NPs modified with the artificial GE11
teins. Cancer is well-known for its EPR effect compared with peptide is different from that of those modified with natural
healthy tissues because of uncontrolled angiogenesis and lack EGF.118 Single particle tracking revealed EGF-modified
of lymphatic drainage. Therefore, passive targeting NPs (see NPs were rapidly internalized into EGFR-expressing cells
Chapter 4) are able to show a significant result in cancer in vitro, showing 80% internalization within 10 min. On the
therapy. However, active targeting can still improve NP de- other hand, GE11-modified NPs took 4 h to reach the same
livery in EPR cancers. A series of studies found that active level of internalization as EGF-modified NPs, while non-
targeting did not increase the accumulation of NPs in EPR targeted NPs reached only 31% internalization. The dif-
tumors, which was dominated by the EPR effect, but distri- ference in internalization between NPs targeted with EGF
bution throughout the tumor and delivery into cells was and GE11 was found to be a result of EGFR activation with
increased with active targeting.8e10,100,101 These results native EGF, which promoted rapid internalization, whereas
showed the importance of active targeting in improving NP EGFR was not activated by GE11.118 This is one example
delivery even in tumors where the EPR effect allows for of how the choice of active targeting agent on the surface of
passive NP targeting. Active targeting is even more critical in an NP can alter target cell uptake.
cancers that do not have an EPR effect such as some hema-
tological malignancies,102e106 small metastatic
107e110 3.4.2 Atherosclerosis
tumors, and circulating tumor cells.111 In these cases,
active targeting is required for specific binding to these cells. Atherosclerosis is the leading cause of cardiovascular dis-
For example, Ab-modified iron oxide NPs targeted to the ease, in which chronic, inflammatory lipid-rich plaques and
HER2/neu receptor were able to bind to breast cancer metas- cholesterol particles accumulate within the artery wall. The
tases to the liver, lungs, brain, and bone marrow in a transgenic inflamed endothelial cells are also prone to leakiness, which
mouse model, whereas IgG control-modified NPs showed no can result in an EPR-like effect. Therefore, NPs are suitable
30 Nanoparticles for Biomedical Applications

to be used in passive drug delivery and diagnosis of disrupted such as in brain cancer132 and TBI,133e138 active
atherosclerosis.119e122 While passive targeting of athero- targeting is required in disorders where BBB integrity is
sclerotic plaques can be achieved with NPs depending on intact. Targets typically include transmembrane ligands to
the permeability of the vessel wall,123 active targeting is act as a Trojan horse to gain access into the brain through
expected to achieve higher penetration into the plaque and receptor-mediated transcytosis across the BBB. These
internalization into target cells. Several ligands have been include receptors for transferrin,3,139e141 lactoferrin,2,142,143
used in active targeting of atherosclerosis, such as vascular and possibly RAGE (receptor for advanced glycation end
cell adhesion molecule-1 (VCAM-1),124,125 monocyte products), which is overexpressed on diseased brain
chemoattractant protein-1 (MCP-1),126 interleukin-10 (IL- microvascular cells,144 or tight junction proteins.
10),127 and E-selectin,128 in hopes of improving therapeutic The rabies virus glycoprotein peptide (RVG29,
delivery and retention to reduce plaque size and inflam- TYIWMPENPRPGTPCDIFTNSRGKRASNG) was devel-
mation. These active targeted NPs showed higher accu- oped to cross the BBB by taking advantage of the pathway
mulation rates within the plaques and significantly longer exploited by the neurotropic rabies virus, either through
retention times to potentially improve therapeutic delivery. nicotinic acetylcholine receptor (nAchR) or GABA recep-
HA NPs have also been used for active targeting of in- tor binding. RVG29 conjugated to generation-5 PAMAM
flammatory cells in plaques since the inflammatory process dendrimers through bifunctional PEG accumulated in all
requires HA-immune cell interactions.129 HA can also act regions of the mouse brain significantly higher than the
as a therapeutic since nanoformulated HA has been shown nontargeting NPs.145 Similar results have been found in
to act as an anti-inflammatory.130 Thus, the HA provided other RVG29 active-targeting NPs where receptor-
the building blocks to achieve the NP size needed for mediated transcytosis across the BBB increased brain de-
passive accumulation, active targeting to promote target livery of various therapeutics to improve treatment in
cell-specific interactions, and therapy by providing athero- mouse models of Alzheimer’s disease and Parkinson’s
protective effects.129 disease.17,146e148
RVG29-conjugated NPs have been utilized for active
3.4.3 Kidney disease targeting toward TBI.15 TBI is an injury that results from a
primary impact to the brain followed by the secondary
Chronic kidney disease is a state of reduced kidney func- release of biochemicals such as reactive oxygen species
tion that can lead to various other health complications such (ROS), glutamate, calcium, and lipid peroxidation products
as stroke, hypertension, and liver dysfunction. Small that can cause long-term neuroinflammation and neuro-
molecule therapeutics have been hindered by poor circu- degeneration for years following the injury. A
lation times, which requires high doses to be delivered transportaneRVG29 peptide complex was developed to
leading to off-target side effects and even lethality. Thus, form a micelle, where anti-caspase-3 siRNA was entrapped
active targeted NPs have been developed using a kidney- inside the cationic micelle. As a result, RVG29 signifi-
targeting peptide (KKEEE)3K to specifically bind to meg- cantly increased the micelle accumulation in a mouse
alin, a cell surface receptor on renal tubule cells, through model of TBI compared withcontrol peptide and resulted in
multivalent display.131 NPs slightly larger than the 10 nm decreased caspase-3 production in target neurons.15
cutoff of glomerular filtration were used and showed se- Neuroinflammation increases expression of vascular
lective accumulation on renal proximal tubule cells in vitro. cell adhesion molecule-1 (VCAM-1) on brain endothelial
In healthy mice, active targeted NPs showed significantly cells, which is an ideal target of active NP targeting. NPs
higher kidney accumulation than non-targeted NPs, sug- targeted to VCAM-1 with Abs or peptides have been used
gesting the use of active targeting for kidney disease.131 for imaging specific regions of neuroinflammation.149,150
Similar strategies have been used with the platelete
3.4.4 Neurological disorders endothelial cell adhesion molecule (PECAM-1), which is
also overexpressed on brain endothelial cells as a result of
Neurological disorders encompass various diseases, neuroinflammation. NPs actively targeted to PECAM-1
including brain cancer, traumatic brain injury (TBI), stroke, show improved brain delivery as compared with control
neuroinflammation, and progressive neurodegenerative NPs.151 This could improve diagnostic information and
diseases such as Alzheimer’s and Parkinson’s disease. The provide a platform for therapeutic delivery.
