CHAPTER - I INTRODUCTION - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5358/10/10_chapter...

Chapter 1 Introduction

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Enhanced Antitumour Effect of Camptothecin Loaded in Long Circulating Polymeric Micelles

CHAPTER - I

INTRODUCTION

1.1 CANCER THERAPY

For over a half century extensive research has been undertaken for the

control of cancer. However, success has been limited to certain malignancies and

surgical intervention is potentially curative for early stage patients. For the

majority of patients with advanced stage of cancer, the treatment is limited to

chemotherapy or radiation. Chemotherapy in particular has limitations due to lack

of selectivity with severe toxicity. Under these circumstances tumour targeted

delivery of anticancer drug is perhaps one of the most important steps for cancer

chemotherapy (Leaf et al., 2004).

Conventional cancer therapy and diagnosis involves the application of

catheters, surgery, biopsy, chemotherapy and radiation. Most anticancer agents

do not greatly differentiate between cancerous and normal cells. Consequently

the systemic application of these drugs often causes severe side effects in other

tissues, which greatly limits the maximal allowable dose of the drug. In addition,

rapid elimination and widespread distribution into non targeted organ and tissues

requires the administration of a drug in large quantities, which is uneconomical

and is often complicated because of non specific toxicity (Matsumura et al.,

2007).

1.2 DRUG DELIVERY SYSTEMS

Drug delivery remains a challenge in management of cancer.

Approximately 12.5 million new cases of cancer are being diagnosed worldwide

each year and considerable research is in progress for drug discovery for cancer.

The newer approaches to cancer treatment not only supplement the conventional

chemotherapy and radiotherapy but also prevent damage to normal tissues and

prevent drug resistance.


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Drug delivery systems (DDS) appeared to be a promising and reliable

approach to deliver potent drugs to the site of action precisely and timely.

Preclinical and clinical studies constantly showed that DDS using natural and

artificial macromolecules resulted in successful cancer therapy with reduced

toxicity and improved efficacy. A mechanism of tumour specific delivery is

explained by characteristic tumour microenvironments. Tumour tissues are

characterized with leaky blood vessels and the premature lymphatic drainage.

Macromolecules owing to the high molecular weight and large hydrodynamic radii

circulate in the blood for a longer period of time than small molecules. These

macromolecules have been observed to preferentially accumulate in the tumour

tissue and retain within the tissues for a prolonged period of time. This

phenomenon is referred to as the enhanced permeability and retention effects,

providing a key rational of DDS using macromolecular drug carriers

(Vincent et al., 2006).

Several innovative methods of drug delivery are used in cancer. These

include water soluble polymers, dendrimers, polymeric micelles and liposomes.

Each carrier has advantageous feature to provide structural flexibility, multiple

functional moieties, sequestered nano depot and robust stability respectively.

They may be injected into the arterial circulation and guided to the tumour by

magnetic field for targeted drug delivery. Polyethylene glycol (PEG) technology

has been used to overcome some of the barriers to anticancer drug delivery.

Encapsulating anticancer drugs in liposomes enables targeted drug delivery to

tumour tissues and prevents damage to the normal surrounding tissues.

Monoclonal antibodies can be used for the delivery of anticancer payloads such

as radio nucleotides, toxins and chemotherapeutic agents to the tumours.


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1.3 NOVEL DRUG DELIVERY SYSTEM (NDDS)

During the last two decades, considerable attention has been given to the

development of novel drug delivery system. The rationale for controlled drug

delivery is to alter the pharmacokinetics and pharmacodynamics of drug

substances in order to improve the therapeutic efficacy and safety. Besides more

traditional matrix or reservoir drug delivery systems, colloidal drug delivery

systems have gained in popularity. The major colloidal drug delivery systems

investigated include liposomes and polymeric nanoparticles. These systems have

been investigated primarily for site specific drug delivery, for controlled drug

delivery and also for the enhancement of the dissolution rate / bioavailability of

poorly water soluble drugs (Davis et al., 2008).

