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CHAPTER 1
NANOPARTICLES IN TARGETED DRUG DELIVERY
SYSTEMS: SURFACE MODIFICATION AND TOXICITY
1.1 INTRODUCTION
Nanomedicine, the application of nanotechnology in medicine,
aims to overcome problems related to diseases at the nano scale where most
of the biological molecules exist and operate. It is an emerging field with
wide range of applications from diagnosis to therapy, which includes targeted
delivery and regenerative medicine. The role of nanotechnology in cancer is
quite significant, enhancing the earlier crude procedures with modern
diagnosis and therapeutic strategies. Nanoparticles are molecular assemblies
that overcome biological barriers (bio-barriers) through their functional
chemistry, and accumulate preferentially in tumors and specifically target the
single cancer cell for detection and treatment. Cancer nanotechnology is an
interdisciplinary field of research that is based in biology, chemistry,
engineering and medicine, and is aiming at a giant leap in cancer diagnosis
and treatment (Wang and Thanou 2010).
1.2 TARGETED DRUG DELIVERY
Targeted drug delivery is a method of delivering the medicine to a
patient in a manner that increases concentration at the diseased parts of the
body. Targeted drug delivery seeks to concentrate the medication in the
tissues of interest while reducing the relative concentration of the medication
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in the surrounding tissues. This improves the efficacy of the drug while
reducing side effects. Drug targeting delivers drugs exclusively to receptors,
organs, or any other specific part of the body to which one wishes to deliver
them. Multi functionalized single walled carbon nanotubes were used for
targeting biological transporters (Yang et al 2008).
The drug’s therapeutic index, as measured by its pharmacological
response and safety, relies in the access and specific introduction of the drug
with its candidate receptor, whilst minimizing its interaction with non –target
tissue. With desired differential distribution of the drug, its targeted delivery
spares the rest of the body and thus significantly reduces the overall toxicity
to the normal cell, while maintaining the drug’s therapeutic benefits. The
targeted or site-specific delivery of the drug is indeed a very attractive goal
because it may to improve the therapeutic index of the drug (Manish and
Vimukta 2011). A schematic diagram of a targeted drug delivery system is
given in Figure 1.1.
Figure 1.1 Schematic diagram of targeted drug delivery system
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1.2.1 Types of Drug Targeting
Drug targeting may be classified into two general methods:
1) Active targeting
2) Passive targeting
1.2.1.1 Active Targeting
Active targeting refers to the delivery of drugs to a target through
the use of specific interactions at the target site where a drug’s
pharmacological activities are required. These interactions include antigen–
antibody and ligand–receptor binding. Alternatively, physical signals such as
magnetic fields and thermal energy that are externally applied to the target
sites may be utilized for active targeting. Active targeting involves the use of
peripherally conjugated targeting moieties for enhanced delivery of
nanoparticle systems. The targeting moieties are important to the mechanism
of cellular uptake. For example doxorubicin, an anticancer drug, is targeted to
cancer cells by entrapping it in folate conjugated liposomes (Lee and Low
1995).
Long circulation times will allow for effective transport of the
nanoparticles to the tumor site through the enhanced permeability retention
(EPR) effect, and the targeting molecule can increase the endocytosis of these
nanoparticles. The internalization of nanoparticle drug delivery systems has
shown an increased therapeutic effect (Kirpotin et al 2006). If the
nanoparticle attaches to vascular endothelial cells via a non-internalizing
epitope, high local concentrations of the drug will be available on the outer
surface of the target cell. Although this has a higher efficiency than free drug
released into circulation, only a fraction of the released drug will be delivered
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to the target cell. In most cases, internalization of the nanoparticle is
important for effective delivery of some anticancer drugs, especially in gene
delivery, gene silencing, and other biotherapeutics (Atobe et al 2007). The
schematic representation of active targeting is given in Figure 1.2.
