Targeted Nano Drugs Improve Disease Treatment

Nanotechnology in
Targeted Drug Delivery
and Therapeutics 14
Diana Sousa, Débora Ferreira, Joana L. Rodrigues, Lı́gia R. Rodrigues
University of Minho, Centre of Biological Engineering, Braga, Portugal
1. INTRODUCTION
Nanotechnology can be defined as a technology that develops materials or devices
within the nanometer scale. This technology can be very useful in targeted drug
delivery and therapeutics for disease prevention, diagnosis, and treatment. Nano-
carriers have been extensively studied for the design of drug delivery systems.
In addition to drug delivery, nanocarriers can be used as biosensors in monitoring
and diagnosis for the detection of biomarkers or pathogens by linking quantum
dots (QDs) and dyes or using magnetic nanocarriers [1].
Targeted drug delivery systems and therapeutics can be advantageous compared to
conventional systems. They can deliver the drug more efficiently to the target site and
improve the therapeutic efficiency, reduce toxicity and side effects [2,3]. Nanocarriers
are tailor-made to protect the drug from destructive environmental and biological
factors (e.g., light, enzymes, oxygen) [1] but also to achieve controlled drug release
in a disease-specific location (Fig. 14.1). For example, in the case of tumors, nanome-
dicines can reach the target passively through leaky vasculature (enhanced permeation
and retention [EPR] effect), whereas active targeting allows the ligands conjugated to
the nanocarriers to bind to overexpressed receptors in cancer cells [4]. These types of
targeting allow nanocarriers to transport chemotherapy drugs to cancer cells without
affecting normal cells. In addition to conventional drugs, nanocarriers can transport
other therapeutic molecules such as therapeutic peptides, proteins, or antibodies, or
even therapeutic nucleic acids like small-interfering ribonucleic acid (siRNA) [5,6].
At the moment, targeted delivery is being studied to treat not only a vast number
of cancers [4] but also human immunodeficiency virus (HIV) [5], Alzheimer’s [7],
Parkinson’s [8] or inflammatory bowel disease [9], among others.
In this chapter, the most promising nanocarrier types are revised (Section 2). In
addition, passive (Section 3.1) and active targeting strategies (Section 3.2) using
different ligands and different conjugation methods (Section 3.3) are discussed. The
advantages and disadvantages underlying each drug delivery system are presented,
as well as the most relevant clinical applications and promising clinical trials.
CHAPTER
357
Applications of Targeted Nano Drugs and Delivery Systems. https://doi.org/10.1016/B978-0-12-814029-1.00014-4
Copyright © 2019 Elsevier Inc. All rights reserved.

2. NANOCARRIERS
The design of nanocarriers for drug delivery offers many advantages including (1)
improvement of hydrophobic drug stability, making them suitable for administration;
(2) enhancement of biodistribution and pharmacokinetics, resulting in improved
efficacy; (3) improvement of the EPR effect, resulting in increased selective targeting;
(4) reduced adverse effects as a consequence of favored accumulation at target sites;
and (5) decreased toxicity by using biocompatible nanomaterials [10,11]. Liposomes,
micelles, polymeric nanocarriers, dendrimers, hydrogels, metallic nanocarriers, QDs,
ceramic nanocarriers, carbon-based nanocarriers, exosomes, and viruses (Fig. 14.2)
have been used as potential nanodelivery platforms [12e26]. The next section will
highlight the benefits and drawbacks of these different nanocarriers as drug nanode-
livery platforms.
2.1 LIPOSOMES
Liposomes are spherical self-assembled artificial vesicles composed of a lipid bilayer,
which encloses an aqueous core, able to deliver several types of biomolecules [27e30].
FIGURE 14.1
Schematic representation of multifunctionalized nanocarriers for drug delivery.
Nanocarriers can deliver a broad range of drugs or therapeutic molecules (payload). The
carrier protects the payload and shields it from the body’s immune system. In active
targeting, the targeting ligands (e.g., aptamer, peptide, antibody) on the nanocarrier
surface guide it to the specific target (e.g., cancer cells) and can be conjugated with a
linker using different strategies. Moreover, the biological surface may be modified to
enhance passive targeting. Depending on the nanocarrier, the drug can be conjugated to
the surface.
358 CHAPTER 14 Nanotechnology in Targeted Drug Delivery and Therapeutics

Depending on the assembly technique used, their size can range from tens
of nanometer to micrometer. The most common classification of these molecules
is based in the number of lipid bilayers present in the colloidal structure, with
unilamellar liposomes containing one lipid bilayer and multilamellar liposomes
containing multiple lipid bilayers.
Liposomes are by far the most clinically established nanosystems for drug
delivery due to several reasons, including general biocompatibility, biodegradability,
and the ability to entrap both hydrophilic and hydrophobic molecules. Furthermore,
their efficacy has been shown in reducing toxicity and systemic side effects, as well
as in attenuating the reticuloendothelial system (RES) clearance [29]. Moreover,
through the addition of agents to the lipid bilayer membrane or surface chemistry
tailoring, liposomes’ biological properties such as surface charge, functionality,
FIGURE 14.2
Schematic representation of different types of nanocarriers used for drug delivery. These
nanocarriers vary in size, shape, and composition. The nanocarrier can be used to
encapsulate or covalently conjugate drugs, imaging agents, and targeting moieties.
2. Nanocarriers 359

specificity, and size, can be straightforwardly tuned [29,31]. For instance, liposomal
surface modification by attaching polyethylene glycol (PEG) units (known as stealth
liposomes) improves the circulation time of liposomes in the bloodstream.
Due to the extensive research being carried out using these drug delivery platforms,
several liposome-based drug preparations are currently commercially available for
human use (e.g., Doxil, AmBisome, and DepoDur) and many others are under different
clinicaltrials [32] (Table 14.1). The first successful US Food and Drug Administration
(FDA)-approved nano drug was the liposome-based drug Doxil (doxorubicin
liposomal), in 1995, for the treatment of ovarian cancer, as well as acquired immune
deficiency syndrome (AIDS)-related Kaposi’s sarcoma [33]. Later, in 1996, a
liposomal product (DaunoXome) for the delivery of daunorubicin was also approved
by the FDA for the management of advanced HIV-associated Kaposi’s sarcoma [34].
Most of the liposome-based products approved for clinical use are for the treatment of
cancer, however, other products are also being developed for other diseases or medical
treatments. For instance, Amphotec and AmBisome were approved by the FDA for
the treatment of fungal infections. Moreover, liposomes have become an important
carrier platform in vaccine development, for example, with the establishment
of Epaxal and Inflexal for vaccination for hepatitis A and influenza, respectively.
Beyond approved agents, several liposome-based drugs are paving the way into
clinical trials [35e38].
Liposomes have also been studied for the delivery of siRNAs. SiRNAs are
therapeutic agents that suppress the expression of targeted genes, for example, in
tumors [39]. They interfere with the expression of specific genes with complementary
nucleotide sequences through the cleavage of the mRNA of interest by an
RNA-protein complex called RNA-induced silencing complex (RISC) [40]. This
results in the mRNA degradation after transcription and, consequently, no translation.
These siRNAs can be introduced directly into cells as synthetic siRNAs or in an
indirect way as long double-stranded RNAs (dsRNAs) or using viral vector systems
that express short-hairpin RNAs (shRNAs) that are subsequently processed to siRNA
by the cellular machinery [41]. It is very important that the nanocarriers are used to
protect siRNA from degradation by nucleases during circulation in the bloodstream.
Therefore, in recent years safe and effective delivery systems have been developed
for siRNAs to be used in the clinical setting, and most of them include lipid-based
delivery vehicles such as liposomes [42]. As reported by Gomes-da-Silva et al.
[17], liposomes were used to encapsulate siRNA molecules against a well-
validated molecular target, polo-like kinase 1 (PLK1), resulting in a substantial
decrease of prostate cancer cells viability. Moreover Guo et al. [6] designed a new
engineered liposomal-siRNA delivery platform toward triple-negative breast cancer
(TNBC), leading to an in vitro and in vivo significant reduction of angiogenesis.
This highly aggressive and metastatic subtype of breast cancer lacks the expression
of estrogen receptor, progesterone receptor, and amplification of the human
epidermal growth factor receptor 2 (HER2). The development of novel targeted
drug delivery therapies is essential for a correct and timely intervention in TNBC
patients [43].