treatment of neurological disorders is especially chal-
lenging because of the sensitivity and importance of the
brain to survival as well as the BBB, which prevents the 3.4.5 Rheumatic inflammation
passive accumulation of delivered therapies into the brain Rheumatoid arthritis (RA) is a chronic inflammatory dis-
(also see Chapter 7). Whereaspassive targeting of NPs is ease where macrophages are chronically activated and
possible for disorders where the BBB is significantly degrade bone and cartilage around joints. While
 Active targeting and transport Chapter | 3 31

methotrexate (MTX) is one of the most common and achieve a therapeutic response.160 To target goblet cells,
effective therapeutics for RA treatment, its prolonged use is which are responsible for mucus production in the in-
accompanied by drug resistance and adverse side effects. testines and transcytosis into the blood, NPs have been
Therefore, active targeted NPs could help improve the site- modified with the CSK (CSKSSDYQC) targeting peptide
specific delivery of MTX. Activated macrophages express and were found to enhance absorption of delivered
the folate receptor,152 FR-b, which opens the opportunity insulin.161e163
for folate surface modification of NPs. As an example, an
MTX-encapsulated folateePEGelipid NP was developed
where folate was covalently conjugated to the PEG on the 3.5 Conclusions and future outlook
surface of the NP to achieve higher uptake into Various actively targeted NPs have been developed using
lipopolysaccharide-activated RAW264.7 cells as compared various NP materials, NP sizes, ligands, conjugation tech-
with non-targeted NPs showing the specificity toward niques, antigens, and targeted tissues. Actively targeted
activated cells. Similarly, folate and MTX surface- NPs have also shown promising results in in vitro and
decorated dendrimers have been used for improved active in vivo studies for improving the selectivity and specificity
in vivo delivery to inflamed joints.153,154 of accumulation and drug efficacy in targeted tissues
As another target, synovial fibroblasts in RA highly compared with those of passive targeting NPs. Some
activate nuclear factor-kappa B (NF-kB), leading to actively targeted NPs have also been found to increase
inflammation. The HAP-1 peptide is able to specifically permeation across biological barriers (described in more
bind to synovial fibroblasts offering the ability for active detail in Chapter 7) such as the BBB and intestinal barrier.
NP targeting. Encapsulated NEMO-binding domain pep- Active targeting is crucial for NP delivery to diseases that
tide, which inactivates NF-kB, within a HAP-1-coated NP, do not show an EPR effect. However, despite the growing
showed accumulation enhancement on SF compared with interest and the prospect of actively targeted NPs in the
non-targeted NP, as well as a reduction in both histological biomedical field, as well as numerous clinical trials, there is
score and pro-inflammatory signaling compared with con- currently no approved actively targeted NP used clinically.
trol NPs.155 The diversity in the surface marker expressed by targeted
tissue between individuals, complicated synthesis of
3.4.6 Diabetes actively targeted NPs, and difficulty in upscaling produc-
tion hinder the translation of actively targeted NPs into
Diabetes is a condition where the body cannot regulate
widespread clinical use. On the other hand, the exponential
blood glucose level, which can be a result of either the
improvement in technologies, such as better diagnostic
pancreas producing little to no insulin (type I diabetes) or
tools for preselection of patients who would respond to the
the body not responding to insulin (type II diabetes). In- therapies, might be helpful in pushing actively targeted NPs
sulin monitoring and injection remain the most effective
into the market in the future.
treatment for diabetes patients to regulate the level of blood
glucose. Therefore, more patient-friendly approaches for
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Chapter 4

Passive targeting in nanomedicine:

fundamental concepts, body interactions,
and clinical potential
Steven M. Narum1, b, Tram Le1, b, Dao P. Le1, Joanne C. Lee1, Nathan D. Donahue1, Wen Yang1 and
Stefan Wilhelm1, 2, a
1
Stephenson School of Biomedical Engineering, University of Oklahoma, Norman, OK, United States; 2Stephenson Cancer Center, Oklahoma City,
OK, United States

4.1 Introduction nanomedicine (see Section 4.2), after which the journey of
administered nanoparticles en route to malignant tissues
Over the past few decades, researchers have been designing and cells in the body is briefly explored (see Section 4.3).
and applying nanoparticles for diagnosis and treatment of Section 4.4 provides a concise description of active and
diseases inside the body. These in vivo biomedical appli- passive nanoparticle targeting strategies. In Section 4.5, we
cations of nanoparticles represent a major research area discuss fundamental concepts of passive nanoparticle tar-
within the continuously growing field of nanomedicine.1 geting, including pathophysiological characteristics of solid
While it is a fascinating concept to use systemically tumors and nanoparticle design rules. Section 4.6 briefly
administered nanoparticles inside the body for medical explores limitations of passive nanoparticle targeting, and
applications, development and clinical translation of Section 4.7 explains nanoparticleebody interactions
nanomedicines are challenging. One of these challenges is and biological barriers. We focus on clinical potential and
delivery.2 Direct and efficient delivery of administered relevance of passively targeted cancer nanomedicines in
nanoparticles to diseased tissues and cells is required for Section 4.8 and conclude our chapter with an outlook on
most nanomedicines to ensure accurate diagnosis and how to further exploit the potential of this technology for
effective treatment. However, biological barriers within the biomedical applications (see Section 4.9).
body, such as the mononuclear phagocyte system (MPS),
limit nanoparticle delivery to diseased sites.3
To address this nanoparticle delivery challenge, re- 4.2 What is “nanomedicine”?
searchers have been working on so-called “targeting” Nanomedicine can be broadly defined as the biomedical
strategies.4 The goal of these strategies is to deliver nano- and clinical applications of rationally engineered nanoscale
particles preferentially to diseased tissues while minimizing materials with typical dimensions between 1 and 100 nm.5
their accumulation in healthy organs and cells. Such tar- Materials in this nanoscale size regime are referred to as
geted delivery approaches may have several clinical bene- nanoparticles. Nanoparticles exhibit unique optical,6 mag-
fits, including (1) reduced treatment-related side effects; (2) netic,7 and biological properties8 that are usually not
improved imaging and diagnosis; and (3) enhanced thera- observed in their corresponding bulk materials. Researchers
peutic outcomes. are able to synthesize nanoparticles from inorganic
In this chapter, we focus on passive nanoparticle tar- (e.g., semiconductor quantum dots,9 upconversion nano-
geting strategies in the context of solid tumor management. particles,10 and iron oxide nanoparticles11) and organic
This chapter begins with a brief introduction of materials (e.g., liposomes,12 dendrimers,13 and polymeric
nanoparticles14) with defined physicochemical properties,
including nanoparticle size, shape, and surface chemistry.
a ORCID: orcid.org/0000-0003-2167-6221.
b Authors contributed equally to this work. Such high tunability of material properties allows

Nanoparticles for Biomedical Applications. https://doi.org/10.1016/B978-0-12-816662-8.00004-7 37

Copyright © 2020 Elsevier Inc. All rights reserved.