The goal of all sophisticated drug delivery systems, therefore, is to deploy

medications intact to specifically targeted parts of the body through a medium

that can control the therapy’s administration by means of either a physiological or

chemical trigger. To achieve this goal, researchers are turning to advances in the

worlds of micro and nanotechnology. During the past decade, polymeric

microspheres, polymer micelles and hydrogel-type materials have all been shown

to be effective in enhancing drug targeting specificity, lowering systemic drug

toxicity, improving treatment absorption rates and providing protection for

pharmaceuticals against biochemical degradation. In addition, several other

experimental drug delivery systems have shown exciting signs of promise,

including those composed of biodegradable polymers, dendrimers (so-called star

polymers), electro active polymers and modified C-60 fullerenes (also known as

“buckyballs”).

Among these carriers only the polymeric micelles undergo dynamic

physicochemical changes during the drug entrapment and release in terms of

molecular assembly and dissociation between block copolymer components. The

polymeric micelles are spiral supramolecular nano assemblies prepared from

self – assembling amphiphilic block copolymers. They feature a sub-100 nm


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core-shell structure, which provides a nano depot for hydrophobic drugs

enveloped with a hydrophilic shell, improving drug solubility. The hydrophilic shell

suppressing protein adsorption allows the polymeric micelles to avoid foreign

body reaction, while improving drug solubility. The property is called stealth

functionality. Because of their characteristic structure and stealth functionality, the

polymeric micelles can stably transport bioactive molecules to the tumour tissues,

suppressing the immune response and non specific drug distribution to the

normal tissues.

1.4 ENHANCED PERMEATION AND RETENTION EFFECT (EPR)

A critical advantage in treating cancer with advanced, non solution based

therapies is the inherent leaky vasculature present in the cancerous tissues. The

defective vascular architecture, created due to the rapid vascularization

necessary to serve fast growing cancers, coupled with poor lymphatic drainage

allows EPR. The ability to target very specific cancer cells also uses a cancers

own structure, in that many cancers over express particular antigens even on

their surface. This makes them ideal targets for drug delivery as long as the

targets for a particular cell type can be identified with confidence and are not

expressed in significant quantities anywhere else in the body (Greish et al.,

2006).

It has been demonstrated that long-circulating polymeric carriers can

preferentially and effectively accumulate in solid tumours. This phenomenon is

explained by the micro vascular hyper permeability to circulating macromolecules

and their impaired lymphatic drainage in solid tumours, which is termed as EPR

effect. Such tumour vascular hyper permeability has been suggested to be due to

over expression of vascular permeability factor (VPF)/vascular endothelial growth

factor (VEGF), as well as secretion of other factors, such as the basic fibroblast

growth factor (BFGF).


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Fig 1: Enhanced Permeation and Retention Effect (EPR)

1.5 ACHIEVING TARGETING BY AVOIDING RETICULO ENDOTHELIAL

SYSTEM (RES)

A major limiting factor to the systemic use of particulate delivery system is

the rapid clearance of carrier from the blood circulation by RES. Following

intravenous administration, the colloidal carriers first come into contact with

plasma/serum protein before they reach the target cells. The opsonins adsorb on

to the surface of the colloidal carriers and render particles recognizable and more

palatable to RES.

Particles with longer circulation times and hence greater ability to target

the site of interest should be 100 – 200 nm in diameter and have a hydrophilic

surface in order to reduce clearance by macrophages. Coating of hydrophilic

polymers can create a cloud of chains at the particle surface which will repel

plasma proteins and work in this area began by adsorbing surfactants to the

nanoparticle surface.


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1.6 DRUG CARRIERS

The requirements of pharmaceutical drug carriers for i.v. administration

include small size, biodegradability and good loading capacity, high content of the

drug in a final preparation, prolonged circulation and ability to accumulate in

required areas. These requirements are reasonably well met by some drug

carriers (microcapsules, liposomes) used predominantly for water-soluble drugs.

Although liposomes can entrap poorly soluble drugs in the hydrophobic

bilayer, their loading capacity is limited because of possible membrane

destabilization. Thus, the development of drug carriers displaying all of the named

properties specifically for the delivery of poorly soluble pharmaceuticals continues

to represent a challenge.

Low solubility in water tends to be an intrinsic property of many drugs,

including anticancer agents, which often represent polycyclic compounds. The

membrane permeability and efficacy of such drugs increases with increasing

hydrophobicity. On the other hand, parenteral administration of those intrinsically

hydrophobic agents is associated with some problems.

