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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.