Table 14.1 Examples of Nanoplatforms and Their Stages in Clinical Use
Trade Name
(Active
Ingredient) Company Indication Status
Liposome
Marqibo
(Vincristine
sulfate)
Talon
Therapeutics Inc.
Acute lymphoblastic leukemia
treatment
FDA
Approved
(2012)
Onivyde
(Irinotecan)
Merrimack Metastatic pancreatic cancer
treatment
FDA
Approved
(2015)
Polymer-based
Plegridy
(Pegylated
Interferon beta-
1a)
Biogen Multiple sclerosis treatment FDA
Approved
(2014)
Adynovate
(Pegylated
Antihemophilic
Factor)
Baxalta Hemophilia A treatment FDA
Approved
(2015)
Micelle
Estrasorb
(Estradiol)
Novavax Menopausal therapy FDA
Approved
(2003)
Dendrimers
(VivaGel) Stapharma Microbicide that inhibits HIV,
HSV-2, and HPV in vitro and in
animal models.
Phase II
Hydrogels
(MuGard) Access
Pharmaceuticals,
Inc.
Mucoadhesive oral wound rinse
for the management of oral
mucositis/stomatitis
Phase IV
Metallic Nanocarriers
Ferrlecit (Sodium
ferric gluconate)
Sanofi Avertis Iron deficiency anemia treatment FDA
Approved
(1999)
Venofer (Iron
Sucrose)
Luitpold
Pharmaceuticals
Iron deficiency anemia treatment FDA
Approved
(2000)
2. Nanocarriers 361

Liposomes have already been adapted into intelligent and switchable nanoplat-
forms by including a wide range of stimuli-responsive functionalities such as pH,
temperature, ultrasound, light, magnetic field, and enzymatic response, highlighting
the effectiveness of liposomes for drug delivery purposes [17,44e48].
2.2 POLYMERIC NANOCARRIERS
Polymeric nanocarriers, one of the most studied drug delivery nanoplatforms
and known as nanospheres (matrix type of structure) or nanocapsules (vesicular
system), are prepared by binding a copolymer to a polymer matrix. These
submicron-sized colloidal carriers can be classified as natural, synthetic,
biodegradable, or nonbiodegradable [49e52]. Among the plethora of synthetic
polymers tested for drug delivery applications, the most outstanding candidates
include PEG, poly-lactic acid-co-glycolic acid (PLGA), polyvinyl alcohol (PVA),
polyvinyl pyrrolidone (PVP), cyclodextrins (CDs). polyethylene (PE),
polyanhydrides, and polyorthoesters [53e55].
Cationic polymers that are able to form complexes with nucleic acids, such as
siRNAs, are also new promising approaches for the therapy of several diseases
[56,57]. For instance Mao et al. [58] demonstrated that their drug delivery system
based on an amphiphilic and cationic triblock copolymer can efficiently deliver
the siRNA targeting acid ceramidase oncogene into breast cancer cells. Moreover,
Zhu et al. [26] reported a nanoplatform composed of a solid polymer/cationic lipid
hybrid core and a lipid-PEG shell that efficiently delivered siRNA into non-small
cell lung cancer cells and thereby induced Prohibitin1 gene silencing effects.
The flexibility of diverse polymer sources enables the modulation of the
polymer sensitivity in response to a specific stimulus, leading to the development
of more accurate drug delivery platforms.
2.3 MICELLES
Micelles, formulated from the combination of water-soluble polymers with phos-
pholipids or long-chain fatty acids and other surfactants, self-assemble into
nanosized colloidal carriers with a hydrophilic shell and hydrophobic
core [59,60]. A micellar structure itself is already advantageous for drug delivery
applications. For instance, due to the hydrophobic core of the micellar structure,
amphiphilic and poorly water-soluble drugs can be loaded and protected by the
hydrophilic shell during transport to the target site [61]. Furthermore, micelles
typically have a diameter less than 100 nm limiting their uptake by the RES
system. Moreover, their hydrophilic surface shields micelles from immediate
recognition and consequently increases circulation times [62].
So far, a large variety of drug molecules have been encapsulated into micelles.
Currently, various polymeric micelles integrated with anticancer agents (e.g.,
SP1049C, NK911, and Genexol-PM) are under clinical trials [63]. Genexol-PM
employs a Cremophor-free polymeric micelle system for the encapsulation of the

anticancer drug paclitaxel (PTX). A Genexol-PM response rate of 58.5% in compar-
ison with plain drugs in clinical trials Phases I and II [65] was observed. More
recently, a series of novel dual-targeting micellar delivery system was developed
based on the self-assembling of hyaluronic acid derivatives conjugated with folate
[66]. PTX was successfully incorporated into the hydrophobic core of the micellar
structure, with an encapsulation efficiency as high as 97.3%. Since this drug delivery
platform is biodegradable, biocompatible, and cell-specific targetable, micelles
became promising nanostructure carriers for the delivery of hydrophobic anticancer
drugs, in specific PTX.
Moreover, multifunctional micelles containing therapeutic and imaging agents
[67,68] and stimuli-responsive drug-loaded micelles [71] are now under active
research.
2.4 DENDRIMERS
Dendrimers are synthetic three-dimensional polymeric macromolecules that are
organized in a well-organized structure [72]. These carriers, composed of various
highly branched monomers that grow radially from the multifunctional central
core, possess low polydispersity indexes and their chemical composition and
molecular weight can be precisely tailored [73e75]. Dendrimers have small size
(up to 10 nm) and a hydrophobic interior enabling the delivery of hydrophobic
drugs [76]. Their dendritic and branching nature also enables drug incorporation
onto the external surface. Moreover, these carriers provide drug delivery advan-
tages due to their enhanced circulation time in the blood, increased bioavailability,
well-programmed release of drug molecules, EPR effect providing uptake of the
nanomaterial by cancer tissues, lack of immunogenicity, and great penetration
ability [77].
Polyamidoamine (PAMAM) is the most common and well-known dendrimer for
drug delivery applications [78e81]. For instance, Kulhari et al. [80] described the
synthesis of trastuzumab-grafted PAMAM dendrimers for delivery of docetaxel to
HER2-positive breast cancer cells. Moreover, Ayatollahi et al. [78] reported the
use of modified PAMAM nanocarriers for efficient delivery of shRNA-plasmid
for specific knockdown of Bcl-xL in a lung cancer cell line.
Other dendrimer delivery vehicles also have been used as drug delivery platforms
such as polyetherhydroxylamine (PEHAM), polyesteramine, polypropyleneimine,
and polyglycerol [75].
The development of intelligent drug delivery platforms based on dendrimers is
currently a challenge since the clinical experience with these types of carriers has
so far been limited.
2.5 HYDROGELS
In recent years, hydrogel nanocarriers have gained important attention as one of the
most promising drug delivery vehicles due to their exclusive features. Hydrogels are
2. Nanocarriers 363

based in hydrophilic polymers organized in three-dimensional cross-linked
networks that can be prepared in a broad range of physical forms including slabs,
nanocarriers, microcarriers, coatings, and films [82]. They are able to protect cargo
from hostile environments, and their porosity allows drug incorporation into the
gel matrix. Moreover, hydrogels can regulate drug release by modifying the gel
structure in response to a particular stimulus such as temperature, pH, and ionic
strength [83,84]. Furthermore, when compared with other types of drug delivery
vectors, hydrogels have the advantage of tunable biodegradability, increased
biocompatibility, proper mechanical strength, among others [84].
Monette et al. [85] reported the encapsulation of primary T cells in a novel
injectable chitosan-based hydrogel. The cells encapsulated in this formulation
retained their anticancer capabilities proving that this vehicle could be further
used as a complement of the existing immunotherapy options.
There are many potential applications of hydrogens that can be explored,
for example, for encapsulating plant secondary metabolites (e.g., curcumin).
Curcumin is a plant natural compound with numerous therapeutic properties.
Although it can be easily obtained from plants, its biosynthetic pathway has
been extensively studied [86e90] with the goal of increasing its bioavailability.
Curcumin’s poor bioavailability is caused by its extremely low-aqueous solubility,
degradation at alkaline pH, rapid clearance, and low-cellular uptake [91,92]. This
limits its application in medicine [91]. However, nanotechnology approaches have
been proven to potentiate curcumin bioavailability, stability, and solubility [91,93].
For instance, Altunbas et al. [13] described a self-assembled peptide hydrogel that
demonstrated to be an effective vector for the localized delivery of curcumin over
sustained periods of time. Furthermore, Songkroh et al. [94] developed injectable
in situ forming chitosan-based hydrogels for curcumin delivery revealing sustained
release profiles of about 3e6 times higher than when compared with other similar
systems.
Hydrogels as drug delivery vehicles are paving their way into clinical trials
[95e97].
2.6 METALLIC NANOCARRIERS
Drug delivery nanoplatforms can also be based on inorganic materials, for instance,
metallic nanocarriers. Metallic nanocarriers such as iron oxide, gold, and silver are a
focus of interest due to their enormous potential for use in targeted drug delivery,
magnetic separation, biotechnology, and diagnostic imaging [98e101]. They can
be synthesized and tuned with several chemical functional groups allowing this
type of nanocarrier to be conjugated with a wide range of payloads and targeting
moieties.
Superparamagnetic nanocarriers (SPION) made from iron oxide (III) particles,
such as maghemite (g-Fe2O3), magnetite (Fe3O4), and hermatite (a-Fe2O3), have
a diameter ranging from 10 to 100 nm and exhibit specific properties like biocom-
patibility and the phenomenon of “superparamagnetism” [76,102]. Vyas et al. [23]