Thus, i.v. administration of aggregates formed by undissolved drug in

aqueous media can cause embolization of blood capillaries (≤5 µm) before drug

penetrates a tumour. Additionally, low solubility of hydrophobic drugs in

combination with excretion and metabolic degradation hinders the maintenance of

therapeutically significant systemic concentrations.

To increase their bioavailability, poorly soluble pharmaceuticals can be

solubilzed by various surfactants. Polymeric micelles demonstrate a series of

attractive properties as drug carriers, such as high stability both in vitro and

in vivo and good biocompatibility.


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1.7 BLOCK COPOLYMERS

Block copolymers are defined as polymers that have two or more blocks

or segments arranging in the main chain and can be classified according to their

architecture as AB-type di block, ABA or BAB-type tri block and multi block,

where A represents the soluble block in a selected solvent and B designates the

insoluble block.

Among block copolymers, linear amphiphilic block copolymers play an

essential role in carrying drugs on a nanoscale level. Amphiphilic block polymers

specifically refer to those having both hydrophilic and hydrophobic blocks in the

same polymer chain, which can then build spherical polymeric assemblies in

aqueous solution called “polymeric micelles” with nanosized and core shell

segregated domains. A drug was conjugated to one segment of the block

polymer to form the core and the other segment remained unmodified as a water

soluble shell.

At present, micelles formed by various amphiphilic block copolymers are

being developed for delivering anticancer, anti-inflammatory, antiviral,

antibacterial, imaging agents and DNA. Overall, block copolymer based drug

delivery systems have been successfully used to target drugs to specific

physiological sites (organs, tissues or cells), solubilize hydrophobic drugs,

increase drug stability and control drug release, realizing the efficiency maximum

and toxicity minimum of drug.

Amphiphilic poly caprolactone - polyethylene glycol (PCL-PEG) block

copolymers form micelles composed of a hydrophobic core and hydrophilic PEG

shell in water. Hydrophobic blocks are segregated from the aqueous exterior to

form an inner core surrounded by a palisade of hydrophilic segments. Block

copolymer micelles are water soluble, biocompatible nano carriers in the size of

10- 200 nm with proven efficacy of delivering hydrophobic drugs.


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The ability of polymeric micelles to target certain cells can also lower the

required dosage. The size and morphology of block copolymer micelles can be

easily changed by adjusting the chemical composition, total molecular weight and

ratio of the block lengths. Various hydrophobic drugs have been incorporated into

the hydrophobic inner core of micelles. These polymers are all strongly

hydrophobic and this has caused some limitations in practical drug formulations.

To add hydrophilic and other physico chemical properties, PEG has been

incorporated into the biodegradable polyesters. PEG is a non toxic, water soluble

polymer with proven biocompatibility. Block co polymers consisting of a

hydrophobic polyester segment and a hydrophilic PEG segment have attracted

large attention due to their biocompatibility and biodegradability. PEG

modification is often referred to as PEGylation, a term that implies the covalent

binding or non-covalent entrapment or adsorption of PEG onto an object.

1.8 POLYMERIC MICELLES – NOVEL FAMILY OF PHARMACEUTICAL

CARRIERS

To minimize premature drug degradation upon administration, prevent

undesirable side effects to normal cells, organs and tissues by cytotoxic drugs, to

increase drug bioavailability and the fraction of the drug accumulated in the

pathological area, various drug delivery and drug targeting system, such as

synthetic polymers, microcapsules, cell ghosts, lipoproteins, liposomes, micelles,

niosomes, lipid particles and many others are currently applied or under

development. To still further increase their performance, all these drug carriers

can be made slowly biodegradable, stimuli-reactive (pH or temperature-sensitive)

and targeted (by conjugating them with lig ands specific towards certain

characteristics components of the pathological area). In addition, drug carriers

should stay in the blood long enough, since prolonged circulation allows for

maintaining the required therapeutic level of pharmaceuticals in the blood for

extended time intervals. In addition to that, long circulating molecular weight

drugs or drug-containing micro particulates can also slowly accumulate in


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pathological sites with affected and leaky vasculature (such as tumours,

inflammation, infarcted area) via the EPR effect and enhance drug delivery in

these areas. The prolonged circulation also allows for achieving a better targeting

effect for specific lig and modified drugs and drug carriers since it increases the

total quality of targeting drug/ carrier passing through the target and the number

of interaction between targeted drugs and their targets, which is especially

important for a successful targeting of pathological areas with diminished blood

supply and/or with low concentration of targeted component (Yokoyama et al.,

1998).