Figure 1.2 Schematic representation of active targeting
1.2.1.2 Passive targeting
Passive targeting is defined as a method whereby the physical and
chemical properties of carrier systems increase the target/non-target ratio of
the quantity of the drug delivered by adjusting these properties to the
physiological and the histological characteristics of the target and non-target
tissues, organs, and cells. Carriers included in this category are synthetic
polymers, some natural polymers such as albumin, liposomes, micro (or nano)
particles, and polymeric micelles. Influential characteristics of passive
targeting are (1) chemical factors such as hydrophilicity/hydrophobicity and
positive/negative charge and (2) physical factors such as size and mass.
Passive targeting can be achieved by minimizing both nonspecific interactions
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and delivery with/to non-target organs, tissues,
and cells, as well as through the maximization of delivery to the target
(Yokoyama 2005). The schematic representation of passive targeting is given
in Figure 1.3.
Figure 1.3 Schematic representation of passive targeting
1.3 NANOPARTICLES IN TARGETED DRUG DELIVERY
1.3.1 Inorganic Nanoparticles
Ceramic nanoparticles are typically composed of inorganic
compounds like silica or alumina. However, the nanoparticle core is not
limited to just these two materials; rather, metals, metal oxides and metal
sulphides can be used to produce a myriad of nanostructures with varying
size, shape, and porosity. Generally, inorganic nanoparticles may be
engineered to evade the reticuloendothelial system by varying size and
surface composition. Moreover, nanocarriers may be porous, and provide a
physical encasement a molecular payload (drug) from degradation or
denaturization. Several functional groups can be introduced onto the surface
of inorganic nanoparticles, ranging from saturated and unsaturated
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hydrocarbons to carboxylic acids, thiols, amines, and alcohols. Inorganic
nanoparticles are relatively stable over broad ranges of temperature and pH,
yet their lack of biodegradation and slow dissolution raises safety questions,
especially for long- term administration.
1.3.2 Polymeric Nanoparticles
Polymeric nanoparticles are biodegradable and biocompatible, and
have been adopted as a preferred method for nanomaterial drug delivery.
They also exhibit a good potential for surface modification via chemical
transformations, provide excellent pharmacokinetic control, and are suitable
for the entrapment and delivery of a wide range of therapeutic agents.
Pertinent nanoparticle formulations include those made from gelatins,
chitosan, poly(lactic-co-glycolic acid) copolymer, polylactic acid,
polyglycolic acid, polyalkylcyanoacrylate, polymethylmethacrylate and
poly(butyl)cyanoacrylate. Furthermore, polymer-based coatings may be
functionalized onto other types of nanoparticles to change and improve their
biodistribution properties. Poorly soluble drugs are encapsulated in a polymer
matrix using layer-by-layer technology and sonication (Agarwal et al 2008).
The biologically inert polymer, polyethylene glycol (PEG) has been
covalently linked onto the surface of nanoparticles. This polymeric coating is
thought to reduce immunogenicity and limit the phagocytosis of nanoparticles
by the reticuloendothelial system (RES), resulting in increased blood levels of
drug.
1.3.3 Liposomes
Liposomes are concentric bilayered vesicles surrounded by a
phospholipid membrane. They are related to micelles which are generally
composed of a monolayer of lipids. The amphiphilic nature of liposomes,
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their ease of surface- modification, and a good biocompatibility profile make
them an appealing solution for increasing the circulating half-life of proteins
and peptides. They may contain hydrophilic compounds, which remain
encapsulated in the aqueous interior, or hydrophobic compounds, which may
escape encapsulation through diffusion out of the phospholipid membrane.
Liposomes can be designed to adhere to cellular membranes to deliver a drug
payload or simply transfer drugs following endocytosis.
1.3.4 Solid Lipid Nanoparticles
Solid lipid nanoparticles are lipid-based submicron colloidal
carriers. They are more stable than liposomes in biological systems due to
their relatively rigid core consisting of hydrophobic lipids that are solid at
room and body temperatures, surrounded by a monolayer of phospholipids.