reported the therapeutic efficacy of doxorubicin-hyaluronan-superparamagnetic iron
oxide nanocarriers (DOX-HA-SPION). By increasing the drug efficacy and
decreasing off-target effects, their results suggested that the use of DOX-HA-SPION
as a drug delivery platform could have an auspicious potential in treating metasta-
sized and chemoresistant breast cancer cells.
The use of magnetic nanocarriers can also be combined with hyperthermia for
the treatment of cancer [103,104]. For instance, Sadhukha et al. [105] highlighted
the potential of an inhalable system based on magnetic targeted hyperthermia for
lung cancer treatment.
Unique chemical, optical, physical, and electronic features of gold-based
nanocarriers combined with their inertness, low toxicity, easy synthesis, high
accumulation in tumors and inflamed tissues by EPR effect, well-established
surface functionalization (thiol functionalization opportunity), and modifiable
stability make these types of nanocarriers promising scaffolds for drug delivery
purposes [106e108]. In recent years, photothermal therapy mediated by
gold-based nanocarriers have emerged as a potential treatment for solid tumors
[109,110]. Kang et al. [19] reported a novel targeted drug delivery vehicle,
constituted by gold nanorods (photothermal agents) coated with porphyrin, as
well as an anti-HER2 antibody (trastuzumab). Their results showed promising
antitumor activity by photothermal ablation of HER2-positive breast cancer cells.
Among the noble metal nanomaterials, silver nanocarriers exhibit a consistent
amount of important features such as simple synthesis, tailorable morphology, and
high surface volume ratio [111,112]. Benyettou et al. [14] described the synthesis
of a silver nanocarrier-ebased drug delivery platform that achieved the effective
synergistic delivery of DOX and alendronate to HeLa cells and improved the
anticancer indices of both drugs.
Potential disadvantages and concerns of general metallic nanocarriers include
weight/weight ratio between functional cargo and the inert platform, the absence
of an inner core, and their release as a nonrecyclable form into the environment [76].
2.7 QUANTUM DOTS
QDs are small-sized nanocarriers ranging typically between 1 and 10 nm that have
unique optical properties, high brightness, and antiphoto bleach features [113].
These plasmonic nanocarriers are constituted by an inorganic elemental core, for
example, cadmium (Cd) and selenium (Se), and a metallic shell, for instance, zinc
sulfide (ZnS) [76]. They can be easily tailored with targeting moieties and also
incorporated in amphiphilic polymers in order to improve specificity, solubility,
size, and visualization properties [114,115].
These semiconductor nanocrystals can be used as drug delivery vehicles or solely
as fluorescent labels for other drug vectors. Cai et al. [116] reported the synthesis of
a pH-sensitive zinc oxide (ZnO) QD-based drug delivery system. Their results
showed an efficient loading and delivery of DOX into lung cancer cells. Moreover,
cadmium sulfoselenide/zinc sulfide QDs-based nanocarriers for delivery of siRNA
2. Nanocarriers 365

against human telomerase reverse transcriptase were developed by Lin et al. [117].
High gene transfection efficiencies were achieved and, more importantly, the
silencing of target gene expression led to suppression of the glioblastoma cells
proliferation.
The QDs combination capability of molecular imaging and therapy can open
new opportunities in the area of cancer treatment. However, the toxicity of some
materials used in their synthesis could be an important concern [118].
2.8 CERAMIC-BASED NANOCARRIERS
Ceramic nanocarriers possess a porous nature and have a particle size less than
50 nm. Usually they are developed from nanoscale ceramic materials such as
hydroxyapatite (HA), silica (SiO2), zirconia (ZrO2), titanium oxide (TiO2), and
alumina (Al2O3) [119]. These nanocarriers can be used as drug delivery vectors
given their great load ability, high stability, and easy integration into hydrophobic
and hydrophilic systems [119].
Among these ceramic-based nanocarriers, mesoporous silica nanocarriers
(MSNs) have attracted attention as a consequence of their biocompatibility, ease
of synthesis, high pore volume, high surface area, high payload ability, and a unique
tailorable structure (silanol functionalization opportunity) [120e122].
Quercetin, one important bioflavonoid, is known for its pharmacological
properties including antiinflammatory, antioxidant, antihypertensive, antiobesity,
and anticancer [123]. However, quercetin’s poor bioavailability, as well as its poor
water solubility limits its therapeutic application. Sarkar et al. [124] reported the
synthesis of a new targeted delivery system to breast cancer cells based on
folate-tagged MSNs loaded with quercetin. Their results ensured an effective
targeted delivery of quercetin toward breast cancer cells with enhanced bioavail-
ability. Furthermore, Wu et al. [24] developed a targeted MSN delivery platform
loaded with arsenic trioxide demonstrating in vivo inhibition of tumor growth in
a TNBC mouse model.
These results demonstrated that the application of target-specific MSNs as
delivery platforms for cancer treatment purposes is promising.
2.9 CARBON-BASED NANOCARRIERS
Carbon-based nanocarriers, including carbon nanotubes (CNTs), fullerenes, and
nanodiamonds, have attracted particular interest given their excellent optoelectronic,
chemical, and mechanical properties. CNTs are hexagonal hollow cylinders made of
one single-walled (SWCNTs) or multiwalled (MWCNTs) sheets of graphene that
have excellent physical strength and exceptional heat and conductivity properties
[76,125]. These nanocarriers can also be chemically modified to display specific
moieties, for instance, functional groups, payloads, and polymers, to confer proper-
ties suited for drug delivery purposes, such as enhanced solubility, increased
biocompatibility, and cellular responsiveness [125,126]. Hassanzadeh et al. [18]

reported a new MWCNTs delivery system of 2-arachidonoyglycerol (2-AG).
This cannabinoid compound possesses proven beneficial effects in the inflammatory
intestinal disorder colitis but its efficiency is limited by its poor solubility and rapid
hydrolysis [127]. Hassanzadeh et al.’s [18] results highlighted the potential
of MWCNTs as carriers for 2-AG, providing a sustained concentration and
longer-lasting therapeutic effects in an experimental model of colitis.
Fullerenes are hydrophobic 1-nm scale spherical closed-cage structures that can
be functionalized by linking hydrophilic moieties to improve aqueous solubility
properties.
Their uniform size and shape, as well as their ability to surpass the cell
membrane and bind, for instance, to mitochondria, led many scientists to develop
fullerenes for drug delivery purposes [125,128]. For instance, Misra et al. [22]
developed a glycine-tethered C60-fullerenes system for the delivery of docetaxel
into breast cancer cells. This cargo vehicle was able to release the anticancer
drug with improved cellular uptake and enhanced efficacy.
Nanodiamonds represent another class of multifunctional carbon-based
nanomaterials, with tunable morphological, chemical, electronic, and optical
properties [129]. They have a tetrahedral arrangement with small overall size
(smaller than 10 nm) and large surface area showing an extraordinary combination
of intrinsic features, particularly remarkable hardness, low friction coefficient, and
thermal conductivity [125,129,130]. Given their high biocompatibility, scalability
in production, and the ability to enhance therapeutic effects [131,132], nanodia-
mond nanocarriers have been developed particularly for advanced tumor therapies.
For instance, Man et al. [21] described the synthesis of nanodiamond-daunorubicin
conjugates with application toward multidrug chemoresistant leukemia.
Nanodiamond-enabled therapeutics showed significant potential to improve cancer
treatment, particularly against resistant strains.
2.10 EXOSOMES
Exosomes, known as “natural nanocarriers,” are nanosized (30e120 nm) membrane
vesicles released by most cell types [133,134]. These extracellular vesicles are
present in almost all biological fluids (e.g., plasma, urine, semen, breast milk, saliva,
serum, and cerebrospinal fluid) [135,136] and function as natural transporters of
cellular components such as proteins, lipids, and nucleic acids between neighboring
and distant cells [137,138]. Several methods have been used for the isolation of these
phospholipid bilayer vesicles including differential ultracentrifugation, density
gradient centrifugation, size exclusion chromatography, immunoaffinity capture,
and PEG-mediated precipitation [136].
Exosomes have gained remarkable interest as potential nanocarriers for drug
delivery [139e142]. They are attractive for drug delivery purposes for several
reasons, including the fact of that they exhibit small size for penetration into deep
tissues, being less likely to be immunogenic or cytotoxic than the other synthetic
delivery systems, possessing slightly negative potential zeta for long circulation,
2. Nanocarriers 367