The development of biocompatible and biodegradable drug carriers

processing small particles size, high loading capacity, extended circulation time

and ability to accumulate in required pathological sites in the body, for the

delivery of poorly soluble pharmaceuticals still has many unresolved issues. The

availability of such carriers is especially important since the therapeutic

application of hydrophobic, poorly water soluble agents is associated with some

serious problems. First, low water solubility results in poor absorption and low

bioavailability, especially upon the oral administration. Second, the aggregation of

poorly soluble drugs upon intravenous administration might lead to various

complications including embolism resulting in side effects as severe as

respiratory system failure and can also lead to high local drug concentration at

the sites of aggregate deposition, which could be associated with local toxic

effects of the drug and its diminished systemic bioavailability.

The use of micelles prepared from amphiphilic copolymers for

solubilization of poorly soluble drugs has attracted much attention recently.

Polymeric micelles are formed by block copolymers consisting of hydrophilic and

hydrophobic monomer units with a length of a hydrophilic block exceeding to

some extent that of a hydrophobic one. If the length of hydrophilic block is too

high copolymers exist in water as unimers (individual molecules) while molecules

with very long hydrophobic block forms structure with non micellar morphology,


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such as rods and lamellae. The major driving force behind self-association of

amphiphilic polymers is again the decrease of free energy of the system due to

removal of hydrophobic fragments from the aqueous surrounding with the

formation of micelle core stabilized with hydrophilic blocks exposed into water

(G.S.Kwon et al., 1999).

Polymeric micelles are nanoscopic core/shell structures usually formed

through the self-assembly of amphiphilic block copolymers (ABCs). ABC micelles

have a hydrophobic core surrounded by a hydrophilic outer shell. The inner core

can be used as a storage site for poorly water-soluble drugs and can act as a

nano-depot for these agents. This drug-loaded inner core is protected by a

biocompatible hydrophilic outer shell. Furthermore, heterogeneous functionalities

can be introduced in each domain to facilitate drug loading and targeting. Over

the past few years, ABC micelles have been used as drug carriers for poorly

water-soluble drugs that result in improved pharmacokinetics (PK) of drugs

(Ho-Chul Shin et al., 2009). Polymeric micelles are prepared from block

copolymers possessing both hydrophilic and hydrophobic chains and they have

received much attention in drug delivery research. Their innate characteristics for

drug targeting include solubilization of hydrophobic molecules, small particle size,

high structural stability, extended drug release and prevention of rapid clearance

by the reticuloendothelial system (Kumi Kawano et al., 2006).

One promising application of this type of aggregates is for drug delivery of

anti cancer drugs. Usually, hydrophobic drugs can be loaded in the cores of the

micelles to lower its toxicity in human body and to prolong their circulation time in

blood. In addition, the nanostructure of micelles may help the aggregates

penetrate through cell membrane to deliver drug at sub cellular level

(Jiaping Lin et al., 2009).


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1.9 MICELLES AS DRUG CARRIERS ARE ABLE TO PROVIDE A SERIES

OF UNBEATABLE ADVANTAGES

a) They can solubilize poorly soluble drugs by hydrophobic core resulting in

the increase of drug stability and bioavailability.

b) They can stay in the body long enough providing gradual accumulation in

the required area. Their size permits them to accumulate in body regions

with leaky vasculature and they can be targeted by attachment of a

specific lig and to the outer surface.

c) The drugs loaded in the micelles can be well protected from possible

inactivation under the effect of biological surroundings and their

bioavailability was usually increased.

1.10 REASONS FOR SELECTING POLYMERIC MICELLES

Core\shell structures account for their qualities as efficient drug delivery

systems.

Core provides a reservoir where hydrophobic drugs can be dissolved and

the corona confers hydrophilicity to the overall system.

Sequestration of anticancer drugs in the inner core can protect them from

premature degradation and allow their accumulation at tumoural sites.

Amphiphilic block copolymers comprising of hydrophilic and hydrophobic

segments are known to form self-assemblies such as lamellas, vesicles

and micelles. The balance between hydrophilic and hydrophobic

segments determines the structures of these self assemblies. Among

them, polymeric micelles have drawn significant attentions in the field of

drug delivery science in that:

The polymeric micelles have clinically relevant particle size for

systemic drug delivery.