These aggregates are further stabilized by the inclusion of high levels of
surfactants. Because of their ease of bio-degradation, they are less toxic than
polymer or ceramic nanoparticles. They have controllable pharmacokinetic
parameters and can be engineered with three types of hydrophobic core
designs: a homogenous matrix, a drug-enriched shell or a drug-enriched core.
1.3.5 Nanocrystals
Nanocrystals are aggregates of molecules that can be combined into
a crystalline form of the drug surrounded by a thin coating of surfactant. They
have extensive uses in materials research, chemical engineering, and as
quantum dots for biological imaging, but fewer uses in nanomedicine for drug
delivery. Magnetic nanoparticles are being used for the targeted drug delivery
of paclitaxel for prostate cancer (Huaa et al 2010). Folate targeted rare earth
nanocrystals were used for the imaging of cancer cells (Setua et al 2010).
Quantum dots were also used for tumor targeting and imaging (Pana and Feng
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2009). Superparamagnetic iron oxide nanoparticles are used for dual drug
targeting (Dilnawaz et al 2010). A nanocrystalline species may be prepared
from a hydrophobic compound and coated with a thin hydrophilic layer. The
biological reaction to nanocrystals depends strongly on the chemical nature of
this hydrophilic coating. The hydrophilic layer aids in the biological
distribution and bioavailability, and it prevents aggregation of the crystalline
drug material. These factors combine to increase the efficiency of overall drug
delivery. High dosages can be achieved with this formulation and poorly
soluble drugs can be formulated to increase bioavailability via treatment with
an appropriate coating layer. Both oral and parenteral deliveries are possible
and the limited carrier, consisting primarily of a thin coating of surfactant,
may reduce potential toxicity (Faraji and Wipf 2009).
1.4 SURFACE- MODIFICATION OF NANOPARTICLES
The association of a drug to conventional carriers leads to
modification of the drug biodistribution profile, as it is mainly delivered to the
mononuclear phagocyte system (MPS) such as liver, spleen, lungs and bone
marrow. Nanoparticles can be recognized by the host immune system when
intravenously administered and cleared by phagocytes from the circulation
(Muller et al 1996). Apart from the size of nanoparticles, nanoparticle
hydrophobicity determines the level of blood components (e.g., opsonins) that
bind this surface. Hence, hydrophobicity influences the in-vivo fate of
nanoparticles (Brigger et al 2002).
Indeed, once in the blood stream, surface non-modified
nanoparticles (conventional nanoparticles) are rapidly opsonized and
massively cleared by the MPS. To increase the likelihood of success in drug
targeting, it is necessary to minimize the opsonization and prolong the
circulation of nanoparticles in in-vivo. This can be achieved by coating
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nanoparticles with hydrophilic polymers/surfactants or by formulating
nanoparticles with biodegradable copolymers that have hydrophilic
characteristics, e.g., polyethylene glycol, polyethylene oxide, poloxamine, and
polysorbate 80 (Tween 80). Studies show that polyethylene glycom (PEG) on
nanoparticle surfaces prevents opsonization by complement and other serum
factors. PEG molecules with brush-like and intermediate configurations
reduce phagocytosis and complement activation, whereas surfaces comprised
of PEG with mushroom-like structures are potent complement activators and
favored phagocytosis (Bhadra et al 2002). The schematic representation of
PEG coated nanoparticle is given in Figure 1.4.