38 Nanoparticles for Biomedical Applications

researchers to engineer nanoparticles with unique capabil- and theranostic nanoparticles are typically administered via
ities for biomedical and clinical use. For example, nano- systemic administration.
particles can be synthesized to function as drug delivery
vehicles for therapeutic applications or as imaging contrast
agents for medical imaging and diagnosis. Application of 4.3 Systemic nanomedicine and the
these rationally engineered nanoparticles for cancer man- journey of administered
agement is referred to as cancer nanomedicine.15
Medical applications of nanoparticles as carriers for
nanoparticles in the body
therapeutic agents require encapsulation and/or surface Systemic administration, i.e., administering nanoparticles
modification strategies. Researchers can load nanoparticles directly into the body’s circulatory system, is a frequently
with a variety of therapeutic agents, including small used approach in nanomedicine. The rationale for systemic
molecule drugs, chemotherapeutics, peptides, antibodies, nanomedicine is that intravenously (i.v.) administered
and nucleic acidebased drugs. This can improve solubility nanoparticles transport directly with the bloodstream
and bioavailability of drugs in vivo and potentially lead to throughout the body and may eventually reach diseased
better therapeutic efficacy against diseased cells compared tissues, such as a primary solid tumor or metastatic lesions.
with administration of free drugs.16,17 Combination of Upon accumulation, nanoparticles will exert their deliberate
therapeutic and diagnostic capabilities into one nanoparticle biomedical function in these diseased tissues (Fig. 4.1A).
is also possible, and such nanoparticles are referred to as Before i.v. administered nanoparticles reach malignant
“theranostic” nanoparticles in the literature.18,19 To exert sites in the body, several key steps occur. First, exposure of
their intended biomedical function, diagnostic, therapeutic, nanoparticles to blood leads to formation of a so-called

FIGURE 4.1 Schematic overview representing biological barriers and transport mechanisms of systemically administered nanoparticles in biomedical
applications. (A) Systemic (intravenous, i.v.) administration of engineered nanoparticles into the circulatory system is a commonly used administration
route in biomedicine. Engineered nanoparticles typically comprise organic and/or inorganic nanoscale core materials. The nanoparticle core is often
surface modified with organic polymers and ligand molecules. (B) Protein corona formation is a dynamic process, which starts immediately upon i.v.
administration into the circulatory system. The nanoparticle protein corona changes dynamically over time. (C) Cells of the mononuclear phagocyte
system (MPS), such as liver macrophages (Kupffer cells), may line the luminal surface of liver sinusoid blood vessels. Kupffer cells have been reported to
remove opsonized nanoparticles quantitatively from the bloodstream. (D) The passive targeting mechanism suggests nanoparticle transport through
interendothelial gaps (paracellular transport) of compromised blood vessels. In cancer nanomedicine, nonspecific interaction with tumor cells may occur
upon paracellular nanoparticle transport. (E) Ligand-coated nanoparticles follow the same transport pathway as passively targeted nanoparticles. In
contrast to passive targeting nanoparticles, ligand-coated nanoparticles may then interact specifically with tumor cells, potentially leading to increased
nanoparticle retention and improved cell uptake.
Discovering Diverse Content Through
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 278 THE APOCRYPHAL ACTS were troubled by unclean
spirits, ^^ brought them to him; and some they laid on the road ^^
by which he was to pass, and he healed all by the power of the
Lord. And those that were healed by him said with one accord and
one voice, " Glory to thee, Jesus, who in like manner hast given
healing to all through thy servant and apostle Thomas! And being in
good health, and rejoicing, we pray thee, that we may become
members of thy flock and be counted among thy sheep. Receive us,
therefore, O Lord, and consider not our trespasses and our former
transgressions, wdiich we have done, because we were in ignorance!
" ^^ 60. And the apostle said, " Glory be to the only-begotten of the
Father; ^^ glory to the firstborn of many brethren; ^^ glory to
thee, the helper and succor of those who take their refuge to thee.
Thou that sleepest not, and raising those that are asleep; that livest
and bringest to life those that are lying in death: O God Jesus Christ,
Son of the living God, redeemer and helper, refuge and rest of all
those that labor in thy work, who affordest health to those who for
thy name's sake bear the burden and heat of the day; ^^ we give
thanks for the gifts given to us by thee, and for the help from thee
bestowed upon us, and thy providential care that has come upon us
from thee. 10 Comp. Luke VL 18. 11 Comp. Acts V, 15. 12 Comp.
Acts UI, 17. 13 Comp. John L I41* Comp. Rom. VIH, 29. 15 Comp.
Alatt. XX, 12.
 ACTS OF THOMAS 279 61. "Perfect these things upon us,
therefore, unto the end, that we may have confidence in thee. Look
upon us (and see), because for thy sake we have left our houses
and our patrimony, and for thy sake we have gladly and willingly
become strangers.^^ Look upon us, O Lord (and see) that for thy
sake we have given up our own possession, that we might obtain
thee for a possession that shall not be taken away. Look upon us, O
Lord, because we have left those related to us by ties of kindred, in
order that we may be united in relationship to thee. Look upon us, O
Lord, who have left our fathers and mothers and providers, that we
behold thy father, and be satisfied with his divine nourishment. Look
upon us, O Lord because for thy sake we left our bodily wives and
our earthly fruit, in order that we may share in that true and lasting
communion and bring forth true fruits, whose nature is from above,
which no one can take from us, in which we abide and they abide
with us." Seventh Deed. concerning the commander, (Aa. pp. 178-
185.) 62. When the apostle Judas Thomas was preaching in India
the word of God, a commander of King Misdai (Masdai) came to him,
and said to him, " I have heard of thee that thou dost receive no 16
Comp. Matt. XIX, 27, 29.
 28o THE APOCRYPHAL ACTS reward, but givest to the poor
what thou hast. For if thou wouldst receive a reward, I should have
sent thee a sufficient sum of money and I had not come myself,
since the king does nothing without me. For my possession is great
and I am rich, one of the wealthy in India. But I never did anything
wrong to anyone. But the reverse I have experienced. I have a wife
and I had a daughter by her, and I love her (the wife )very much, as
nature demands it, and I had no intercourse with another woman.
And it happened that there was a wedding in our city, and those
which made the wedding were good friends of mine. So they came
and asked me (my consent) by inviting my wife and daughter. Being
well befriended, I could not refuse it. So I sent her, though she did
not care, and I sent also many slaves with them. So they went away,
decked with much jewelry, she and her daughter. 63. And when it
was evening, and the time had come to come home from the
wedding, I sent lamps and torches for to meet them, and I stood by,
looking out when they came, and I could see her and my daughter.