They have excellent stability in low concentrations.


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The hydrophobic core of the polymeric micelles provides a nano

depot for hydrophobic drugs sequestered from the aqueous

solution.

The surface of the polymeric micelles can be used for the

installation of targeting molecules achieving active cancer

targeting and multiple drugs can be incorporated in the same

micelles concurrently for effective combination therapy.

1.11 COMPOSITION OF POLYMERIC MICELLES

Usually, amphiphilic micelle forming unimers include poly

(ethylene glycol) blocks with a molecular weight from 1-15 kDa as hydrophilic

corona forming blocks. This polymer is inexpensive, has low toxicity, serves as an

efficient steric protector of various biologically active macromolecules and

particulate delivery systems and has been approved for internal applications by

regularity agencies. Still, some other hydrophilic polymers may be used

as hydrophilic blocks. Among possible alternatives to PEG, poly

(N-vinyl-2-pyrrolidone) is frequently considered as a primary alternative to PEG.

Similar to PEG this polymer is highly biocompatible and was used in formulations

of such particulate drug carriers as liposomes, nanoparitcles, microspheres and

diblock polymer micelle. Another hydrophilic c andidate is poly (vinyl alcohol) and

poly (vinyl alcohol- co-vinyl oleate) co-polymer was used to prepare micelles

enhancing transcutaneous permeations of retinyl palmitate. Poly vinyl alcohol

substituted with oleic acid was also used for carrying lipophilic drug

(V.P.Torchillin et al., 2006).

1.12 POLYMERIC MICELLE: STRUCTURE AND COMPOSITION

Polymers that have two or more blocks or segments arranging in main

chain is meant to be block copolymer, classified according to their architecture as

AB type diblock, ABA or BAB type triblock and multiblock, where A represents the

soluble block in a selected solvent and B designates as insoluble block

(Y. Nishiyama et al., 2001). Among block copolymers, linear amphiphilic block


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copolymer play an essential role in carrying drugs on a nanoscale level

(Chun-Liang Lo et al., 2009). Amphiphilic block polymers specifically refer to

those having both hydrophilic (water) and hydrophobic (oil) blocks in the same

polymer chain, which can then build spherical polymeric assemblies in aqueous

solution, called “polymer micelle”.

Block-copolymers consist of at least two, covalently bound, segments or

blocks of different homopolymers. For instance, a triblock-copolymer can have a

general form An-Bm-Cp, with A, B, C, being different monomer types constituting

the different blocks. The subscripts n, m and p stands for the degree of

polymerization, i.e. the average number of each monomer present in each

respective block. Branched structures can also be found among copolymers, graft

copolymers. Graft copolymers can be considered as a special case of

block copolymers, a comb-like structure in which several blocks of homopolymers

B are grafted as branches to a main chain of homopolymers A, known as the

backbone.

Fig 2: Structure of Polymeric Micelle


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Fig 3: Structure of Polymeric Micelle

1.13 TYPES OF POLYMERIC MICELLES

Polymeric micelles can be classified according to the type of

intermolecular forces driving the segregation of the core segment from the

aqueous milieu. In past few decades, at least two main categories were identified,

viz.

I) Amphiphilic Micelle : Formed by Hydrophobic interaction.

II) Poly ion Complex Micelle : Formed by Electrostatic interaction.

1.13.1 Amphiphilic Micelle

The self assembly of amphiphilic block copolymers in water is based on

non-polar and hydrophobic interactions between the lipophilic core forming

polymer chains. The process is concomitantly driven by a gain in entropy of

solvent molecule as the hydrophobic components withdraw from the aqueous

media (Masato Watanabe et al., 2006). Most amphiphilic copolymers employed

for drug delivery purposes contain either polyester or a poly (amino acid)

derivative as the hydrophobic segment. Poly (lactic acid) (PLA); poly

(ε-caprolactone) (PCL); Poly (glycolic acid) are all biocompatible and


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biodegradable polyesters approved by the FDA for biomedical application in

humans. Poly (L-amino acid) (PAA), commonly used in drug delivery include poly

(aspartic acid) (PASP), poly (glutamic acid) (PGLU), poly (L-lysine), poly

(histidine) (PHIS). However, for these polymers to self assemble in to amphiphilic

micelles, the PAA segment must either be electro statically neutral or conjugated

to hydrophobic moieties. Amino acid-based block copolymers are being studied

extensively in the field of drug delivery because of their biodegradability,

biocompatibility and structural versatility. By varying the chemical structure of

PAA, it is possible to tailor their enzymatic degradability and degree of

immunogenicity. Polyesters constitute another class of polymers that can be

employed to prepare amphiphilic micelles.