Figure 1.4 Schematic representation of PEG coated nanoparticles
Prolonged circulation can help to achieve a better effect for targeted
(specific ligand-modified) drugs and drug carriers, allowing more time for
their interaction with the target because of the increased number of passages
through it with the blood. Chemical modification of pharmaceutical
nanocarriers with PEG is the approach most frequently used to impart in-vivo
longevity to drug carriers (Torchilin 1996). PEGylation of hyaluronic acid
nanoparticles have improved the tumor targatability (Choi et al 2011). Lipka
et al (2010) have shown increased blood circulation time for PEG modified
gold nanoparticles. The term “steric stabilization” has been introduced to
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describe the phenomenon of polymer-mediated protection. The layer-by-layer
method is used for surface- modification to prepare biofunctional
nanoparticles (Labouta and Schneider 2010). Multifunctional nanocarriers,
combining several useful properties in one nanoparticle, can significantly
improve the efficiency of the therapeutics (Torchilin 2006). On the biological
level, coating nanoparticles with PEG sterically hinders interactions of blood
components with their surface and reduces the binding of plasma proteins
with the PEGylated nanoparticles (Senior et al 1991). This approach prevents
drug carrier interaction with opsonins and slows down their capture by the
RES (Senior 1987). The mechanisms by which PEG prevents opsonization
include shielding of the surface charge, increased surface hydrophilicity,
enhanced repulsive interaction between polymer-coated nanocarriers and
blood components, and the formation of the polymeric layer over the particle
surface, which is impermeable for large molecules of opsonins even at
relatively low polymer concentrations (Gabizon and Papahadjopoulos 1992).
As a protecting polymer, PEG provides a very attractive combination of
properties: excellent solubility in aqueous solutions; high flexibility of its
polymer chain; very low toxicity, immunogenicity and antigenicity; lack of
accumulation in RES cells; and minimal influence on specific biological
properties of modified pharmaceuticals (Pang 1993). PEG molecules with a
molecular weight below 40 kDa are readily excretable from the body via the
kidneys (Veronese 2001).
1.5 TUMOR-TARGETED SPECIFIC LIGANDS ON
LONG-CIRCULATING NANOCARRIERS
Targeting ligands were attached to nanocarriers to achieve better
selective targeting by PEG-coated nanoparticles via the PEG spacer arm, so
that the ligand extends outside of the dense PEG brush, excluding steric
hindrances for its binding to the target receptors. With this in mind, potential
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ligands such as transferrin, folic acid (FA), lectins, epidermal growth factor
(EGF), antibody were attached (Torchilin et al 2001, Das et al 2009). One
challenge of targeting cancers and tumors is that the defective cells are often
very similar in characteristics to their surrounding healthy tissue. To
differentiate such cells, the ligands can be designed to have specificity for
receptors that are overexpressed on cancerous cells, but are normally or
minimally expressed on normal, healthy cells. These molecules should have
high affinity to their cognate receptors, plus have innate abilities to induce
receptor-mediated endocytosis. The targeting layer poses as the outmost
exterior of the nanoparticle delivery system, where targeting ligands are
generally presented on top of the stealth layer. Structures such as antibodies,
antibody fragments, proteins, small molecules, aptamers and peptides have all
demonstrated abilities to induce nanoparticle-targeting to cancer cells
(Wang et al 2008). Antibodies against the HER-2 receptor, the transferrin
receptor (TfR) and the prostate specific antigen receptor are all common
examples of receptor targets, due to their over-expression of such receptors on
cancer cells (Wagner et al 1994).
Folate, one of a number of targeting ligands, has many unique
advantages such as presumed lack of immunogenicity, unlimited availability,
functional stability, defined conjugation chemistry and a favourable non-
destructive cellular internalization pathway (Brzezinska et al 2000). It was
also reported that the folate receptor (FR), known as the high affinity
membrane folate- binding protein, binds to folic acid (an oxidized form of
folate) with high affinity (Kd~10−10 M) (Sudimack and Lee 2000). While
elevated expression of FR has frequently been observed in various types of
human cancers, the receptor is generally absent in most normal tissues (Zhao
and Lee 2004, Weitman et al 1992). The selective amplification of FR
expression among human malignancies suggests its potential utility as a
cellular marker that can be exploited in targeted drug and gene delivery
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(Sirotnak and Tolner 1999). Folate-PEG-coated liposomes were used for
tumor targeted drug delivery (Wang et al 2010). Therefore, several studies
recently have reported that folate itself has a great potential as a targeting
moiety for the FR instead of monoclonal antibodies against the FR
(Oh et al 2006). Figure 1.5 gives the schematic representation of folate
receptor on target cancer cell.