showing deformable cytoskeleton, and their similarity to cell membranes [133].
Moreover, due to EPR effect, exosomes preferably accumulate at solid tumor sites
or inflamed tissues [136,137]. In addition, this class of vesicles can be engineered
with targeting peptides, proteins, aptamers, or antibodies for precise therapeutic
delivery [136].
Alvarez-Erviti et al. [143] were the first to harness the potential of exosomes
for the delivery of siRNA in a targeted manner to neurons, microglia, and oligoden-
drocytes in the mouse brain. The potential clinical applications of exosome-
mediated drug delivery have been explored and spread rapidly. For instance,
Tian et al. [141] described a new effective exosome-based delivery system of
curcumin for cerebral ischemia therapy. After administration of peptide-tagged
exosome loaded with curcumin, the inflammatory response and cellular apoptosis
in the lesion region was suppressed more effectively than when curcumin or
exosomes that were administrated alone. Furthermore, Agrawal et al. [12] reported
the use of milk-derived exosomes for oral delivery of the chemotherapeutic drug
PTX. Their results exhibited a PTX sustained release, a significant inhibition of
tumor growth in human lung tumor xenografts, and also excellent lower systemic
and immunogenic toxicities compared with PTX treatment alone.
Exosome-based vehicles for drug delivery purposes appear to be a promising
direction for therapeutics, however, there are still some issues and challenges that
need to be addressed including production of exosomes in large scale for clinical
use, which cell type to use for exosome derivation, and determination of in vivo
exosome potency and toxicology [133].
2.11 VIRUS-BASED NANOCARRIERS
Viruses, present ubiquitously in the environment, can infect mammals, bacteria, or
plants and have been used to develop virus-based nanocarriers [144]. These natural
carrier systems evolved to package, protect, and deliver nucleic acids to host cells
and can be subverted for the delivery of other cargos [144,145]. Moreover, viruses
have replication ability allowing inexpensive manufacturing on an industrial scale
level [144].
Virus particles typically consist of regular arrays of virus coat protein mole-
cules, which self-assemble to form a highly defined three-dimensional structure.
These biodegradable and biocompatible particles range in sizes from approxi-
mately 10 nm to over a micron and can come in many different shapes and surface
properties [145,146].
Several engineered mammalian viruses are under study as drug and gene
carriers [147e149]. Glybera, an orphan medicine, was the first viral product
derived from adeno-associated virus (AAV) to be approved by the European
Medicines Agency (EMA) for treatment of rare disease lipoprotein lipase
deficiency [150]. Plant-based viruses such as cowpea mosaic virus (CPMV) and
red clover necrotic mosaic virus (RCNMV), and bacteriophages such as MS2
and M13, are regarded as safer delivery vectors than mammalian systems because

they are nonintegrative in mammalian systems and are less prone to trigger a
negative effect [144,151]. The structure of these virus-based nanocarriers can be
tailored using genetic engineering approaches, chemical modifications, or
self-assembly/encapsulation strategies. These virus-based nanocarriers can be viral
nanocarriers or a nonreplicative subclass of viral nanocarriers, the virus-like
nanocarriers. The virus-like nanocarriers are homogenous nanocarriers derived
from the coat proteins that lack their natural genome and are noninfectious
[152e154].
These nanocarriers, which can be designed to release their payload in response to
physical alterations, for instance in pH, chemical stimuli, temperature, or redox
status, are highly promising scaffolds among the many nanomaterials that are being
developed as “smart” drug delivery platforms [152]. For instance, Aljabali et al.
[155] described the use of an engineered plant virus, CPMV, for drug delivery of
the chemotherapeutic drug DOX. CPMV covalently decorated with 80 DOX
molecules showed higher toxicity than free DOX against cancer cells. Regarding
the use of bacteriophages as delivery vehicles DePorter and McNaughton [156]
reported the use of a genetically and enzymatically engineered M13 bacteriophage
for intracellular delivery of exogenous proteins to human prostate cancer cells.
Moreover, Galaway and Stockley [16] described the use of an MS2 virus-like
nanocarrier for targeted delivery of anti-Bcl2 siRNA toward HeLa cells.
It is clear that there is growing interest in the potential applications of virus-based
nanocarriers in the medicine field. However, some challenges still need to be
overcome such as understanding their behavior in vivo.
3. TARGETING
Recent advances in nanocarrier technology for effective targeted cellular delivery
led to the development of a variety of novel therapeutic and diagnostic platforms
[157]. Effective targeted drug delivery systems require drug accumulation within
a target zone and specific interactions with the target receptor at a molecular
level [158].
The first generation of nanocarriers for drug delivery systems was based on a
passive targeting mechanism to increase efficiency over traditional free-drug formu-
lations [159]. Afterwards, a new concept was introduced, consisting of active
targeting through the incorporation of specific ligands to enhance drug delivery to
the target sites [160]. This improvement was possible due to the attachment of
receptor-specific ligands to the outer surface of nanocarriers using conjugation
strategies.
To design an effective nanocarrier, it is necessary to consider several parameters
including the type of ligand, the ligand conjugation chemistry, and the administra-
tion route. The optimization of all these parameters requires a high amount of
time and resources, which explains the fact that only a few developed targeted
nanocarrier formulations have reached the clinic [161].
3. Targeting 369

3.1 PASSIVE TARGETING
Passive targeting, also referred to as physical targeting, is based on the transport of
nanocarriers by convection or passive diffusion within the body. Convection refers to
the movement of molecules in fluids, allowing the transport of large molecules. On
the other hand, low-molecular-weight compounds are predominantly transported by
passive diffusion, which is defined as a physical process where molecules move
across the cell membrane, according to the difference in concentration and without
the need of energy input [162].
Through this approach, the nanocarriers’ circulation in the blood stream to the
target receptor depends on features like charge, molecular size, or shape. Passive
targeting results in nanocarrier accumulation in areas with leaky vasculature
[163]. Therefore, most nanocarriers are expected to accumulate in tumors due to
the pathophysiological characteristic of tumor blood vessels [164]. Tumor blood
vessels are irregularly shaped, leaky, and dilated due to rapid growth and abnormal
blood flow. These features allow nanocarriers access to the tumor interstitium
through the endothelial gap junctions [165]. In a normal vasculature, endothelial
junctions range from 5 to 10 nm in size, while in tumor tissue, these junctions range
from 100 nm to 2 mm, depending on the tumor type [166,167]. These characteristics
provide an EPR effect that allows the selective accumulation of nanocarriers in the
tumor interstitium (Fig. 14.3) [168]. The EPR effect is the gold standard in cancer-
targeting drug designing, because nanocarriers can be specifically designed to take
advantage of passive targeting. Through the use of passive targeting it is possible to
FIGURE 14.3
Schematic illustration of the passive targeting via the enhanced permeability and
retention (EPR) effect. Nanocarriers can extravasate through the gaps between
endothelial cells and accumulate in tumor cells.

achieve 10 to 100 times higher local concentrations of drug-loaded nanocarriers at
the tumor site than when using the free drug alone [169]. In addition, the EPR effect
also promotes prolonged drug retention due to poor lymphatic drainage [170].
Despite its advantages, passive-targeting approaches also possess several limita-
tions. The results of passive targeting are inconsistent due to the fact that tumor
vascularization and angiogenesis depend on cancer type and status [166,170].
Moreover, the absence of specificity in cell uptake leads to a poor internalization
of nanocarriers, which introduced the need to attach ligands to the nanocarriers’
surface to enhance drug delivery efficiency [160,171].
A heightened knowledge of the barriers encountered by passive targeting of
nanocarriers led to the optimization of several nanocarrier design parameters,
such as size, shape, and surface characteristics, aiming at overcoming these barriers.
3.1.1 Nanocarrier Size
The nanocarriers’ size is extremely important for their performance as a drug
delivery system and can be tailored for directing the nanocarrier distribution
in vivo. Size range affects several biological events that include circulation
half-life, extravasation through leaky vasculature, and macrophage uptake [172].
Therefore, the nanocarriers’ size should be large enough to prevent their clearance
from the system, and should be small enough to escape macrophages capture.
A nanocarrier should be at least 10 nm in diameter to prevent clearance through
renal excretion [173,174]. The largest size of nanocarrier to be used for drug delivery
depends on the “cut-off” size of the permeabilized vasculature to allow a successful
uptake toward the target. The size of the gap junction between endothelial cells of
the leaky vasculature can vary mainly between 100 and 600 nm, thus the nanocar-
riers’ size should be up to 100 nm [175]. The larger nanocarriers (>200 nm) tend
to be retained in the liver and spleen (150e200 nm) [176]. In order to be an effective
drug carrier, the nanocarrier should have a diameter between 10 and 150 nm to
ensure longer circulation time and increased accumulation in the desired location.
Cabral et al. [177] tested a variety of sub-100-nm polymer micelles of different sizes
(30, 50, 70, and 100 nm) in highly and poorly permeable tumors. In this study, all
polymer micelles penetrated well within highly permeable tumors, but only
small-sized nanocarriers (<50 nm in diameter) were able to accumulate in poorly
permeable tumors and achieve an antitumor effect.
Drug release is also affected by the nanocarrier size. Smaller nanocarriers allow a
faster drug release due to the proximity of the encapsulated drug to the carrier
surface, while larger nanocarriers allow a higher number of encapsulated drug
molecules and a slower release [178]. Therefore, the nanocarrier size provides the
means of adapting drug release rates.
3.1.2 Nanocarrier Shape
Shape-related factors affect the nanocarrier transport into blood, particularly in
small capillaries, influencing the cellular uptake and the ability to overcome biolog-
ical barriers [179].
3. Targeting 371