1.13.2 Poly Ion Complex Micelle

Beside hydrophobic interactions, electrostatic interaction between two

oppositely charged poly electrolyte’s can also allow the formation of polymeric

micelles, which are termed “polyion complex (PIC) micelles”

(Kataoka K et al.,2001). This system has been successfully used for the delivery

of plasmid DNA (Itaka K et al., 2003), proteins (Jaturanpinyo et al., 2004), heparin

(Nishiyama N et al., 2003). As with conventional micelles, the PIC micelles

possess many advantages such as a simple preparation, the good structural

stability, high drug loading capacity, prolonged circulation in the blood, targeted

delivery, low toxicity, etc. In addition, PIC micelles have their unique

characteristics. For example, the micelles can encapsulate a variety of

therapeutic agents such as hydrophobic compounds, hydrophilic compounds,

metal complexes and charged macromolecules; the preparation of micelles are

carried out in aqueous solutions without introducing any organic solutions, which

can eliminate the side-effect caused by residual solvents.

1.14 MECHANISM OF MICELLE FORMATION

Micelle formation occurs as a result of two forces. One is an attractive

force that leads to the association of molecules while the other one, a repulsive


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force, prevents unlimited growth of the micelles to a distinct macroscopic phase.

Amphiphilic copolymers self-associate when placed in a solvent that is selective

for either the hydrophilic or hydrophobic polymer.

The micellization process of amphiphilic copolymers is similar to the

process described for low molecular weight surfactants. At very low

concentrations, the polymers only exist as single chains. As the concentration

increases to reach a critical value called the critical micelle concentration (CMC),

polymer chains start to associate to form micelles in such a way that the

hydrophobic part of the copolymer will avoid contact with the aqueous media in

which the polymer is diluted. At the CMC, an important quantity of solvent can be

found inside the micellar core and micelles are described as loose aggregates

which exhibit larger size than micelles formed at higher concentrations. At those

concentrations, the equilibrium will favor micelle formation; micelles will adopt

their low energy state configuration and the remaining solvent will gradually be

released from the hydrophobic core resulting in a decrease in micellar size.

Amphiphilic copolymers usually exhibit a CMC much lower than that of low

molecular weight surfactants. Amphiphiles with high CMC may not be suitable as

drug targeting devices since they are unstable in an aqueous environment and

easily dissociate upon dilution.

1.15 ARCHITECTURE- PROPERTIES RELATIONSHIP

1.15.1 Shape

Micellization is a procedure that minimizes the free energy of an

amphiphilic polymer solution through the formation of selectively ordered

structures. A large number of diblock polymer AB or triblock polymer ABA have

been proven to self assemble micelles in a good solvent for the A block. In

general, when the soluble block exceeds the length of the insoluble block, the

particle assumes a core/shell spherical form, evidenced by atomic force

microscopy (AFM), dynamic light scattering (DLS) and regular and cryo-

transmission electron microscopy (TEM). On the other h and, highly asymmetric


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diblocks containing long insoluble blocks and very short soluble blocks can be

hardly dissolved in water to form micelle. A special preparation is required that

involves copolymer dissolution in a certain organic solvent, followed by the

gradual addition of water. A transfer to ellipsoid, rod and lamellar micelles may

occur when altering the copolymer concentration, type and concentration of

electrolytes in the medium, temperature, organic solvent and the method of

micelle preparation.