And as I stood, I heard a lamentation. Woe to her! was heard from
every mouth. And the slaves returned with torn garments and told
me what had happened. We saw, said they, a man and a boy with
him ; the man had his hand upon thy wife, the boy upon thy
daughter. But they ran away from them. And we wounded them with
swords, but the swords fell to the ground and they (the women)
also, gnashing with their teeth and
 ACTS OF THOMAS 281 knocking their heads against the
ground. And when we saw this, we came to tell thee. Upon hearing
this, I tore my garment, and struck my face with the hands, and ran
all the way like mad. And having gone away, I found them
prostrated in the market. And I took them and brought them into my
house, and having regained their senses after a while, they rose and
sat down. 64. " I now began to ask my wife. What is it that had
happened to thee? And she said, Dost thou not perceive what
happened to me? For I asked of thee not to let me go to the
wedding, since I did not feel very well. And as I walked by the way
and came to the aqueduct, I saw a black man before me and his
head shaking a little to me and a boy like him, standing by his side.
And I said to my daughter, Look at these two ugly -looking men,
whose teeth are like milk and whose lips are like soot. And we left
them at the aqueduct and went on. After sunset, having broken ofif
from the wedding, and gone with the slaves through (the city), and
when near the acqueduct, my daughter noticed them first and she
came to me. And after her I saw them also, coming towards us, and
we ran away from them. And the slaves also, which were with us ran
away. (And they) beat us, and threw us down. And as she told me
this, the demons came near again and threw her down. And since
that hour they can go out no more, being locked up in one or in
another house. And on their account I suffer much and am troubled.
For
 282 THE APOCRYPHAL ACTS wherever they are they throw
them down and uncover them. I ask thee, therefore, to pray to God
: help me and have mercy upon me ! For since three years no table
(for the meal) has been set up in my house, and my wife and my
daughter sat at no meal. Especially (I ask thee) for my unhappy
daughter, which has not seen anything good yet in this world. 65.
When the apostle heard this from the commander, he felt very sorry
for him. And he said to him, ''Believest thou that Jesus heals her?"
And the commander said, " Yes." And the apostle said, " Commend
thyself to Jesus, and he will heal and help her," Said the commander,
" Show him to me, that I may ask him and believe on him." And the
apostle said, " He appears not to these bodily eyes, but is only found
with the eyes of the mind." And the commander lifted up his voice
and said " I believe on thee, Jesus, and I beseech and ask of thee,
help my little faith, which I have toward thee." The apostle
commanded the deacon Xenophon ^ to assemble all in one place.
And when the multitude was assembled, the apostle spoke, standing
in the midst : 66. " My children and brethren, which believe on the
Lord, remain in this faith, by preaching Jesus, who has been
preached to you by me, and by putting your hope in him. Forsake
him not, and he shall not forsake you. When you sleep in this 1
Syriac : Xanlliippus.
 ACTS OF THOMAS 283 sleep weighing down the sleepers,
he sleeps not and watches. And when you travel by sea and are in
danger and there is none to help, he walks upon the waters and
helps. I am now about to go from you, and it is uncertain whether I
shall see you again in my body. Be not like the people of Israel,
which fell when left alone for a short time by its shepherd.^ I leave
with you in my place deacon Xenophon, for he also preaches Jesus
like myself. For neither am I something, nor he, but Jesus. For I also
am a man, clothed with a body, a son of man, like one of you. I
have also no riches, as some, which convince also the possessors of
their entire uselessness, since they are left behind on earth, whence
it came. But the trespasses which men take upon themselves on
their account, and the filth of sin, they take with them. The rich are
seldom found in the practice of mercy. But the merciful and the
meek of heart — they shall inherit the Kingdom of God. Even beauty
remains not with man. For they which rely upon it, when old age
comes, shall suddenly be confounded. Everything has its time. There
is a time to love, a time to hate.^ Let the hope, therefore, be on
Jesus Christ, the Son of God, who is always loved and desired, and
remember us, as we remember you. For we also (Thomas and
Xenophon), unless we carry the burden of the commandments, we
are not 2 Comp. Exod. XXXII. 3 Eccles. Ill, I, 8.
 284 THE APOCRYPHAL ACTS worthy to be preachers of that
name, and shall be punished there afterward." 67. And having
prayed with them and remained a long time in prayer and
supplication, he commended them to the Lord and said : " Lord, the
Lord of each soul, which dwelleth in a body ; Lord, Father of the
souls which hope in thee and wait for thy mercy, who redeemeth thy
men from error, and freest from servitude and corruption those who
are subject to thee and take refuge with thee, come to the fold of
Xenophon, anoint them with holy oil, heal their wounds and keep
them from the grievous wolves." And he laid his hands upon them
and said, " The peace of the Lord come upon you and go also with
us!" Eighth Deed. about the wild asses. {Aa. pp. 185-197) 68. And
the apostle went forth to go on his way. And all accompanied him
with tears and adjured him to remember them in his prayers and not
to forget them. And when he had mounted the wagon and all
brethren were left behind, the commander came, ordered the driver
to rise, and said, " I pray and supplicate to be deemed worthy to sit
under his feet and to become his driver on this way, that he may
become my companion on that way, in which only a few walk."
 ACTS OF THOMAS 285 69. And having gone about two
miles, the apostle bade the commander to rise and sit beside him,
allowing the driver to take his own seat. And as they went on it
happened that on account of the great heat the beasts of burden
became tired and could move no more. And the commander became
very sad and discouraged, and thought of running by foot to fetch
other beasts of burden for the wagon. But the apostle said, " Let not
your heart be troubled, neither let it be afraid ;^ but believe on
Jesus Christ, whom I have preached to thee, and thou shalt see
great wonders." And looking about he saw a herd of wild asses,
grazing by the way. And he said to the commander, "If thou
believest on Jesus Christ, go to the herd of wild asses and say, Judas
Thomas, the apostle of the Messiah of the new God, saith. Let four
of you come, because we need you !" 70. And the commander went,
seized by fear, because they were so many. And as he went, they
came to meet him. And having come near, he said to them, " Judas
Thomas, the apostle of the new God, commands you that four of
you should come, because I need them!" And the wild asses upon
hearing this, came to him running with one accord ; and having
come, they fell upon their knees.^ And the apostle said to them, "
Peace be with you ! Yoke four in place of these beasts of burden put
aside !" And every one of them came and crowded 1 Comp. John
XIV, 27. 2 The Syriac inserts here a hymn of the apostle.