The nanocarriers’ shape affects their circulation in the blood stream due to
hemodynamic forces opposing a particle movement and differences of density
between the nanocarrier and the blood [179]. Goldman et al. [180] verified that
hydrodynamic forces increase as the radius of a spherical carrier increases; whereas
nonspherical carriers experience tumbling and rolling dynamics that favor vessel
wall interactions significantly more than spherical carriers. Geng et al. [181] demon-
strated that filamentous polymer micelles have long-circulation lifetimes (>1 week
after intravenous injection) compared to spherical nanocarriers (2e3 days).
Moreover, surface curvature affects interactions between cell and nanocarrier
surfaces. Champion and Mitragotri [182] demonstrated the importance of the
carriers’ shape for interactions with cells and, consequently, for the carriers’
internalization. The authors verified that carriers with a curvature lower or equal
to 45 degrees exhibited faster internalizations than the ones with a curvature higher
than 45 degrees. These results reinforce the exploration of carriers with different
shapes such as ellipsoidal, cylindrical, and discoidal that present minimal regions
of curvature in order to improve cell internalization and drug accumulation of
therapeutics.
3.1.3 Nanocarrier Surface Characteristics
The immune system can recognize nanocarriers when they are administrated and
eliminates them from the bloodstream by macrophage phagocytosis [183]. Apart
from an appropriate size and shape, nanocarriers should have a hydrophilic surface
to escape macrophage phagocytosis. This is achieved by coating nanocarriers with
hydrophilic polymers, such as PEG, polyethylene oxide, polyoxamer, and
poloxamine, or surfactants, such as polysorbate 80 (Tween 80) [184]. Studies
demonstrated that the presence of PEG on nanocarriers’ surface increases the
half-life circulation of the PEGylated nanocarrier because it prevents opsonization
by mononuclear phagocyte system (MPS) and other serum components [185].
Nanocarrier surface charge represents another parameter that can be manipulated
to prolong circulation lifetimes. Nanocarriers with neutral and negative surface
charges have been shown to reduce the aggregation of serum proteins in longer
circulation [186]. The study by Yamamoto et al. [187] demonstrated that negatively
charged nanocarriers resulted in lower accumulation in the liver and spleen, while
positively charged nanocarriers have a higher rate of nonspecific uptake in the
majority of cells. On the other hand, other studies showed that cationic nanocarriers
are preferentially internalized by tumor cells [188] and sites of chronic inflammation
[189]. Hence, zwitterionic nanocarriers have emerged to switch their charge based
on environmental stimulus, allowing a higher efficiency in drug delivery to reach
target sites [190].
3.2 ACTIVE TARGETING
The concept of active targeting was introduced in 1906 by Paul Ehrlich, and it was
described as a “magic bullet” needed to target specific drug delivery within the

body [160]. Active targeting involves the use of a targeting ligand on the surface of
the nanocarriers in order to facilitate uptake by the diseased cells through the target
recognition of the ligand [191,192]. These ligands include proteins, peptides,
antibodies, nucleic acids, among others [193,194]. The targeted molecule is
typically selected based on its selectivity or overexpression in diseased organs,
tissues, cell surfaces, or subcellular domains and can be a protein, sugar, or
lipid [195].
Active targeting promotes the internalization of the ligand-conjugated drug car-
rier on the target cell through specific receptor-mediated endocytosis. After
receptor-mediated internalization, an endosome is formed and the intracellular
drug release occurs into the endosome, due to the decrease of pH [163,196]
(Fig. 14.4). This approach increases the drug dose delivered to diseased cells, which
is important for the therapeutic efficacy of anticancer drugs and other
biotherapeutics, including gene delivery and gene silencing. In addition, it allows
systematic administration of smaller doses [197]. The internalization ability on
surface receptors is the base for the design of targeted delivery systems. However,
the ligand-nanocarrier design is complex due to their architecture. It is important
FIGURE 14.4
Schematic illustration of active targeting drug delivery using antibodies (A), aptamers (B),
or proteins (C) as ligands. The nanocarrier is internalized via receptor-mediated
endocytosis. Afterward, nanocarrier depolymerization occurs in the lysosome and the
drug escapes and diffuses to the nucleus. Tf, transferrin; TfR, transferrin receptor; PSMA,
prostate-specific membrane antigen.
3. Targeting 373

to take into consideration the ligand conjugation chemistry and the ligand function-
ality in biological environments. To that end, it is vital to ascertain the ligand stabil-
ity in the administration route or the nonspecific binding of proteins during the
ligands’ journey through the bloodstream. Other factors such as ligand affinity to
the target, immunogenicity, and reproducibility can affect the choice of the targeting
ligand [195].
There are many different types of targeting moieties that may be used as a basis
for targeted drug delivery (Table 14.2). The following section will describe the most
widely used active targeting ligands for therapies including antibodies, aptamers,
peptides, proteins, and other small molecules.
3.2.1 Antibody-Based Targeting
An antibody is a large Y-shaped glycoprotein produced by B lymphocytes as a
component of the immune system that recognizes foreign substances. Monoclonal
Table 14.2 Examples of Molecular Targets and Their Targeting Ligands
Type of
Ligand
Targeting
Moiety
Targeted
Ligand
Targeted Cell
Type References
Antibodies mAb anti
EGFR
EGFR Cancer cells [198,199]
mAb anti
VEGF
VEGF Angiogenesis in
tumor environment
[332,333]
Aptamers Aptamer
against
PSMA
Prostate-
specific
membrane
antigen
Prostate cancer
cells
[200,201]
Aptamer
against E-
selectin
E-selectin Inflamed cells [202,203]
Peptides RGD
peptide
Integrin avb3 Tumor
neovasculature
[204e206]
Ab1e42
peptide
Amyloid-b Amyloid plaques [207,208]
Proteins Tf TfR Cancer cells [209,210]
Lf LfR Brain cells [211]
Vitamins Folate Folate receptor Cancer cells and
activated
macrophages
[212e215]
Riboflavin Riboflavin carrier
protein
Cancer cells [216e218]
Carbohydrates Galactose ASGPR Hepatocytes [219,220]
ASPGR, asialoglycoprotein receptor; EGFR, epidermal growth factor receptor; Lf, lactoferrin; LfR,
lactoferrin receptor; mAb, monoclonal antibody; PSMA, prostate-specific membrane antigen; RGD,
arginylglycylaspartic acid; Tf, transferrin; TfR, transferrin receptor; VEGF, vascular endothelial growth
factor.

antibodies (mAbs) are antibodies produced by identical immune cells that recognize
and bind to a specific antigen [221]. Because they have high affinity and specificity,
mAbs have been conjugated to the surface of nanocarriers for targeting specific
antigens present on the cell membrane [195]. The antibody-antigen interaction
can induce multiple mechanisms including interference with the ligand-receptor
binding or suppression of protein expression [62]. Currently, mAbs are usually
chimeric, humanized, or fully human antibodies. The chimeric antibodies are 70%
human, while humanized antibodies are 85%e98% human and are less immuno-
genic than chimeric antibodies. These antibodies are derived from nonhuman
species, usually mice, whose protein sequences have been modified to increase their
similarity to antibodies produced in humans [222]. Fully human antibodies have no
murine sequences and are produced by two different strategies, namely phage
display technologies and transgenic mice [223].
One of the first molecules targeted by mAbs was the epidermal growth factor
receptor (EGFR), which plays a crucial role in cell proliferation, differentiation,
survival, angiogenesis, and metastasis [224]. Overexpression of EGFR protein is
frequently found in many different tumors such as lung, breast, gastric, colorectal,
prostate, bladder, pancreatic, ovarian, and renal cancer [225]. Since the FDA
approved EGFR immunotherapy, researchers have been using anti-EGFR mAbs,
such as cetuximab (IMC-C225) and panitumumab (also known as ABX-EGF or
Vectibix), for creating EGFR-targeted nanocarriers [226]. A variety of nanocarriers
including gold nanocarriers, liposomes, and polymeric nanocarriers have been used
for targeted delivery and therapy. Anti-EGFR-ILS-DOX is a doxorubicin-loaded
anti-EGFR immunoliposome, in Phase II clinical trials, to provide maximal drug
delivery and internalization to cancer cells via a targeted receptor (Fig. 14.4A).
Based on clinical studies, the EGFR-specific antibody enhances the specificity
and efficiency of chemotherapy, while the encapsulation of the cytotoxic drug within
PEGylated liposomes decreases its toxicity at the same time [227].
Despite the advantages of mAbs as ligands, there are also several challenges that
limit their application in vivo [228]. The large size of mAbs (150 kDa) prevents the
effective surface conjugation on nanocarriers and causes a notable increase in the
nanocarriers’ diameter that leads to poor tissue penetration in order to reach target
cells [195,229]. Moreover, the high production costs of mAbs limits their use as tar-
geting moiety. Their production requires the use of large cell cultures of mammalian
cells followed by an extensive purification process [222].
The advances in protein engineering and expression have allowed the generation
of novel classes of antibody fragments to circumvent mAbs problems. These frag-
ments, such as antigen-binding fragments (Fab) and single-chain variable fragments
(scFv), have been exploited as a part of nanocarrier-antibody fragment conjugates
due to their smaller size. It was concluded that they induce a smaller immune
response, while maintaining the function of active targeting [230]. To date, fragment
conjugates have shown great potential and several promising candidates entered
clinical trials. MCC-465 and MM-302 are two examples of antibody fragment-
targeted nanocarriers. MCC-465 is a Fab-decorated doxorubicin-encapsulated
3. Targeting 375