1.15.2 Critical Micelle Concentration and Size

A critical micelle concentration value is the minimum concentration of a

copolymer that will result in micelle formation. This parameter is a very critical

indicator of micellization ability and micelle stability: the lower the CMC value,

easier the formation of micelle and more stable is the micelle. Micelles are

subject to extreme dilution upon intravenous injection into humans. If kinetically

stable, slower dissociation allows polymeric micelles to retain their integrity and

perhaps drug content while circulating in the blood above or even below CMC for

some time. Thus a lower CMC can warrant the micelle to retain its original

morphology until reaching the target site, which is a significant advantage of

amphiphilic polymers over small molecular surfactants. CMC can be effectively

measured using the fluorescent probe method. The most popular probe is pyrene

owing to its very low solubility in water, its long life time and its sensitivity of

emission and excitation spectra to the polarity of its environment.

1.15.3 Drug Encapsulation

There are two methods to load drugs: physical and chemical

encapsulation. Compared to chemical encapsulation, the physical encapsulation

of drugs within the polymeric micelle core is more attractive because many

polymers and drug molecules do not bear reactive functional groups and the

pharmacological effectiveness of the drug is maintained without chemical

modification.


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Physical modification usually operates through dialysis or O/W emulsion

methods. Parameters including solvent type, concentration and duration can

affect the morphology of the micelles and its drug encapsulation.

Physical entrapment of drugs is generally done by the dialysis or oil-in-

water emulsion procedure. The dialysis method consists in bringing the drug and

copolymer from a solvent in which they are both soluble (e.g. ethanol, N-N-

dimethylformamide) to a solvent that is selective only for the hydrophilic part of

the polymer (e.g. water). As the good solvent is replaced by the selective one, the

hydrophobic portion of the polymer associates to form the micellar core

incorporating the insoluble drug during the process. Extending the dialysis over

several days may ensure the complete removal of the organic solvent.

The oil-in-water emulsion method consists in preparing an aqueous

solution of the copolymer to which a solution of the drug in a water insoluble

volatile solvent (e.g. chloroform) is added in order to form an oil-in-water

emulsion. The micelle-drug conjugate is formed as the solvent evaporates.

Entrapment efficiency depends on the initial amount of drug added. Going

over the maximum loading capacity results in precipitation of the drug and lower

yield. Evidence of drug incorporation can be obtained by GPC or DLS since both

methods can detect a change in micellar size which usually increases in the

presence of drugs.

1.15.4 Biodistribution

Biodistribution in the body is an integrative problem related to the size,

CMC, surface charge and the targeting moiety of the micelle. Critical micelle

concentration and size can ensure shape integrity to retain the drug and extend

the circulation time of micelle, which facilitates the accumulation of drug loaded

micelle and subsequent drug release at the target site. In addition, surface

charge is another predominant factor that affects micelle biodistribution. Having


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an electrically neutral surface on the micelle should suppress the unspecific

uptake. Incorporating drugs into stealthy micelles, which present a hydrophilic

shell such as PEG, is the most effective method to prolong plasma half-lives of

the drugs by reducing interactions with the blood components and RES uptake.

PEG coating also keeps polycation/DNA complexes in the blood for a longer time

period and also enhances the micelle stability, leading to improved gene delivery

efficiency.

1.15.5 Drug Release

Typically, a drug exerts its action only after it releases from the micelle

core. When drugs are physically encapsulated in stable polymeric micelles, the

drug release rate is controlled by the diffusion out of the micelle core and /or by

dissociation of the micelles. The diffusion rate may be quiet low if the drug prefers

to interact with the core forming block. The design of block copolymer micelles

with glassy cores under physiological conditions (37 ˚C) also favors release in a

sustained manner. The release rate of the encapsulant from the micelle is

accelerated with an increased content of PEG but delayed with more hydrophobic

chains (Li Yan Qiu et al., 2006).

1.16 PHARMACEUTICAL APPLICATIONS

Theoretically, polymeric micelles may find practical applications in a

variety of pharmaceutical fields, from oral delivery to sustained release and site-

specific drug targeting. However, until now polymeric micelles have been almost

exclusively evaluated for the parenteral administration of anticancer drugs.

1.16.1 Passive Drug Targeting

Unlike hydrophilic drugs that can be delivered easily using a number of

different systems, delivery of hydrophobic drugs are more complicated. They are

more difficult to dissolve and incorporate into the more common drug delivery

systems. However, the use of polymeric micelles has been found to be effective

in delivering hydrophobic molecules. When amphiphilic block co-polymers


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(i.e. have hydrophilic and hydrophobic segments) are placed in an aqueous

environment, the large solubility difference between the hydrophilic and

hydrophobic segments drives the formation of polymeric micelles. The

hydrophobic segments form an inner core, where hydrophobic drugs can be

loaded, while the hydrophilic segments (ex. Poly (ethylene glycol) surround the

core to stabilize and increase the solubility of the device. Polymeric micelles are

currently used in the delivery of tumour-targeting drugs, like Doxorubicin.