Figure 1.5 Schematic representation of folate receptor on target cancer
cell
1.5.1 Folate Conjugate Uptake Via Receptor-Mediated Endocytosis
The mechanism of FR transport of FA into cells is clear, in that
folate conjugates are taken up nondestructively by mammalian cells via
receptor-mediated endocytosis. Figure 1.6 shows the schematic representation
of FR-mediated endocytosis. After binding to FR on the cancer cell surface,
folate conjugates, regardless of size, are seen to internalize and traffick to
intracellular compartments called endosomes. Intracellular transport of
protein is facilitated through folate receptor-mediated endocytosis (Zheng et
al 2010). Folate conjugate containing endosomes has been shown to have pH
values between 4.3 and 6.9 (most frequently, pH 5.0) due to a process called
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endosome acidification. Since the binding of FA to its receptor is pH
dependent, decreasing dramatically at pH values < 5, it can be anticipated that
some of the folate conjugates will dissociate from their receptors and remain
inside the cell. Direct measurements of the efficiency of folate conjugate
unloading reveal that only 15 to 25% of the receptor bound conjugates are
released inside the cell, while the remainder apparently recycle back to the
cell surface attached to FR (Lu and Low 2002).
Figure 1.6 Schematic representation of FR-mediated endocytosis
1.6 NANOTOXICOLOGY
The immune system serves as our primary defence against foreign
invasion. Antigen-presenting dendritic cells, macrophages and other
phagocytic cells are equipped with specialized machineries to recognize and
respond to foreign stimuli including particles. Therefore, nano–immuno
interactions are important to consider when engineered nanomaterials are
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devised for in vivo administration (Dobrovolskaia and McNeil 2007).
Cellular uptake may occur through several different pathways, depending on
the properties of the nanoparticles (such as primary particle size, shape,
surface charge, etc), but also on the specific cell type in question: for instance,
macrophages in the lung do not necessarily utilize the same repertoire of
recognition molecules as macrophages in the bone marrow or peritoneum.
This is also relevant for the biodistribution of nanomaterials. Biodistribution
of gold nanoparticles is found to be dependent on particle size and surface
charge. Understanding the mechanism of uptake and the subsequent
biodistribution of nanomaterials is not only important for our understanding
of potential adverse effects, but will also enable the optimization of
nanoparticle design for future biomedical applications.
The interaction between cells and nanoparticles is influenced by
plasma proteins, which have been shown to coat nanoparticles instantly once
they get in contact with plasma (Nel et al 2009). Biodegradation of particles is
another important factor that has to be considered. Adverse effects may occur
when particles are not biodegraded or readily eliminated (excreted) from the
body, and long-term in-vivo studies in model organisms are needed to address
the consequences of the accumulation in different organs and tissues of
administered nanoparticles.
Surface- modification is an important aspect of nanoparticle design
for biomedical applications. Modulation of nanoparticle surfaces can
influence particle uptake, biological responses, and biodistribution (Jiang et al
2008). After every modification performed on nanoparticles, the
biocompatibility of the particles has to be assessed. Surface functionalization
can be utilized to increase circulation time in blood, reduce non-specific
distribution or specific targeting of tissues or cells by using a targeting ligand
(Shubayev et al 2009).
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Many aspects of nanoparticle architecture and composition influence
systemic toxicity. Care must be taken regarding the relative size difference
between nanoparticles and the vasculature diameter. Particles >5 µm diameter
may embolize these vessels. Moreover, <100 nm particles have a high
likelihood of aggregating; thus forming a cluster that can embolize and
occlude blood flow. In fact, this property has been used to intentionally
occlude the vasculature of tumors in the clinical setting, such as with the
transarterial chemoembolization of hepatocellular carcinoma and other meta-
static neuroendocrine tumors of the gastrointestinal tract. Alternatively,
undesired consequences may also result, including lodging of these
aggregates in various organs. For example, intravenous administration of
nanoparticles prone to aggregation can result in a pulmonary embolism,
strokes, myocardial infarctions and other micro infarctions at distant sites and
organs. Particles up to 4–5 µm in size could be injected directly into the
carotid arteries of mice without producing detectable problems, with a caveat
that very large quantities were not tested. Thus, nanoparticle administration
should result in no adverse embolic phenomena, provided the nanoparticles
do not aggregate (Kohane et al 2002).