 286 THE APOCRYPHAL ACTS to be yoked. But there were
four stronger than the rest, and these were yoked. Of the others,
some went before, some followed. And having gone a short
distance, he dismissed them, saying, " To you, the inhabitants of the
desert, I say, Go to your pastures! For if I needed all, you would all
go with me. But now, go to your place, where you were." And they
quietly went away, till they disappeared. 71. While the apostle, the
commander, and the driver went on, the wild asses walked quietly
and evenly, in order not to disquiet the apostle of God. And when
they had come near the gate of the city, they turned aside and
stopped before the house of the commander. And the commander
said, " It is not possible to tell what happened, but I will await the
end and then I will speak." And the whole city came, having seen
the wild asses yoked. And the fame also spread that the apostle
intended to remain here. The apostle asked the commander, "
Where is thy dwelling, and whither art thou bringing us ?" And he
said to him, " Thou knowest thyself that we are at the door, and
these which had come along at thy behest know it better than
myself." 72. Having said this, they alighted from the wagon. And the
apostle began to say, " Jesus Christ, whose knowledge is despised in
this country; Jesus Christ, of whom nothing has been heard in this
country; Jesus, who receivest all apostles in every country and every
city, and by whom all
 ACTS OF THOMAS 287 worthy of tliee arc giorilied ; Jesus,
who has taken thee a form and becamest like a man and didst
appear to all of us in order not to separate us from thy love; Lord,
thou art he who hast given himself for us and has bought us with a
price by his blood, as a precious possession. But what have we.
Lord, to offer in exchange for thy life which thou hast given for us?
For what we have is thy gift. than this, that we ask thee and
(thereby) have life," 73. And having spoken thus, many came from
all sides to see the apostle of the new God. And the apostle said
again, " Why do we stand idle ? Lord Jesus, the hour has come.
What wishest thou that should be done? Command, therefore, that
this be accomplished what must be done now." And the wife and
daughter of the commander were very troubled by the demons, in
such a wise that the inmates of the house thought that they would
rise no more. For they would not allow them to eat anything at all,
but threw them on their beds, and they recognized no one till the
day on which the apostle came. The apostle said to one of the wild
asses, which were yoked on the right side, " Go into the court, and
there standing call the demons and say unto them, Judas Thomas,
the apostle and disciple of Jesus Christ, says, Come out hither! For
for your sakes and against your relatives have I been sent to destroy
you and to persecute you to your place, till the time of
consummation comes and you go down into your dark depth."
 288 THE APOCRYPHAL ACTS 74. The wild ass,
accompanied by many people, went in and said, " I say to you, the
enemies of Jesus the Christ, I say to you which close the eyes not to
see the light — since the worst nature cannot be changed for good
— to you I say, the children of hell and destruction, (the children) of
him who unceasingly does evil, who always renews his operations
and that which becometh his nature, to you I say, most impudent,
who shall be destroyed by yourselves — but what I should say
concerning your destruction and end and what I should advise, I
know not. For it is much and immense to hear it. But your
trespasses are greater than the punishment which- is reserved for
you. But to thee, demon, and thy son, which follows thee — I say —
for now I have been sent for your sakes — but why make many
words about your nature and origin, which you know yourselves and
are nevertheless impudent? Judas Thomas, the apostle of Jesus the
Messiah, who has been sent hither out of much love and kindness,
commands you. Go out in the presence of all the people here and
tell me of what family you are!" 75. And immediately the woman and
her daughter came forth, like dead and dishonored. And when the
apostle saw them, he was sad, especially on account of the girl, and
said to the demons, " Let no forgiveness and forbearance fall to your
lot, for you also know no forbearance or compassion ! But in the
name of Jesus, leave them and go aside!" When the apostle had
said this, the women fell
 ACTS OF THOMAS 289 down and died. For they had neither
breath, nor did they speak. And the demon began and said with a
loud voice, " Hast thou come hither again, mocker of our nature and
kindred? Hast thou come again to deface our tracks. And as I think,
you win not suffer us at all to remain upon earth. But this you
cannot do at this time." The apostle, however, supposed that this
demon was the same which was driven out from that woman. 76.
And the demon said, " I beseech thee, suffer me to go and to dwell
where thou wishest, and command me for that purpose, then I shall
not fear liim who has power over me. For as thou hast come to
preach, so have I come to destroy. As he who sent thee punishes
thee for not fulfilling his will, so, unless I do the will of him who has
sent me, I am sent before the time and appointed season into my
nature (exist no more as an individual). And as the Messiah helps
thee in thy work, so helps me my father in that which I do. And as
he prepares for thee the vessels, worthy that he dwell in them, so
selects he (my father) vessels, by which I accomplish his deeds. And
as he nourishes and provides his subjects, thus he (my father)
prepares for me and those in which I dwell punishments and
torments. And as he gives thee eternal life as reward for thy work,
so he (my father) offers me as recompense for my works everlasting
destruction. And as thou enjoyest thy prayer and good works and
thy spiritual hymns, thus I enjoy murders and adulteries and the
wine-offerings offered
 290 THE APOCRYPHAL ACTS Upon the altars. And as thou
turnest men over to everlasting life, I turn those which obey me to
everlasting damnation and punishment. Thou receivest thy reward, I
mine." J'j. The demon having spoken this and the more, the apostle
said, " Jesus commands thee and thy son through me, that you no
more enter into a human dwelling but go out and go and dwell
altogether outside of the dwelling of men!" And the demons said to
him, " Thou hast given us a hard order. But what wilt thou do to
those which are now hidden from thee? For the makers of idols (of
wood and stone) enjoy them (the demons dwelling in them) more
than thou, and the multitude worships them and does their will,
bringing offerings to them and offering wine and water libations as
food and presenting gifts." And the apostle said, " they shall now be
destroyed with their deeds." And suddenly the demons became
invisible. But the women did lie like dead upon the ground, having
no voice. 78. And the wild asses stood together and separated not.
But the wild ass which by the power of God was able to speak said
to the apostle, whilst all were silent and looked on what they would
do, " Why standest thou idle, apostle of the Most High, who waits
that thou beseech him for the greatest knowledge? What dost thou
delay? For thy teacher wishes to show his great deeds by thy hands.
What standest thou, herald of the hidden One? For thv Master will
make known through thee the
 ACTS OF THOMAS 291 secret, reserving it for those, whom
he deems worthy to hear it. What restest thou, who performs great
deeds in the name of the Lord? For thy Lord encourages thee, by
giving thee courage. Be not afraid therefore. For he will forsake no
soul which according to kindred belongs to thee. Begin, therefore, to
call upon him, and he shall willingly hear thee. What standest thou
and admirest all his deeds and effects! For these things are small
which he has shown by thee. And what wilt thou say of his great
gifts? For thou shalt not be able to tell them fully. What dost thou
wonder at his bodily healings, which pass, especially as thou
knowest the true and lasting healing which he gives to those who
belong to him? And why dost thou look at this temporal life, and
thinkest not of the eternal ? 79. " And to you, multitudes standing
here and waiting that the prostrated women shall be raised, I say,
Believe the apostle of Jesus Christ ; believe the teacher of truth;
believe him who shows you the truth ; believe on Jesus ; believe on
the Messiah which was born; that the born have life through his life ;
who also became a child and was educated, that the perfect
humanity appear through him. He taught his own teacher,^ because
he is the teacher of truth and the wisest of the wise ; he offered
sacrifice also in the temple, to show that every offering is hallowed
(by him).^ This here is his apostle, the 3 Comp. Thomas' Gospel of
the Infancy, ch. 6. 4 Comp. Matt. XVII, 27.