liposome that showed positive results in preclinical studies with adequate
biodistribution and highly efficient delivery of doxorubicin to stomach cancer cells
[231]. MM-302 is an anti-HER2 scFv conjugated with a PEGylated liposomal
formulation of doxorubicin that showed encouraging efficacy results in HER2-
positive breast cancer [232].
3.2.2 Aptamer-Based Targeting
Aptamers are small nucleic acid ligands containing between 25 and 50 bases length
that are generated by molecular evolution to bind with high affinity and specificity to
a variety of targets due to the ability of the molecules to fold into unique conforma-
tion with three-dimensional structures [233,234]. Aptamers’ selection was first
described in 1990 from an in vitro process called Systematic Evolution of Ligands
by Exponential Enrichment (SELEX) through combinatorial libraries of 1015
random oligonucleotides [235,236]. Subsequent studies led to variations based on
the conventional SELEX such as Cell-SELEX [237], Counter SELEX [238], or
Toggle SELEX [239] to adapt the selection process to the intended applications.
This class of ligands is particularly interesting due to their unique ability to bind
to a variety of targets including peptides, enzymes, antibodies, various cell surface
receptors, and other small molecules [240,241]. Moreover, aptamers have potential
advantages over other targeting moieties, such as a relatively small size (15 kDa),
low immunogenicity, high affinity and selectivity, and easy scale-up preparation
without variations, which makes them an attractive alternative to antibodies and
peptides [242].
However, aptamers also have several limitations, such as rapid blood clearance
due to DNases or RNases degradation. To make them more resistant to nuclease
degradation, they are typically chemically modified with PEG [243] or at the 20-fluo-
rine position [244] to enhance the bioavailability and pharmacokinetic properties.
Another important modification is the use of locked-nucleic-acids (LNAs), which
hold great promise to stabilize aptamers due to their thermostability and resistance
to nuclease degradation [245,246]. Even though the aptamers selected in vitro
present high affinity to the target, in biological systems this affinity could be
completely different. For that reason, optimization or variations based on the
conventional SELEX emerged using whole living cells, pathogens, or animal
models [247].
To date, a number of aptamers targeting specific receptors have been successfully
adapted for targeted drug delivery, including anticancer drugs, toxins, enzymes, and
siRNAs [248]. The best-characterized aptamer for targeted delivery is 20-fluoro-
pyridine-RNA aptamer, also known as A10-RNA aptamer, generated against the
extracellular domain of prostate-specific membrane antigen (PSMA) that is highly
overexpressed in prostate cancer cells (Fig. 14.3B) [249]. This aptamer has been
used to target prostate cancer cells to deliver DOX [250] and gene silencing systems
[251] in liposomes [201] and QDs [200]. The enhanced efficacy of PSMA-aptamer
nanocarrier is attributed to the intracellular drug release upon PSMA-mediated
endocytosis, causing efficient delivery into prostate cancer cells. Zhen et al. [201]

developed an aptamer-liposome-CRISPR/Cas9 system that specifically binds to
prostate cancer cells expressing PSMA. The aptamer-liposome-CRISPR/Cas9
system provides cell typeespecific CRISPR/Cas9 delivery and promotes PLK1
silencing that results in tumor regression.
Besides the use of aptamers as targeting moieties, there are several aptamers that
can be used for therapeutic purposes in the same way as mAbs [252,253]. In addi-
tion, they can be used in diagnosis due to their ability to selectively identify different
types of cells or differentiate cancer cells from normal cells based on molecular
differences of membrane proteins [254].
3.2.3 Protein-Based Targeting
The three-dimensional shape of proteins provides affinity for specific substrates,
and, therefore, nonantibody proteins can be used as targeting moieties. Numerous
naturally occurring proteins have endogenous targets that can be exploited for
therapeutic applications [195]. For example, transferrin (Tf) is a serum glycoprotein
that transports iron through blood and into cells by binding to the Tf receptor (TfR).
The TfR is a vital protein involved in iron homeostasis and cell growth regulation
[175,255] and is overexpressed in malignant cells due to their need for iron [256].
Therefore, this receptor is an attractive target for the delivery of anticancer
therapeutics. Moreover, the presence of Tf-ligands was found to be essential for
the intracellular delivery and gene silencing efficiency of siRNA nanocomplexes
[257]. The strategy of using Tf to target TfR is currently under clinical investigation
for various nanocarriers [258].
Guo et al. [209] focused their attention on developing Tf-conjugated DOX-
loaded nanocarriers (Fig. 14.4C). The nanocarriers were made using PLGA, and
the conjugated and loaded nanocarriers were tested on human lung cancer cell lines.
The Tf-conjugated carriers exhibited evident antitumor effects in vitro. Lactoferrin
(Lf) is also an iron-binding protein present in many tissues and biological fluids
[259]. Lf is involved in several physiological functions, including regulation of
iron absorption [260], immune response [261], antimicrobial effects [262], antiin-
flammatory properties [263], and anticancer effect [64,264]. Studies demonstrate
that lactoferrin receptors (LfR) are expressed in the brain and they are able to
transcytosis through bloodebrain barrier, making the Lf a suitable targeting ligand
[265e267]. Huang et al. [211] exploited Lf as a ligand conjugated to PAMAM for
efficient gene delivery to the brain. The results on primary brain capillary endothe-
lial cells showed the potential of Lf in the design of gene delivery systems for brain
targeting.
Synthetic proteins can also be exploited as targeting ligands. For example,
affibodies [268] or ankyrin repeat proteins [269] were developed to decorate nano-
carriers. These approaches possess the advantage of using high-affinity artificial
ligands, which do not have to compete against highly abundant, naturally occurring
proteins. Nevertheless, proteins share some limitations with antibodies such as their
large size that results in an increase of nanocarrier diameter, and their patterning on
nanocarriers’ surface that may induce the immune system activation [195].
3. Targeting 377

3.2.4 Peptide-Based Targeting
In order to deliver drugs to the target site, peptides with high affinity for membrane
receptors overexpressed on target cells emerged as attractive targeting moieties.
These moieties are molecules consisting of 2e50 amino acids linked by peptide
bonds [270]. Due to their small size, they allow a higher penetration efficiency to
the target cells, low immunogenicity, and ease of preparation at small costs
[271,272].
The main strategy to select peptide ligands is to screen peptide libraries produced
by phage display [273,274]. Phage display is a selection technique that allows the
creation of libraries that contain up to 1010
different sequences expressed as a
genetic fusion to bacteriophage coat protein. This method can be used to identify
peptides that target a specific receptor or certain cell types, even if the receptors
are unknown [275]. Moreover, phage display is adaptable to both in vitro and
in vivo conditions.
Colorectal cancer is one of the most common cancers worldwide [276]. Wu et al.
[70] used phage display technology to identify peptides that could bind to colorectal
cancer cells with high affinity and specificity. The authors developed a conjugated-
liposomal drug carrier by incorporating these peptides into liposomes for delivering
chemotherapeutic agents to colon cancer cells. This approach resulted in a higher
dose of drugs accumulated at the tumor site, which increased the tumor inhibition
ability [70].
One of the true drawbacks of peptides is the susceptibility to proteolytic cleavage
compared to other targeting moieties [277]. This issue may be ameliorated by the
incorporation of positively charged amino acids, especially at the terminal position,
to improve cell and tissue penetration of peptides and help in vitro and in vivo
bioavailability [278].
Currently, peptide-based targeting delivery applications have already entered
clinical trials. The results of clinical trials have been encouraging in terms of ther-
apeutic efficiency. The most widely used peptide is arginylglycylaspartic acid
(RGD), which binds to cell surface receptors known as integrins. Integrins have
key roles in cell adhesion, migration, and proliferation. Therefore, their overexpres-
sion in cells is highly associated with cancer progression [204e206]. Given its
utility as a cancer-targeting agent, RGD has been widely used to create targeted
therapeutic and imaging platforms. Furthermore, many other peptides have been
identified and used for targeted delivery of oligonucleotides, drugs, imaging agents,
and viruses [272].
3.2.5 Small Molecules
Small molecules have a very high selectivity and avidity to their target receptors
making them attractive tools for targeting cells. Moreover, small-molecular-weight
compounds have properties that strongly contrast with the targeting ligands
presented earlier, including smaller sizes, low production cost, low immunogenicity,
and improved stability. In this section, small molecules such as vitamins and
carbohydrates will be discussed in more detail.