1.7 ANTITUMOR ACTIVITY
In the continuing search for effective treatments for cancer, an
emerging paradigm is the use of nanotechnology to uncap the full potential of
existing chemotherapy agents (Ferrari 2005). Midkine-antisense
oligonucleotide-loaded nanoparticles have been shown to supress
hepatocellular carcinoma growth (Dai et al 2009). Integral physicochemical
properties of nanovectors can be modulated to improve the antitumor efficacy
of chemotherapeutic agents (Moghimi et al 2001). For example, the shape and
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size of nanostructures can play a deterministic role in the biological outcome
(Decuzzi et al 2009, Chaudhuri et al 2010).
Surface-modifications to increase hydrophilicity can mask the
nanovectors from the reticuloendothelial system, thereby increasing
circulation time and altering the pharmacokinetics of the active agents
(Moghimi et al 2001). Such nanovectors accumulate preferentially in the
tumors due to the unique leaky tumor vasculature coupled with impaired
intratumoral lymphatic drainage, which contributes to an enhanced
permeation and retention (EPR) effect (Yuan et al 1995). Indeed, nanovectors
were shown to deliver between 5–11 X more doxorubicin to Kaposi sarcoma
lesions than to normal skin (Northfelt et al 1996). Cisplatin loaded
PLGA-PEG nanoparticles are well tolerated and have a higher anticancer
activity compared to pure cisplatin (Mattheolabakis et al 2009). Similarly, the
tumor paclitaxel concentration-time area under the curve was found to be
33% higher when administered as an albumin-paclitaxel nanoparticle, and is
currently approved for use in metastatic breast cancer (Desai et al 2006).
Paclitaxel-loaded nanoparticles show drastically enhanced cytotoxicity
compared to pure paclitaxel (Li et al 2009).
Most nanoparticles are expected to accumulate in tumours due to
the EPR effect. Nanoparticle tumour-accumulation is deemed possible due to
the highly permeable blood vessels of the tumours as a result of rapid and
defected angiogenesis. Cisplatin loaded gelatin-polyacrylic acid nanoparticles
accumulate well within the tumor due to EPR effect (Ding et al 2011, Desai
2012). In addition tumors are characterised by dysfunctional lymphatic
drainage that helps the retention of nanoparticles in the tumour long enough to
allow local nanoparticle disintegration and release of the drug in the vicinity
of tumor cells. The phenomenon has been used widely to explain the
efficiency of nanoparticle and macromolecular drug accumulation in tumors
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(Wang and Thanou 2010). Figure 1.7 gives the schematic representation of
EPR effect (Yokoyama 2005).
Figure 1.7 Schematic representation of EPR effect
1.8 SCOPE OF THE PRESENT INVESTIGATIONS
The thesis mainly focuses on the application of hydroxyapatite and
titanium dioxide nanoparticles for targeted drug delivery application. Detailed
investigations were carried out on the size dependent cytotoxicity of
hydroxyapatite and titanium dioxide nanoparticles on the human hepato
carcinoma cells. Hydroxyapatite–alginate nanocomposites were studied for
their sustained drug delivery application. Surface- modification was done for
hydroxyapatite and titanium dioxide nanoparticles with PEG and FA.
Paclitaxel, an anticancer drug, was attached, and its drug release profile was
studied. Acute and sub-chronic toxicity analysis was done to find the toxicity
of surface-modified paclitaxel-attached hydroxyapatite and titanium dioxide
nanoparticles. In vivo anticancer activity was done to evaluate the anticancer
property of surface-modified paclitaxel attached hydroxyapatite and titanium
dioxide nanoparticles.