 292 THE APOCRYPHAL ACTS revealer of truth. It is he who
does the will of him who sent him. But lying apostles and prophets
of lawlessness shall come,^ whose end shall be according to their
deeds, which indeed preach and give laws that one should flee
lawlessness, but they are found at all times in sins. They are clothed
indeed in sheep's clothing, but inwardly they are ravening wolves ;
they are not satisfied with one wife, but corrupt many women; they
say that they despise children, yet ruin many children and suffer for
them; they are not satisfied with what they have, but wish that
everything useful should serve them alone, whereas they pretend to
be his (Christ's) disciples; they say one thing with their mouth, but in
their hearts they think otherwise; they command others to refrain
from wickedness, but themselves they do nothing good; they are
regarded as temperate and command others to abstain from
fornication, theft, and avarice, but in secret they do all these things
themselves, teaching others not to do these things." 80. While the
wild ass was thus talking, all looked at It. And when it was silent, the
apostle said, " What I am to think of thy beauty, O Jesus, and what
to say about it, I know not ; rather I cannot. For I am not able, O
Christ, to utter it completely, O thou that restest and only One who
art wise, who alone knowest what is in the heart and the contents of
thought; — glory be to thee, merciful and tranquil ; glory be to thee,
wise word ; glory 6 Comp. Matt. VH, 15 ; 2 Pet. U, 1.
 ACTS OF THOMAS 293 to thy mercy, which is shed over us
; glory to thy compassion which is spread over us; glory to thy
majesty, who didst come down for our sakes; glory to thy highest
kingdom, which humbled itself for our sakes ; glory to thy strength
which became weak for our sakes; glory to thy deity which for our
sakes appeared in the image of man; glory to thy humanity, which
died for our sakes, to make us alive; glory to thy resurrection from
the dead, for by it our souls shall share in the resurrection and rest;
glory and praise to thy ascension into heaven, for by it thou didst
show us the way to the highest after thou didst promise that we
shall sit on thy right hand and judge with thee the twelve tribes of
Israel. Thou art the heavenly word of the Father ; thou art the
hidden light of the mind; thou art he that shows the way of truth; O
persecutor of darkness and destroyer of error." 81. When the apostle
had spoken thus, he went to the women and said, " My Lord and my
God, I doubt not in thee, nor do I call upon thee in unbelief, who art
always our helper and assistance and restorer who givest us thy
strength, encouragest us, and givest thy servants freedom in love. I
beseecli thee, let these women rise up healed, and become again as
they were ere the demons struck them.'' Having spoken thus, the
women turned and sat down. And the apostle ordered the
commander that his servants take them and bring them in. An^ 1
when they had come in, the apostle said to the wild asses, " Follow
me." And they followed him till
 294 THE APOCRYPHAL ACTS outside of the gates. And
having come out, he told them, " Go in peace to your pastures !"
And the wild asses went away willingly, the apostle standing and
seeing to it that no harm be done to them by anyone, till they had
become invisible in the distance. And the apostle returned with the
people into the house of the commander. Ninth Deed. about the wife
of charis. (A a. pp. ip/-2^p.) 82. It came to pass that a woman, (the
wife) of Charis, the near relative of the king, named Mygdonia, came
to see and to behold the new appearance of the new God, who was
preached, and the new apostle, who abode in their country. And she
was carried by her slaves, but could not be brought to him on
account of the many people and the narrow space. So she sent to
her husband for more servants. They came and went before her,
pushing and striking the people. When the apostle perceived this, he
said to them, " Why do you make these go away who come to hear
the word and show willingness for it? You wish to be near me,
whereas you are far — as it has been said of the people, which
came to the Lord, Having eyes, and ye hear not.^ And to the
multitudes he said, He 1 Mark Vni, 18.
 ACTS OF THOMAS 295 that hath ears to hear, let him hear ;
^ And, come unto me, all ye that labor and are heavy laden, and I
will give you rest." ^ 83. And looking at her (Mygdonia's) carriers, he
said to them, this beatitude, which was given to them, is now also
given to you who are heavy laden. Ye are those which carry
burdens, grievous to be borne,'^ and are driven onward by her (the
woman's) behest. And whereas you are men they lay burdens upon
you, as upon the unrational beasts, because your lords think that
you are not men like themselves. whether they be bond or free.
whether bond or free, poor or rich. For neither shall the riches help
the rich, nor will poverty save the poor from judgment. For we
received not a commandment which we cannot fulfill, nor did he put
upon us heavy burdens grievous to be borne. He did neither put
upon us such a building, as men build it, nor stones to be hewn and
houses to be established, as your artists make up by their
intelligence, but we received the commandment from the Lord, that
what is displeasing to us when done unto us by another, we should
not do to another. 84. " First of all abstain from adultery, for it is the
cause of every evil,
 296 THE APOCRYPHAL ACTS which account the curse came
upon Cain;> also of theft, which induced Judas Iscariot and caused
that he hung himself; for those that are given to avarice see not
what they do; and of ostentation and all dirty deeds, especially of
the carnal the end of which is eternal damnation. For this
(uncleanliness) is the starting point of every evil. In like manner, it
also leads those which are proud into servitude, drawing them down
into the depth and subjecting them to their hands, that they see not
what they do, on which account their deeds are unknown to them.
85. "Ye, however, and become ye thereby well-pleasing to God, and
gives life eternal and despises death. And (walk) in kindness
(meekness), for it overcomes the enemy and alone obtains the
crown of victory. And in goodness and in helping, the poor and
satisfying the want of the needy, by bringing (from your possession)
and communicating to the needy. Especially walk in holiness, for
before God it is the starting point of every good thing. The holiness
is of God, destroying fornication, overcoming the foe, well-pleasing
to God. It is an invincible athlete, it is highly esteemed of God and
 ACTS OF THOMAS 297 is glorijEied by many. It is the
messenger of peace, by preaching peace. < Moderation, however >
if one acquires it, he is without cares, because he pleases the Lord
and waits for the time of redemption. For it does nothing which is
wrong, and gives Hfe and rest and joy to all which obtain it. 86. "
And meekness has subdued death, bringing it under its power.
Meekness has overcome the enemy. Meekness is a good yoke.