3.2.5.1 Vitamins
One of the most widely studied small molecules as a targeting moiety for the
delivery of agents is folate. Folate is a water-soluble vitamin and is required for
essential cell functions [279]. The folate receptor, located at the apical surfaces of
polarized epithelia, is significantly upregulated on many cancer cells compared to
normal tissue. Its overexpression was detected in ovarian, lung, brain, head and
neck, renal, and breast cancer [193,280]. Besides, several studies have found that
activated macrophages, which are implicated in inflammatory pathologies such as
rheumatoid arthritis, psoriasis, Crohn’s disease and lupus, express high levels of
folate receptors [281]. The folate receptor has a very high binding affinity for folate
(Kd ¼ 109
M), which allows the targeted delivery of imaging and therapeutic
agents to tumors [280,282]. In addition, folate ligands are extensively used for
targeting because they are inexpensive, nontoxic, nonimmunogenic, easy to
conjugate to carriers, and stable in storage and in circulation [283]. A wide array
of nanoplatforms including liposomes, gold nanocarriers, dendrimers, iron oxide,
and QDs have been targeted using folate. Bilthariya et al. [284] have been studying
the use of folate receptor for targeting activated macrophages in rheumatoid arthritis
treatment. They developed a folate-conjugated albumin nanocarrier loaded with
Etoricoxib, which is a drug that is used to treat pain and inflammation. The
folate-conjugated albumin nanocarrier loaded with Etoricoxib showed higher
accumulation on inflamed tissues when compared to Etoricoxib-nanocarriers
without folate.
Riboflavin, also known as vitamin B2, is essential for normal cell functions,
growth, and development, since it performs key metabolic functions in biological
oxidation-reduction reactions [285,286]. Riboflavin transporters (RFTs) and the
riboflavin carrier protein (RCP) are highly upregulated in metabolically active can-
cer cells (e.g., breast cancer, prostate cancer, and hepatocellular carcinoma), which
makes riboflavin an attractive small moleculeetargeting ligand for nanomedicines
[287]. This feature was demonstrated in several studies with a variety of nanocarriers
that can play a role in the design of novel therapeutics in cancer. For this purpose,
dendrimers have been used in conjugation with riboflavin. Thomas et al. [218] devel-
oped a methotrexate-riboflavin PAMAM dendrimer conjugate able to undergo
cellular binding and uptake in KB cells, inhibiting the cell growth in vitro.
Based on the results from recent years, it is clear that there is a great hope for
vitamin-based conjugates to provide a useful moiety for targeted drug delivery
systems.
3.2.5.2 Carbohydrates
Carbohydrates form another class of small moleculeetargeting ligands that selec-
tively recognize cell surface receptors like lectin [288]. Carbohydrate moieties,
including mannose [289], glucose [290], galactose [291], and their derivatives,
have been widely used for delivery of therapeutic agents.
Galactose is a simple sugar that is also frequently used for targeted delivery,
and it has a high affinity for asialoglycoprotein receptor (ASGPR) found on
3. Targeting 379

hepatocytes [289]. Ding et al. [219] developed a PEG-stabilized gold nanocarrier
with galactose as a targeting ligand exhibiting an increased uptake in a HepG2 liver
cell line. Sato et al. [292] ascertained that galactose-modified liposomes can be
highly effective at hepatic siRNA delivery compared with the bare nucleic acid.
For imaging purposes, iron oxide nanocarrier functionalized with a galactose-
containing polymer displayed increased accumulation in the liver, demonstrating
their utility as contrast agent for liver magnetic resonance imaging (MRI) [220].
Mannose is highly expressed in cells of the immune system, which makes this
targeting moiety a useful strategy for improving the efficacy of vaccines and chemo-
therapeutic agents [293]. Thus, different strategies have been used to develop drug
delivery systems able to target the mannose receptors. Targeting of dendritic cells,
which highly express mannose receptors, has been used to enhance the activity of
nanocarrier vaccine formulation. Xu et al. [294] developed a lipid-calcium-phos-
phate (LCP) nanocarrier loaded with peptides and functionalized with mannose to
create a therapeutic anticancer vaccine. The nanoformulation was able to reduce tu-
mor growth in in vivo models.
3.3 MOIETIES CONJUGATION STRATEGIES
Moieties conjugation strategies on the nanocarriers’ surface can influence the ligand
functionality and consequently the recognition of the target site. Moreover, due to
the difference of physicochemical properties that depend on nanocarrier type, the
choice of an appropriate conjugation strategy is not trivial. The strategies can be
characterized as noncovalent interactions, covalent interactions, and click chemistry
[295]. In all cases, the ligand-nanocarrier stability will dictate the conjugation strat-
egy to use. In the following section, the advantages, drawbacks, and examples of
each strategy will be discussed.
3.3.1 Noncovalent Conjugation Strategies
Noncovalent conjugation approaches encompass physical interactions that include
electrostatic, hydrophobic, and affinity. Electrostatic and hydrophobic interactions
are used for the assembly of therapeutic agents onto nanocarriers but their binding
mode is not appropriate for immobilizing targeting moieties onto nanocarriers’
surface. On the other hand, affinity interactions are effective for conjugating ligands
to nanocarriers [296].
Affinity interaction between avidin and biotin is one of the oldest known cross-
linker conjugations and also represents one of the strongest bonds in nature with a Kd
around 1015
M [297]. Other biotin-binding proteins have been developed based on
the avidin-biotin complex such as streptavidin and neutravidin [298]. Interaction of
biotin with avidin/streptavidin/neutravidin, which is almost an irreversible bond, has
been commonly used to conjugate nanocarriers to targeting ligands. Moreover, these
proteins have small molecular sizes, which do not influence the ligand functionality
[299]. This conjugation strategy has been applied to antibodies, peptides, and
aptamers. Meirinho et al. [300] developed an electrochemical aptasensor to detect

osteopontin, which is a protein related to breast cancer progression. A biotinylated
RNA aptamer with affinity for osteopontin was immobilized on a streptavidin-
modified gold surface, and through cyclic voltammetry the aptasensor was able to
detect osteopontin in standard solutions [300e302]. Moreover, liposomes biotin/
avidin-decorated are widely used to allow the attachment of a variety of biotinylated
ligands of interest for targeting therapy [303,304]. Hu et al. [305] conjugated RGD
peptides to the surface of microbubble particles by biotin-avidin linkage and
explored the viability of their use in assessing avb3 integrin expression in in vivo
models. The weakness of this conjugation strategy is the potential immunogenicity
due to the presence of the exogenous protein on the surface, which limits their use in
in vivo targeting [306].
3.3.2 Covalent Conjugation Strategies
Various covalent strategies have been employed to link ligands with reactive groups
of the nanocarriers’ surface. This conjugation strategy involves nanocarrier surface
functionalization with amine, thiol, and aldehyde groups. The features of covalent
strategies conjugation are presented in Table 14.3.
3.3.2.1 Amide Groups
The carbodiimide chemistry is the most frequently used strategy to modify free
carboxylic acids (COOH) with primary amine groups (NH2) to functionalize the
nanocarriers’ surface. The 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
is the most common carbodiimide. It activates the free carboxyl groups on the mole-
cule allowing it to bind with primary amine groups of the other molecule, thus
creating an amide bond. EDC reacts in the presence of N-hydroxysulfosuccinimide
(sulfo-NHS) to accelerate the reaction rate and the final coupling efficiency. The
EDC-NHS reaction produces a strong covalent bond between the compounds
[307]. This strategy is efficient for conjugation of proteins, antibodies, and peptides
because they are composed of amino acids that contain primary amine and carbox-
ylic groups. Also, DNA and RNA molecules, such as aptamers, can be chemically
synthesized with free carboxylic acid or primary amine groups [308]. Polymeric
nanocarriers [309], QDs [310], magnetic [311], and gold nanocarriers [312], among
other nanocarriers, can use this strategy to couple ligands to their surface.
A drug delivery system composed of DOX-loaded PLA nanocarrier and AP1
peptide that could specifically bind to interleukin-4 receptor, highly expressed on
glioma cells, was also developed [313]. The AP1 peptide was functionalized through
EDC-NHS linkage on nanocarrier surface and the results demonstrated that AP1-
nanocarrier-DOX exhibited therapeutic effect on tumor-bearing mice compared
with the unmodified nanocarriers and free doxorubicin.
However, proteins, antibodies, and peptides have numerous amine functional
groups available, which makes it difficult to control their conformation and function-
ality. For this reason, strategies with other functional groups are employed in these
ligands, such as thiol-maleimide chemistry due to low abundance of cysteine groups
compared to amine groups on proteins, antibodies, and peptides.
3. Targeting 381

Table 14.3 Conjugation Reaction, Linkages Formed and Their Diagram Representation
Type of Conjugation Linkage Diagram
NH2/COOH Amide bond
Thiol/Maleimide Thioether bond
Gold/Thiol Thiolesulfur bond
Hydrazide/Aldehyde Hydrazone
382
CHAPTER
14
Nanotechnology
in
Targeted
Drug
Delivery
and
Therapeutics