Polymeric micelle incorporated drugs may accumulate to a greater extent than

free drug into tumours and show a reduced distribution in non-targeted areas.

Several in vivo studies showed that polymeric micelles were able to improve the

efficiency of anticancer drugs against leukemia (M.Yokoyama et al., 1990,

X.Zhang et al., 1997) and solid tumours .(M.Yokoyama et al., 1991 and X. Zhang

et al., 1997).

1.16.2 Active Drug Targeting

The EPR effect is considered as a passive targeting method, but drug

targeting could be further increased by binding pilot molecules such as antibodies

or sugars or by introducing a polymer sensitive to variation in temperature

(S.Cammas et al., 1997, J.E.Chung et al., 1998). Thermo-response may be

utilized to enhance drug release and vascular transport by local temperature

change. pH sensitive micelles could serve for the delivery of drugs to tumours,

inflamed tissues or endosomal compartments, since they are all associated with a

lower pH than normal tissue.


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Fig 4: Targeted Polymeric Micelle

1.16.3 Intracellular Delivery of Polymeric Micelle

To improve the efficiency of drug loaded micelles by enhancing their

intracellular delivery thus compensating for excessive drug degradation in

lysosomes as a result of endocytosis mediated capture of therapeutic micelles by

cells. One approach to achieve this is by controlling the micelle charge. It is

known that the net positive charge enhances the uptake of various nanoparticles

by cells. Cationic lipid formulations such as lipofectin (an equimolar mixture of

N-[1-(2,3-dioleyloxy)propyl]- N,N,N-trimethyl ammonium chloride-DOTMA and

dioleoyl phosphatidylethanalamine-DOPE), noticeably improve the endocytosis

mediated intracellular delivery of various drugs and DNA entrapped in to

liposomes and other lipid constructs made of these composition. After

endocytosis, the lipofectin-based particles are believed to escape from the

endosomes and enter a cell’s cytoplasm through disruptive interaction of the

cationic lipid with endosomal membranes. Some PEG Based micelles, such as

PEG-PE micelles, have been found to carry a net negative charge, which might


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hinder their internalization by cells. The compensation of this negative charge by

the addition of positively charged lipids to PEG-PE based micelles could improve

their uptake by cancer cells .It is also possible that after the enhanced

endocytosis, drug loaded mixed micelles made of PEG-PE and positively charged

lipids could escape from the endosomes and enter the cytoplasm of cancer cells.

Fig 5: Intracellular Delivery of Polymeric Micelle

1.17 BIOLOGICAL SIGNIFICANCE OF POLYMERIC MICELLE

A major objective of using polymeric micelles as a drug vehicle is to

modulate drug disposition in the body for better therapeutic efficacy. For

successful drug targeting, the achievement of a prolonged blood circulation of

polymeric nanocarriers might be of primary importance, because polymeric

carriers are delivered to the target tissue through the bloodstream and the

extravasation process is generally considered to be slow and in a passive

manner. However, there are several obstacles to the long circulation of polymeric

carriers, which include glomerular excretion by the kidney and recognition by the

RES located in liver, spleen and lung. The glomerular excretion can be avoided


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by using polymeric carriers with a larger size than its threshold value. On the

other h and, RES recognition may be avoidable by designing polymeric carriers to

have a size smaller than 200 nm as well as an excellent biocompatibility. It is

known that non-biocompatible nanoparticles are recognized by the RES via the

complement activation, followed by elimination from the circulation; however, the

surface modification of nanoparticles with hydrophilic and biocompatible

polymers, such as PEG, can impair or even avoid RES recognition. A highly

flexible and hydrated PEG chain attached to the nanoparticles surface is

assumed to have an effective protein-resistant property due to its steric repulsion

effect. Therefore, it is likely that polymeric micelles, nanoscale colloidal carriers

covered with a high density of PEG shells, might circumvent the aforementioned

obstacles, thus showing a stealthy property during blood circulation.

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