Meekness fears none and resists not. Meekness is peace and joy and
enjoying of rest. Remain, therefore, in holiness and take the freedom
from care (proceeding from moderation) and approve meekness. For
in these three main parts the Messiah is described, whom I preach
to you. Holiness is the temple of the Messiah, and whoever lives in it
obtains him as inhabitant. For he fasted forty days and forty nights,
without tasting anything. And whoever keeps it shall live in it as
upon a mountain. Meekness, however, is his glory, for he said to our
fellow-apostle Peter, Turn back thy sword and put it up again into its
place. For if I would do this could I not put more than twelve legions
of angels from my Father on my side?"'' 87. When the apostle spoke
thus and the whole multitude heard it, they crowded and came near.
But the wife of Charis, the relative of the king, started up from the
palanquin, threw herself to the oComp. Matt. XXVI, 52, 53; John
XVIII, u.
 298 THE APOCRYPHAL ACTS ground before the apostle,
took hold of his feet, beseeching and saying, " Disciple of the living
God, thou hast come into a desert country. For we live in a desert,
because by our life we are like the irrational animals ; but now we
shall be saved through thy hands. I beseech thee, therefore, care for
me and pray for me, that the mercy of God, whom thou preachest,
come upon me and I become his maid, and have part in the prayer
and in the hope and in the faith on him, and receive also the seal
and become a holy temple and he dwell in me." 88. And the apostle
said, " I pray and ask for you all, brethren, which believe in the Lord,
and for you, sisters, which hope in the Messiah, that the word of
God may rest on you all and dwell in you ; for we have no power
over you." And he began to speak to the woman Mygdonia : " Rise
up from the ground and consider. For this ornament which thou hast
on will not help thee any, nor the beauty of thy body, nor thy
garments. Neither the fame of the authority which surrounds thee,
nor the power of this world, nor this filthy intercourse with thy
husband will be of use to thee, if thou art deprived of the true
intercourse. For the exhibition of jewelry is destroyed, and the body
ages and changes, and the garments wax old, and the power and
dominion pass by, accompanied by the responsibility for each's
behavior in it (the rule). And the communion of begetting children
also passes by, since it is an object of contempt. Jesus alone always
remains and they which hope in him." When he had spoken this, he
 ACTS OF THOMAS 299 said to the woman, '' Go in peace,
and the Lord will make thee worthy of his mysteries." And she said,
" I am afraid to go away, fearing lest thou lea vest me and goest to
another people." And the apostle said to her, " Though I go away, I
shall not leave thee alone, but Jesus will be with thee because of his
compassion." And she fell down, worshipped him, and went to her
house. 89. And Charis, the relative of King Misdai, after having
bathed, went up to recline at dinner. And he incjuired after his wife,
where she was. For she had not come as usual from her chamber to
meet him. And her servants said to him, " She is unwell." And he
started up, went to the chamber and found her on the couch and
covered. And he unveiled her, kissed her, and said to her, " Why art
thou so sad?" And she said, "I am unwell." He said to her, " Why
hast thou not observed the decency becoming a free woman and
stay at home, but didst go and listen to the idle words and look at
works of sorcery? But rise, dine with me, for I cannot eat without
thee." But she said to him, " Excuse me for today, for I am very
much afraid." 90. Upon hearing this from Mygdonia, Charis would
not partake of the meal, but ordered his servants to bring her to eat
with him. And having brought her, he demanded that she should eat
with him. And she excused herself. As she would not, he ate alone,
saying to her, " On thy account I refused to eat with King Misdai,
and thou wouldst not eat with me? " And she said, " Because I am
 300 THE APOCRYPHAL ACTS unwell." Having risen up,
Charis intended to associate with her after his custom. But she said,
" Have I not told thee, that I refused for to-day? " 91. Upon hearing
this he went away and went to sleep on another couch. When he
awoke from his sleep he said, " My mistress Mygdonia, hear the
dream which I have seen. I saw myself at meal near King Misdai and
besides us stood a table containing everything. And I saw an eagle
coming down from heaven taking away two partridges from the
place before me and the king, which he carried into his nest. And he
came near again fluttering about us. And the king ordered a bow to
be brought to him. The eagle took again a dove and a turtle from
the place before us. The king threw an arrow after him which
pierced him from one side to the other without hurting him. And he
flew to his nest. And raised from the sleep, I am frightened and very
sad because I had tasted the partridge and he would not suffer me
to bring it to my mouth." And Mygdonia said to him, *' Thy dream is
beautiful, for thou eatest partridges daily, whereas this eagle has till
now not tasted a partridge." 92. When it was morning, Charis went
and put the left shoe on the right foot. And stopping he said to
Mygdonia, "What does this mean? For behold, the dream and this
act ! " Mygdonia said to him, " This also is not bad, but seems to me
very beautiful: from a bad thing comes the better." Having washed
his hands, he went to salute King Misdai.
 ACTS OF THOMAS 301 93. In like manner also Mygdonia
went early in the morning to salute the apostle Judas Thomas. She
met him talking to the chief commander and the multitude. And he
exhorted them by speaking of the woman which had received the
Lord into her soul, and asked whose wife she was. The chief
commander said, " She is the wife of Charis, the relative of King
Misdai. And her husband is very severe and the king obeys him in
everything which he says. And he allows her not to remain in the
knowledge which she professes. He has also often praised her in the
presence of the king by saying none were so good for love as she.
[Everything of which thou speakest to her is strange to her."] And
the apostle said, "If the Lord has truly and indeed risen in her soul
(as sun) and she has received the sown seed, she will neither care
for the earthly life nor fear death, nor will Charis be able to do any
harm to her. For he whom she has received into her soul is greater, if
indeed she has truly received him." 94. When Mygdonia heard this,
she said to the apostle, " In truth, my lord, I have received the seed
of thy words and shall bring forth fruits which are like the seed."
Says the apostle, " Lord, these souls which are thine own, praise and
thank thee ; the bodies which thou didst deem worthy to be
habitations of thy heavenly gift thank thee." And he also said to
those about him, " Blessed are the saints, which have never been
condemned by their souls (conscience) ; for because they have ob 
 302 THE APOCRYPHAL ACTS tained these (not condemning
consciences), they doubt not in themselves. Blessed are the spirits
of the saints which have safely received the heavenly crown for the
fight commissioned to them. Blessed are the bodies of the saints,
because they were deemed worthy to become temples of God, that
Christ may dwell in them. Blessed are ye, because ye have power to
remit sins. Blessed are ye, if ye lose not that which is committed to
you, but take it with you with joy and gladness. Blessed are ye,
saints, because it is given to you to ask and to receive. Blessed are
ye, meek ones, because God has deemed you worthy to become
heirs of the heavenly kingdom. Blessed are ye, meek ones, for ye
have overcome the wicked one. Blessed are ye, meek ones, because
ye shall see the face of the Lord. Blessed are ye who hunger for the
Lord's sake, for rest is preserved for you, and your souls rejoice from
now on. Blessed are ye quiet ones (because ye were found worthy)
to be delivered from sin." When the apostle had said this in the
hearing of the whole multitude, Mygdonia was more strengthened in
the faith and in the glory and majesty of the Messiah. 95. And
Charis, the king's relative and friend came to the breakfast and
found not his wife at home. And he asked all in his house, " Whither
has your mistress gone?" And one of them said, " She went to the
stranger." Upon hearing this from the one servant, he was angry at
the others, because they did not announce to him at once what
 ACTS OF THOMAS 303 had happened. And he sat down
and waited for her. And when it was evening and she entered into
the house, he said to her, " Where hast thou been ? " She answered
and said, " To the physician?" He said, "Is the stranger a physician?"