3.3.2.2 Thiol Groups
The strong interaction between thiol compounds and noble metal surfaces allows the
nanocarriers’ surface functionalization. The most popular thiol linkage is
thiol-maleimide due to its high efficiency in aqueous environments. Maleimide
groups have high selectivity toward the thiolate group of cysteine, which is one of
the least abundant residues present in proteins, antibodies, and peptides, making
this strategy an attractive conjugation approach [314]. The thiol-maleimide reaction
occurs between thiol group (-SH functional group) and C1 carbon of maleimide form-
ing a thioether bond, which is stable within 24 h in human serum [315]. This type of
conjugation has been widely explored in immunoliposome formulation. Lee et al.
[213] developed an efficient siRNA delivery system to target metastasized tumors
in the lungs. Anticancer siRNAwas encapsulated in maleimide-containing liposomes
conjugated with thiolated antibodies against the EGFR. The liposomal complex was
efficiently transfected in cancer lung cells, resulting in cancer cell death.
Apart from thiol-maleimide, thiol-gold strategy conjugation has been extensively
used for conjugation of moieties onto gold surfaces. The forces between thiol and
gold surfaces originate a goldesulfur bond (AueS) [316]. The inert nature of
gold has enabled the formation of a wide range of functional groups on gold
surfaces. Thiolate groups are assembled on gold surfaces by submerging a noble
metal substrate into a solution of the desired thiol chemical. The strength of
AueS interaction formed between thiols and gold groups depends on the quality
of the thiol compounds, as well as the gold surface properties [308]. The assembly
requires a clean surface, which can be achieved with several washes of the gold
material in acetone, methanol, or piranha solution or with treatments such as
ultraviolet/ozone, electrochemical oxidation, and oxygen plasma [317]. The
covalent binding of the thiol group to gold is widely used for aptamer attachment,
since these moieties can be modified with thiol groups. An aptamer-conjugated
gold nanocarrier that specifically recognized overexpressed breast cancer protein
HER2 was described by Zhu et al. [318]. The synthesized gold nanocarriers were
mixed with 30-thiolated HER2-RNA aptamer obtaining aptamer-conjugated gold
nanocarriers through the formation of AueS bond. The bioconjugated nanocarriers
were combined with an electrochemical immunosensor that was able to differentiate
between HER2-positive and HER2-negative breast cancer cells.
3.3.2.3 Aldehyde Groups
Lysine is a residue that is described as a representative target of aldehydes [319]. The
functionalization of aldehyde groups consists in the utilization of hydrazide
chemistry to immobilize ligands via aldehyde groups on the nanocarrier surface.
However, biomolecules do not possess the aldehyde groups in their structure,
making necessary the incorporation of aldehyde groups via periodate oxidation
[320] or galactose oxidase [321]. The hydrazide-functionalized nanocarriers are
used for coupling several moieties such as proteins, antibodies, and peptides. The
major advantage of this method is the control of ligand modification, although the
conjugation efficiency in this strategy is very poor [295]. A tumor-targeted
3. Targeting 383

multifunctional viral nanocarrier was synthesized based on an efficient hydrazine
reaction. The viral nanocarrier was functionalized to convert exposed lysine residues
to benzaldehyde groups via oxidase, and then VEGF receptor-1 antagonist peptide
was assembled using hydrazide chemistry. The viral nanocarriers demonstrated
ability to recognize vascular endothelial growth factor receptor (VEGFR) on
endothelial cell lines and tumor in mice, thus validating this system as a nanocarrier
platform in vivo [322].
3.3.3 Click Chemistry
“Click chemistry” was first described in 2001 by Kolb’s group as a reaction between
alkyne and azide groups in the presence of copper (Cu(I)) as catalyst, resulting in the
formation of a stable triazole linkage [323] (Fig. 14.5). This chemistry offers the
advantage of a one-step reaction and has been characterized as holding high effi-
ciency, stereospecificity, harmless side products, and compatible with both organic
and aqueous reaction conditions. These features provide highly orientated linkage.
Therefore, click chemistry is suitable for the conjugation of targeting moieties to
the nanocarriers’ surface [324,325]. Polymeric nanocarriers [326], liposomes
[327], and magnetic nanocarrier [328] surfaces have been widely coupled to
different moieties using this chemistry.
Shen and collaborators [329] used the click reaction to conjugate folate onto
superparamagnetic nanocarriers. The authors synthesized Fe3O4eAu nanocarriers
functionalized with azide group on the surface for posterior encapsulation of fluores-
cence dyes. Folate was conjugated with alkyne and then immobilized on the
azide-terminated Fe3O4eAu nanocarrier through Cu(I)-catalyzed “click chemistry.”
Fe3O4eAu-Folate showed ability to target leukemia cell line K562 that was used as a
folate receptor model and demonstrated potential to be useful for MRI of cells [329].
However, Cu(I)-catalyzed alkyne-azide click reactions can result in undesirable
modification or loss of functionality in biological molecules such as proteins or
aptamers, due to the presence of the Cu(I) catalyst. Recently, new developments
in Cu(I)-free “click chemistry” have attracted much attention for application to
biological systems [330,331].
FIGURE 14.5
Copper (Cu(I)) catalyst is employed in bioconjugation strategies where functionalized
nanocarriers containing azide groups on the surface capture alkyne-modified targeting
ligands.

4. FUTURE PERSPECTIVES
As discussed in this chapter, the use of nanocarriers for drug delivery and therapeu-
tics has enormous potential. There are numerous nanocarriers that can be
functionalized with different molecules to increase the treatments’ efficiency. As
described, each of these nanocarriers exhibits advantages or drawbacks depending
on the specific application. However, although several studies highlight
nanocarriers’ versatility and preclinical potential, most of them offer only minor
performance improvements over conventional treatments and very few nanocarriers
progress to clinical trials. Due to these low performance improvements, pharmaceu-
tical companies do not invest in the production of these nanocarriers. There is a need
for more efficient nanocarriers that deliver the drug to a target site at a specific
concentration and period of time. This site-specific delivery of nanocarriers still
remains unrealized. A nanocarrier that proves to be very efficient in in vitro
experiments may not behave that well in vivo. The nanocarrier design needs to
account for all the biological barriers that it crosses after intravenous administration.
Researchers are learning from the failed clinical trials and are applying the lessons
learned to develop a new, more efficient generation of nanomedicines.
LIST OF ABBREVIATIONS
a-Fe2O3 Hermatite
g-Fe2O3 Maghemite
AAV Adeno-associated virus
Al2O3 Alumina
AIDS Acquired immune deficiency syndrome
ASGPR Asialoglycoprotein receptor
Au Gold
Cd Cadmium
CD Cyclodextrin
COOH Carboxylic acids
CNT Carbon nanotube
CPMV Cowpea mosaic virus
Cu(I) Copper
DNA Deoxyribonucleic acid
DOX Doxorubicin
DOX-HA-SPION DOX-hyaluronan-superparamagnetic iron oxide nanocarrier
dsRNA Double-stranded RNA
EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EGF Epidermal growth factor
EGFR EGF receptor
EMA European Medicines Agency
EPR Enhanced permeability and retention
Fab Antigen-binding fragments
FDA Food and Drug Administration
List of Abbreviations 385

Fe3O4 Magnetite
HA Hydroxyapatite
HER2 Human epidermal growth factor receptor 2
HIV Human immunodeficiency virus
Kd Dissociation constant
Lf Lactoferrin
LfR Lf receptor
LCP Lipid-calcium-phosphate
LNA Locked-nucleic-acids
mAb Monoclonal antibody
MPS Mononuclear phagocyte system
MRI Magnetic resonance imaging
mRNA Messenger RNA
MSN Mesoporous silica nanocarrier
MWCNT Multi-walled CNT
NH2 Amine group
NHS N-hydroxysuccinimide
PAMAM Polyamidoamine
PE Polyethylene
PEG PE glycol
PEHAM Polyetherhydroxylamine
PLA Poly(lactic acid)
PLGA Poly(lactic-co-glycolic acid)
PLK1 Polo-like kinase 1
PSMA Prostate-specific membrane antigen
PTX Paclitaxel
PVA Polyvinyl alcohol
PVP Polyvinyl pyrrolidone
QD Quantum dot
RCNMV Red clover necrotic mosaic virus
RCP Riboflavin carrier protein
RES Reticuloendothelial system
RFT Riboflavin transporter
RGD Arginylglycylaspartic acid
RISC RNA-induced silencing complex
S Sulfur
scFv Single-chain variable fragments
Se Selenium
SELEX Systematic evolution of ligands by exponential enrichment
shRNA Short-hairpin RNA
siRNA Small interfering RNA
SiO2 Silica
SPION Superparamagnetic nanocarrier
sulfo-NHS N-hydroxysulfosuccinimide
SWCNT Single-walled CNT
Tf Transferrin
TfR Tf receptor
TiO2 Titanium oxide

TNBC Triple-negative breast cancer
VEGF Vascular endothelial growth factor
VEGFR Vascular endothelial growth factor receptor
ZnO Zinc oxide
ZnS Zinc sulfide
ZrO2 Zirconia
2-AG 2-Arachidonoyglycerol
ACKNOWLEDGMENTS
This study was supported by the Portuguese Foundation for Science and Technology (FCT)
under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020
(POCI-01-0145-FEDER-006684) and under the scope of the Project RECI/BBB-EBI/0179/
2012 (FCOMP-01-0124-FEDER-027462). The authors also acknowledge financial support
from BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European
Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional
do Norte. D. Ferreira is recipient of a fellowship (UMINHO/BD/21/2016) supported by a
doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund
under the scope of Norte2020. J. L. Rodrigues acknowledges the post-doctoral grant
(UMINHO/BPD/37/2015) funded by FCT.
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