She said, *' Yes, a physician of souls. Most physicians heal the
bodies, which decay ; but he heals the souls, which do not perish."
When Charis heard this, he was inwardly very wroth at Mygdonia on
account of the apostle. But he answered nothing, for he was afraid,
as she was superior to him in riches and intelligence. He went to the
meal, but she went to her chamber. And he said to one of his
servants, " Call her to the meal." But she would not. 96. When he
heard that she would not leave her chamber, he went in and said to
her, " Why wilt thou not eat with me ? And wilt thou not also have
intercourse with me according to custom? And in this respect I am
more suspicious. For I heard that that sorcerer and deceiver teaches
that none should cohabit with his wife, and he reverses what nature
demands and the deity has ordered." WHien Charis said this,
Mygdonia held her peace. Again he says to her, " My lady and wife
Mygdonia, be not deceived by deceitful and foolish words, nor by the
works of sorcery which this man, as I heard, does in the name of the
Father, of the Son, and of the Holy Ghost. In this world it has never
been heard that one has raised the dead. But as I hear, he is
reported to raise the dead. And as he neither eats
 304 THE APOCRYPHAL ACTS nor drinks, think not, that he
neither eats nor drinks for righteousness' sake. He rather does it
because he has nothing. For what should he do who has not even
the daily bread ? And he has only one garment because he is poor.
And as concerns this that he receives nothing from anyone he does
it because he is aware that none has been made well by him." 97.
When Charis said this, Mygdonia was dumb like a stone. She prayed,
however, for daylight, that she might go to the apostle of the
Messiah. He left her and sadly partook of his meal, for he was
anxious to have intercourse with her. When he had left, she bent her
knees and prayed thus : " Lord God, merciful Father, and Redeemer
Christ, give me strength that I overcome the boldness of Charis, and
grant unto me to keep the holiness which is well-pleasing to thee,
that by it I may also find eternal life." Having thus prayed, she
betook herself to her bed, being clothed. 98. Having eaten, Charis
came near her. And she cried, " Henceforth thou hast no place
beside me, for my Lord Jesus who is with me and rests in me is
better than thou." And laughingly he said, " Well dost thou mock
with these words at that sorcerer, and nicely dost- thou laugh at him
who says. You have no life with God unless you sanctify yourselves !
" Having said this, he tried to go into her. But she would not suffer it,
but cried out with a piercing voice, " I call upon thee, Lord Jesus,
forsake me not! I have taken refuge in thee! As I
 ACTS OF THOMAS 305 have perceived that it is thou who
seekest those which are in ignorance and savest those which are
kept in error, so I pray thee now of whose report I heard and in
whom I believed, Come to my assistance and save me from Charis's
insolence, that his impurity have no power over me." And she put
her hands (to her face), and ran away uncovered. And upon leaving
she tore down the curtain of her chamber, put it around her, went to
the nurse, and slept with her. 99. And Charis spent the whole night
in sadness, striking his face with the hands. And he thought of going
immediately to the king and to report to him of the power which
surrounds him. But considering he said within himself, " If the great
sadness which now fills my heart obliges me to go to the king, who
will introduce me to him? For I know that, had not evil report thrown
me down from my pride and vaunt and greatness and brought me
into this smallness and separated my sister Mygdonia from me, I
should not have come out at this time (of the night) even if the king
stood at the door and give him an answer. But I shall wait till it is
day. I know that the king will grant what I ask of him. And I will
speak of the madness of the stranger, whose tyranny throws the
great and illustrious into the abyss. For it pains me not that I am
deprived of her intercourse, but I sorrow for her, because her noble
soul has been humbled. She, a woman of comeliness, in which none
of the servants has ever detected an unseemliness, ran uncov 
 306 THE APOCRYPHAL ACTS ered from her chamber, and I
know not whither she went. But it is possible that having been made
mad by that sorcerer, in her madness she went to the market to
seek him. For nothing seems to her lovely but that man and his
words." 100. Having spoken thus, he began to moan and say, " Woe
to me, wife, and woe to thee also! For too soon have I been
deprived of thee ! Woe to me, most beloved, for thou art better than
my whole kindred. For I had neither a son nor a daughter from thee
that I could enjoy them. Thou hast not even lived with me a year,
and an envious eye has torn thee from me. Had the power of death
taken thee away, I should have counted myself a king and leader !
But that I should suffer this at the hand of a stranger! And, possibly,
he is a slave, who ran away to my harm and to that of my most
unhappy soul. But let nothing come in my way till I have destroyed
him and avenged this night. And let King Misdai not (again) find
pleasure in me unless he gives me revenge in the head of the
stranger and in the commander Si for, who became her cause of
destruction. For through him he came here and stays with him. And
many go in and out there whom he teaches a new doctrine by
saying that none can live unless he frees himself from all his
possessions and like himself becomes an abstainer. And he
endeavors to get many friends. loi. As Charis was considering this, it
became day. And having passed the night waking, he put
 ACTS OF THOMAS 307 on a cheap dress, and shoes on his
feet, and, looking sad and dejected, he went to sakite the king.
Upon seeing him, the king said, " Why art thou so sad, and why
didst thou come in such an attire? And thy face is also changed."
Charis answered and said to the king, " I have to tell thee of
something new, and of a new devastation which the commander
Sifor has brought upon India: a Hebrew magician whom he has in
his house and who leaves him not. Many go to him, whom he
teaches a new God and gives them new laws, of which no one has
ever heard, by saying. ' It is impossible that you enter into the
eternal life which I preach to you, unless you give up your wives,
and the wives also give up their husbands. It happened that my
unhappy wife also went to him and heard his words. And she
believed them, left me during the night, and ran to the stranger. But
let Sifor and the sorcerer hidden in his house be brought to thee,
and punish them, that not all of our people perish." 102. When his
friend Misdai heard this, he said to him, " Be not sad and
discouraged ! I will have them brought here, and I will avenge thee,
and thou shalt have thy wife again. For if I avenge others who
cannot avenge themselves, I will avenge thee above all." And the
king went out and sat upon the seat of judgment. Being seated, he
ordered to call Sifor, the commander. And having come into his
house, they found him at the right hand of the apostle, and
Mygdonia at his feet, listening with the whole people. And the king's
messengers came
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